Abstracts |
ABSTRACTS
1. In vitro Propagation of Pointed Gourd - M.J. Alam, M.A. Mamun1 and I. Ahmad2, Department of Genetics, Shahjalal University of Science and Technology, Sylhet, Bangladesh. 1Department of Biotechnology, Shahjalal University of Science and Technology, Sylhet, Bangladesh. 2Department of Food and Tea Technology, Shahjalal University of Science and Technology, Sylhet, Bangladesh.
In vitro propagation of pointed gourd (Trichosanthes dioica Roxb.) locally known as 'patal' has been studied. Nodal segments from field grown mature plants were cultured aseptically on MS and modified (with 3% maltose) MS (MMS) medium supplemented with different concentrations of cytokinins (BA and Kn) and their combinations with NAA and CW. Almost all of the combination treatments on MS were responded to root and callus formation whereas combination treatments on MMS only responded to callus formation. Treatments of BA and Kn alone were better on MMS than MS. But the combination treatments on MS were better than those of MMS. Overall, BA responded better than Kn. The frequency of multiple shoot formation was 100% for nodal segments when cultured on MS containing 1.0 mg/l Kn + 0.05 mg/l NAA while 90% explants produced shoots on MMS containing 1.0 mg/l BA with an average number of shoots per explant was 3.44 ± 0.61. The longest shoot was found at MS + 2.0 mg/l Kn + 10% CW and that was 10.25 ± 0.18. IBA was found to be superior to NAA for root induction. Cent per cent shoots formed roots in MS containing 2.0 mg/l IBA. After hardening, in vitro raised plantlets were transferred to potted soil and survival rate was found to be 70%.
2. In vitro Mass Propagation of Stevia reboudiana and Utilization of Steviosides as a Natural Sweetner - Sumeet Ranjan and Anjanii Kumar, Thar Biotech Pvt. Ltd., 2/202 Rakshak House, South City I, Gurgaon-122001, Haryana, India
This communication describes the importance of mass multiplication of a herbal plant called Stevia reboudiana. This plant contains a compound known as steviosides, having lots of therapeutic importance. It is an excellent diabetic aid, nourishes pancreas and regulates blood glucose level. It also prevents tooth decay and checks oral infection. The need of mass multiplication arises due to more market demand of this compound, which can be utilized further as a natural sweetener. In this globalization era most of the pharmaceutical company as well as food industry is demanding for the plant extract. The present cost of a single plant is about Rs. 20 each. But the cost can be reduced by its mass multiplication. A protocol has been generated for its multiplication in vitro and for organogenesis as well. Leaf disc explants have been cultured on MS supplemented with 1.0 mg/l NAA + 2.0 mg/l BAP callus has been observed and were transferred on the same medium for organogenesis. Rhizogenesis and embryogenesis has been observed on that medium.
3. In vitro propagation of Coleus forskohlii Briq : An attempt to conserve this endangered species - Sambhavy and Anjanii Kumar, Thar Biotech Pvt. Ltd., 2/202, Rakshak House, South City I, GGN-122001, Haryana, India.
The presentation is focused on the conservation of threatened Coleus forskohlii Briq (Lamiaceae) having huge medicinal values through tissue culture. A simple and efficient protocol for in vitro micropropagation of Coleus forskohlii which is at verge of getting endangered is developed leading to complete plantlets from apical and axillary buds within 20 - 25 days and callus from leaf discs within 20 - 25 days. Ninety per cent shoot induction was achieved, when both apical and axillary buds were cultured on MS fortified with 1.0 mg/l NAA and 1.5 mg/l BAP. Optical callus was observed, when leaf discs were cultured on MS fortified with 1.0 NAA and 0.5 mg/l BAP. The experiment is also designed to conserve its germplasm.
4. In vitro Plant Regeneration Protocol of Tossa Jute (Corchorus olitorius) - K.M.K Huda, M.E. Hoque1 and A. Khatun2, Department of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh. 2Department of Biotechnology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh. 3Cytogenetics and Genetic Engineering Lab., Bangladesh Jute Research Institute, Dhaka, Bangladesh.
Seeds of C. olitorious germinated on both agar-solidified hormone free MS and cotton supported hormone free liquid MS. The percentage of seeds germinated on cotton-supported medium was found to be much higher (97.55) than on agar-solidified (45.80) medium. The seedling grown on cotton-supported medium was healthier than agar solidified medium and growth rate also higher in the same culture. In vitro germinated cotyledonary petiole used as explants for plant regeneration. Three varieties of C. olitorious such as O-9897, O-72 and OM-1 were used as experimental material. Different concentration of BAP (1, 2, 3 and 4 mg/l) and 0.5 mg/l IAA was applied with MS for in vitro regeneration. Among the hormonal combination highest regeneration (6.18 per explant) was achieved in the combination of 3 mg/l BAP and 0.5 mg/l of IAA. Plant regeneration was also observed at different concentrations (28, 56, 84 and 112 mg/l) of FeSO4 and it was noticed that MS containing 56 ml/l FeSO4 gave highest regeneration (81.94%) of tossa jute. The effect of surfactant on regeneration in tossa jute was also conducted in different concentration. Pluronic F-68 non-ionic surfactant (0.01 - 0.08%) increases the mean percentage of shoot induction. Satisfactory root induction occurs in hormone free MS. The regenerated plantlet transfer to the soil and it survives in normal environment without any morphological changes.
5. Mass Propagation of R. serpentina through Plant Tissue Culture Techniques - Deepak Kumar, Darakhshaan Akhtar Olayee and Anjanii Kumar, Thar Biotech Pvt. Ltd. 2/202, Rakshak House, South City-I, Gurgaon, Haryana-122001, India
Rauwolfia serpentina Benth. and R. vomitoria Afz. are of commercial significance internationally. It is an erect, evergreen shrub, 0.6 to 1m high, and grows in the Indian subcontinent, Burma, ShriLanka, Thailand and Indonesia. It is usually propagated from seeds although stem and root cuttings can also be used. Rauwolfia species contains more than 50 alkaloids. Reserpine and rescinnamine are the most active hypotensive agents. Other alkaloids of commercial importance are ajmalicine, ajmaline, and serpentine. The natural reserves of this plant are declining as a result of over harvest. IUCN has kept this plant under endangered status and it is listed in CITES (the Convention of International Trade in Endangered Species of wild fauna and flora). Including it among 32,500 species of appendix II means it is not threatened with extinction, but may become so unless trade in specimens of species is subject to avoid utilization compatible with their survival. Roots of Rauwolfia serpentina, the Indian snake root, or sarpagandha has long been in use as antipyretic, emmenagogue, sedative and antidote for snake poison. It needs to be propagated seeing its valued requirements and declining wild type. The mass propagation has been made possible through cell and tissue culture techniques. We have already obtained responses in MS and Gamborg’s Media.
6. The effect of drought stress pretreatment on anther culture response of wheat (Triticum aestivum L.) - S.M. Shahinul Islam, M.A. Bari and J.E. Schmid1, Institute of Biological Sciences, Rajshahi University, Rajshahi-6205, Bangladesh. 1Geschäftsleiter, Diakonie Nidelbad, Eggrainweg 3, CH-8803 Rüschlikon, Switzerland
Drought stress potency was studied on three wheat varieties, namely Barkat, Kanchan and Pavon-76 through anther culture. Anthers containing microspores were collected at early to mid uninucleate stage and harvested from spikes cold treated at 4°C for 3 - 15 days. Precise anthers were pretreated for different durations (1 - 9 hrs) to accomplish drought stress. Drought stress was found to pose a significant effect on anther culture. Regeneration potentials of the varieties were determined by estimating the percentage of anther responses on embryo induction, regeneration, production of green and albino plants. Three - five hour’s drought stress showed highest percentage of embryoids induction and green plantlets. One and two hours also gave significantly better results from control. All the genotypes produced embryos and green plantlets and among them Barkat showed best response followed by Kanchan and Pavon-76. Genotypes, under this study, produced green plants in addition to albino but seven and nine hours drought stress showed three - fourfold higher albino plant production in comparison to green plants.
7. Effect of selective subculturing on plant regeneration via anther culture of coconut - P.I.P. Perera1,4, V. Hocher2, J.L. Verdeil3, D.M.D. Yakandawala4 and L.K. Weerakoon1, 1Tissue Culture Division, Coconut Research Institute, 61150 Lunuwila, Sri Lanka, 2Institute for Research and Development (IRD), UMR 1098 BEPC, IRD, BP 64501 - 911 Av. Agropolis, 34394 Montpellier Cedex 1 - France, 3CIRAD, TA40/02 Avenue Agropolis, 34398 Montpellier Cedex 5-France, 4Post-Graduate Institute of Science, University of Peradeniya, Peradeniya, Sri Lanka.
Embryogenic calli/embryos were produced by culturing the anthers at three weeks Before splitting stage, pre-treated at 38ºC for six days, into Eeuwens Y3 liquid medium containing 9% sucrose, 100 µM 2,4-D and 0.1% activated charcoal. The embryos and calli were categorised into three groups and different plant regeneration protocols were applied. Very immature embryos with a diameter of 0.05 - 0.1 cm (group one), were subcultured again into the above medium followed by somatic embryo induction medium (with reduced 2,4-D), maturation medium (devoid of growth regulators) and germination medium (containing 5 µM BAP, 0.1 µM 2,4-D and 0.35 µM GA3). The calli and translucent embryos of 0.1 - 0.5 cm in diameter (group two) were sub-cultured into somatic embryo induction medium, maturation- and germination medium. Well defined, opaque embryos (group three), irrespective of the size, were subcultured into the maturation- and germination medium. Only 12 and 13% of the embryos in the groups one and two gave rise to shoots, respectively while the majority got vitrified. Embryos in group three gave rise to 43% shoots. Two types of embryos (with or without a germination point in the haustorium) could be observed. The germinated embryos developed into plantlets with single, double or multiple shoots.
8. Cloning of Three Medicinal Plant Species (Holarrhena antidysenterca Wall., Wedelia chinensis (Osb.)Merr. and Woodfordia fruticosa (L.) Kurz.) through in vitro Culture - S.A.M. Nurul Islam, Saiful Alam M. Tareq, Haradhan Banik and Mahbubur Rahman, Bangladesh Forest Research Institute, P.O.Box 273, Sholoshahar, Chittagong, Bangladesh.
Apical shoot tips and nodal buds were used as explants for initiating in vitro cloning and mass propagation of Holarrhena antidysenterca, Wedelia chinensis and Woodfordia fruticosa. MS was suitable for establishing culture and multiple shoots induction. In vitro multiple and axillary shoots were induced in supplemented with BAP (0.5 - 3.0mg/l and Kn (0.5 - 1.0) but sometimes GA3 was suitable for shoot elongation and healthy shoot production. Woodfordia fruticosa produced huge multiple shoots with aerial tiny roots in the culture medium containing BAP. Excised shoots of Wedelia chinensis and Woodfordia fruticosa were rooted after culturing in half strength MS containing IBA (1.0mg/l) but Holarrhena antidysenterca did not rooted in it. Though it was rooted in the half strength MS with NAA (1.0 - 3.0mg/l). The rooted shoots of the three species were successfully acclimatized and established in soil. They were vigour in nature, Wedelia chinensis and Woodfordia fruticosa produced much branching and Wedelia chinensis flowered within 12 weeks in April.
9. Seed potato production through tissue culture by aman seeds - A Sister Concern of aman group : Prospect and problems - M.A. Momin, N. Nahar and M. Hossain1, Aman Seeds, Narikel Baria, Rajshahi, Bangladesh. 1Department of Botany, Rajshahi University, Rajshahi-6205, Bangladesh.
Potato is one of the most important vegetables cum cash crop in Bangladesh. Owing to the vegetative mode of propagation potato is very much susceptible to viral and other microbial diseases. Plant tissue culture is an alternative technique for the production of disease free planting materials of potato. Aman Seeds, a tissue culture based Agro - Industry, has been producing disease indexed seed potato for the last five years. The authors present here the activities of Aman Seeds with special emphasis on the prospect and problems in establishing tissue culture based Agro-Industry in Bangladesh.
10. Collection, Identification and Biochemical Analyses of Different Sea Weeds (Marine Algae) from Saint Martin’s Island - K.M. Formuzul Haque, Shamima Yesmin Chy, Shahina Akter, M. Abdul Wahab, K.K. Nath and Habibur Rahman Vuiyah, BCSIR Laboratories, Chittagong, Bangladesh.
Five species of algae were collected from Saint Martin’s island, identified and biochemical analyses were carried out in BCSIR Laboratories, Chittagong. Biochemical composition were analysed to evaluate its food value and also to find out monthly variation in composition during the period of investigation. The protein content of Sargassum coriifolium was 16.07 % whereas in Padina tenuis that was estimated at 8.32. The percentage of fat in Sargassum coriifolium along with other seaweeds was 0.5% that does not agree with the result of Bird and Benson. In comparison with the study of “ Greentech Greenhouse Bangladesh Ltd.” with Spirulina, it was found that except the protein contents the biochemical parameters of these seaweeds were higher than that of Spirulina. The carbohydrate content in Dictyota dichotoma (38.94%) was lower among these seaweeds but more than that of Spirulina. Carbohydrate contents was higher (56.29%) in Hypnea musciformis. Mineral contents as well as other parameters especially carbohydrate contents were higher in these algae than that of Spirulina. Biochemical composition was analysed to evaluate its food value and also to find out monthly variations in composition during the period of investigation.
11. Sustained and accelerated in vitro mass multiplication with pure genetic identity in anthurium - S. Gantait, N. Mandal, S. Bhattacharyya1 and P. K. Das1 - Department of Biotechnology, Instrumentation and Environmental Science, B.C.K.V., Mohanpur, W.B.-741252, India. 1Department of Genetics, B.C.K.V., Mohanpur, W.B.-741252, India.
Anthurium andreanum, the herbaceous perennial member of Asteraceae, is most accepted both for its gorgeous long-lasting flowers and handsome foliage. Shoot tip of 30 days old plantlet was collected and inoculated in MS+BAP+NAA with 3% sucrose for shoot bud induction. First shoot bud was induced within a week in MS with 0.1 mg/l NAA and 0.25 mg/l. BAP under artificial environmental condition whereas many as six buds from single explant appeared within next 20 days. For multiple shoot proliferation MS with 0.5 mg/l BAP and 60 mg/l adenine sulphate proved to be the best resulting 10 multiple shoots per inoculated shoot bud within 50 days. Sixty single-shooted microplants were then transferred in rooting medium. Simultaneously subculture also continued for sustained shoot multiplication. As many as 11 roots per microplant were produced in 27 days using MS supplemented with 0.5 mg/l IAA and 2 g/l activated charcoal. Autoclaved sand and intermittent water spraying ensuring high humidity optimized the primary hardening period of 15 days. The semi-hardened plantlets were transferred to earthen pots filled with sand, soil, charcoal and coconut fibre mixture (2 : 2 : 1 : 1) where 51 well hardened plantlets out of 60 were recovered in next 30 days showing 85% success. From explant inoculation to hardening the total period took around 150 days. The genetic identity of both ex vitro hardened cloned and in vitro sustained clones (three months old) with their mother were tested using 10 ISSR primers. Clonal fidelity was proved as they displayed reproducible identical bands.
12. Hairy root cultures of Plumbago indica for increased production of plumbagin - Moumita Gangopadhyay and Sabita Bhattacharya, Medicinal plant laboratory, Department of Botany, Bose Institute, 93/1 A.P.C Road, Kolkata-700009, India.
Plumbago indica is the richest source of 'plumbagin' amongst all other plumbagin-yielding species, belonging to the same genus or other genus of different taxa. This toxic naphthoquinone compound, synthesized in plants roots has several pharmaceutically important properties like anticancer, antifertility, antimicrobial, in particular antituberculosis. Unsustainable medicinal use of the roots by destructing whole plant poses severe threat to their survival in several parts of India, where this species is now rarely available. Techniques supporting root growth at high rate, in particular of the hairy roots, for indefinite period in culture, retaining its medicinal properties is now not merely a state of the art laboratory method, but also effective enough in industrial application. Present study describes production and culture of hairy roots of P. indica using Agrobacterium rhizogenes ATCC 15834, confirmation of the transformed nature of the roots at molecular level and evaluation of their potential in respect of plumbagin prodution. Attempts on augmentation of this metabolite by using heavy metals as elicitors were done. Quantitative assessment of plumbagin was carried out by using high performance liquid chromatography.
13. Cell suspension culture and evaluation of cell growth in Abrus precatorious - L.A. Banu , N. Nahar and M. A. Bari, Biotechnology Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
Abrus precatorious is a very important medicinal plant, well-known for the production of lectin (Abrin) highly toxic in nature and widely used for antifertility purposes. It is relatively difficult to regenerate the plantlets in tissue culture media instead callus is produced profusely from any explants in a culture medium supplemented with hormones. Profuse callus production is rather an added advantage to develop cell suspension culture in liquid medium. In vitro grown calli were taken in MS liquid medium in agitated condition, fortified with 0.5mg/l BAP. Cells were harvested by filtration. Growth curve was prepared from cell weights taken for nine times each after two days intervals. The cells continued to grow until 12 days and growth period from 6 - 8 days was noticed as the peak period of cells growth for Abrus precatorius plant. Isolated cell produced highest 8.2% calli when suspended on MS supplemented with 0.5 mg/l BAP + 0.1 mg/l NAA. Callus derived from single cells produced highest number of embryo (25 - 28%) when cultured on MS with 0.5mg/l BAP.
14. Establishment of cell suspension culture and plantlet regeneration of Brinjal (Solanum melongena L.) - M.J. Hossain, M. Raman and M.A. Bari1, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh. 1Biotechnology Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
The aim of this study to show an efficient protocol for establishment of cell suspension culture and plantlet regeneration through cell culture from the cotyledonary explants of Brinjal (Solanum melongena L.). In this investigation we used three varieties of Brinjal cv. Loda, China and Jhotika. In first step, the somatic embryogenic calli formation was done using MS medium supplemented with different concentrations of auxin and cytokinin singly or in combination. Cells of the three varieties were isolated from the rapidly growing embryogenic and friable calli using orbital shaker. For callus induction the isolated cells were transferred to MS liquid medium containing different hormonal concentrations and after 37-63 days of incubation the micro-calli were appeared. The Loda and China varieties showed the best result (8.0 and 8.2%, respectively) in 2 mg/l NAA + 0.05 mg/l BAP and 2 mg/l 2,4-D + 0.05 mg/l BAP. For embryo formation, micro-calli were subcultured on MS solid medium and the Loda variety showed the best result (21%) in the medium containing 1.0 mg/l BAP + 0.05 mg/l GA3. The bipolar embryos were selected and cultured in MS with different combinations and concentrations of NAA and BAP and IBA for shoot and root formation. Optimum shoot and root formations were recorded in MS supplemented with 0.75 mg/l NAA+1.5 mg/l BAP and 2.0 mg/l NAA + 0.5 mg/l IBA, respectively. The plantlets appeared in the embryo mass were cultured and acclimatized.
15. In vitro regeneration protocol established in Elephant yam, Amorphophallus campanulatus Blume - a medicinal aroid - K.K. Paul , M.A. Bari and Abul Mandal1, Institute of Biological Sciences, Rajshahi University, Rajshahi-6205, Bangladesh. 1School of biology, skovde, sweden.
An experiment was conducted to investigate different plant hormonal effects on axillary cormel bud explants of elephant foot yam, Amorphophallus campanulatus Blume cv. madras for in vitro regeneration. For direct regeneration highest percentage (87) was noticed in MS medium supplemented with O.5 mg/l Kn + 2.0 mg/l NAA and followed by 84% regeneration obtained in MS containing 1.0 mg/l Kn +1.5 mg/l NAA. Highest percentage of callus induction (67) was obtained on MS supplemented with 2.0 mg/l Kn +1.50 mg/l NAA followed by 49% in MS supplemented with 1.0mg/l Kn +1.5 mg/l NAA .The calli were of compact in nature, protocorm like, varied from friable to semi friable and pinkish or off white in color. Efficient callus regeneration was observed in MS supplemented with 0.5 mg/l Kn with 1.0 mg/l NAA. Regenerated plantlets with unstable fibrous rooting were transplanted on plastic pot with fertile loamy soil in shady place and subsequently acclimatized in natural soil after two months of observation.
16. In vitro callogenesis and specific features of callus grown from three cultivars of banana in Bangladesh - N. Nahar and M.A. Bari, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
Callus induction and cell suspension culture has been a challenging work for long time in Bangladesh. Extensive research work has been done on three cultivars of banana in Bangladesh for callus induction and cell suspension culture. Three modified MS media were used for this investigation and immature male flowers were taken from 1st to 18th position in bud. Callus induction initiated within 5 - 8 weeks of inoculation and continued to grow for 5 - 6 months. Analysis of their structure, color and embryonic nature callus were categorized into six distinct types but ideal callus appeared with translucent proembryos. Among the three media used MA1 (solid) was proved to be the best performer in callus induction for banana. The growth hormone 2, 4-D played a vital role in callus initiation in banana with optimum strength 4 mg/l 2, 4-D.
17. Evaluation of Arsenic Hyperaccumulation of Fern Species in Bangladesh - F.M. Antara and M.A. Bari, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
Contamination of soils with arsenic is both toxic and carcinogenic. The genus Pteris (Pteridiaceae) is remarkable in that it has several species, including Pteris vittata , that hyperaccumulate arsenic. Ma and co-workers discovered Pteris vittata (Brake fern) is extremely efficient in extracting arsenic from soils and translocating it into its above ground biomass. When grown in areas with elevated levels of arsenic , more than 1% of the dry weight of Pteris vittata frond is arsenic. In all arsenic stricken countries screening has been made among the fern species leading to identify the most hyperaccumulating candidate in the country. An attempt to evaluate the fern species on their hyperaccumulation potentialities against arsenic contamination. Six fern species and all of them were subjected to arsenic stress under both artificial and pot culture. Different hormonal media compositions were tested for artificial culture of fern species available in Bangladesh. Two species - Pteris vittata and Dryopteris chrysocoma responded well in in vitro regeneration. MS supplemented with 1.0 mg/l BAP + 0.1 mg/l NAA was proved to be the efficient media composition for shoot multiplication from rhizome tip and prothallus development and proliferation. MS supplemented with 2.0 mg/l BAP + 0.1 mg/l NAA appeared as the most suitable media composition for callus induction but highest regeneration was obtained in MS supplemented with 0.5 mg/l BAP + 0.5 mg/l Kn. Arsenic was treated in different concentrations: 100, 200, 625,1200, 2500 and 3000 ppm to prothallus and callus and the harvested data are under the process for chemical analysis.
18. In vitro propagation of Stemona tuberosa Lour., a rare medicinal plant of Bangladesh - Animesh Biswas, M.A. Bari, Mohashweta Roy, S.K. Bhadra1, Institute of Biological Sciences, Rajshahi University, Rajshahi-6205, Bangladesh. 1Department of Botany, University of Chittagong, BangladeshAn efficient and handy repeatable protocol for in vitro large scale micropropagation of Stemona tuberosa, a rare medicinal plant of Bangladesh was developed using nodal segments on to MS supplemented with 3.0 mg/l BA and 0.5 mg/l NAA. Under continuous dark condition the nodal segments swelled without any differentiation. After 15 - 20 days exposure to dark the cultures were transferred to a daily cycle of 16/8h light/dark photoperiod and the swollen nodal segments underwent direct organogenesis producing huge number (average more than 25 shoots/culture) of shoot buds. The shoot buds went through rapid elongation on a range of BAP (0.1 - 1.5 mg/l) and IBA (0.1 - 1.0 mg/l) supplemented MS. Rooting of elongated shoot buds was successfully achieved (90%) in half strength MS with 1.0mg/l NAA. After two weeks of acclimatization, 80% of the plantlets survived in outside natural condition.
19. In vitro propagation of Plumbago indica L. - a rare medicinal plant through direct organogenesis in nodal explants - Animesh Biswas, M.A. Bari, Mohashweta Roy and S. K. Bhadra1, Institute of Biological Sciences, Rajshahi University, Rajshahi-6205, Bangladesh. 1Department of Botany, University of Chittagong, Chittagong, Bangladesh
protocol for the micropropagation of Plumbago indica L., has been developed using nodal segment explants. Multiple shoots were formed in high frequency on to MS fortified with 3.0 mg/l BAP with 0.5 mg/l NAA with in two weeks of culture. An average of 11 shoots were obtained from a single explant after two successive subcultures at 14 days interval. Microshoots were elongated in the same medium along with subcultures. Shoots were rooted on half strength MS supplemented with 1.0 mg/l NAA. In vitro grown plantlets with strong root system were successfully established in natural environment through successive phages of acclimatization. The survival rate of regenerated plants was 100%.
20. Callus Induction and High Frequency Plant Regeneration of Scoparia dulcis Linn., a Perennial Medicinal Herb, through Axillary Shoot Proliferation - A.K.M.S. Howwan, F. Afroz, L.S. Bari, J.L. Munshi, M.A.A. Jahan and R. Khatun, Biological Research Division, BCSIR Laboratories, Dhaka-1205, Bangladesh.
An efficient protocol was established for in vitro propagation of the perennial medicinal herb Scoparia dulcis (Scrophulariaceae) through indirect organogenesis in nodal segment. Green compact nodular callus was observed from nodal segments on MS basal medium supplemented with 1.5 mg/l BAP + 0.2 mg/l NAA within three weeks. The callus produced large number of shoots when subcultured on MS with 0.5 mg/l BAP + 0.1 mg/l NAA. In vitro raised shoots rooted on half strength of MS with 1.0 mg/l IBA + 1.0 mg/l NAA. For acclimatization and transplantation, the plantlets in the rooting culture tubes were kept in normal room temperature for seven days before transplanting in pots where plantlets were reared for three weeks. The survival rate of plantlets was found to be 85%. Regenerated plants were morphologically uniform having normal leaf shape and growth.
21. In vitro regeneration and genetic transformation in chickpea (Cicer aritinum) - Tanjina Akhtar Banu1, R.H.Sarker and M.I. Haque, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh. 1Tissue Culture Section, BCSIR laboratory, Dhanmondi, Dhaka-1205, Bangladesh.
In vitro regeneration system was developed based on directly organogenesis from decapitated matured embryo explants of four chickpea varieties, namely, Barichhola-4, Hyprochhola, Binachhola-3 and Binachhola-4. Best response towards multiple shoot regeneration was obtained on MS supplemented with 0.5 mg/l Kn, 0.5 mg/l BAP, 0.2 mg/l NAA along with double concentration of calcium chloride and ammonium nitrate. However, good shoot health and expanded leaves was found on MS medium containing 1.0 mg/l Kn. Apart from these, a few experiments were conducted with cotylidonary nodal explants. Highest frequency of multiple shoots were obtained on MSB medium containing 4× micronutrients of MS with 3.0 mg/l BAP and 0.04 mg/l NAA in all four varieties. Shoots regenerated on 1.0 mg/l Kn supplemented medium showed good response towards rooting medium supplemented 0.2 mg/l IBA. For genetic transformation two genetically engineered Agrobacterium tumefaciens (LBA4404/ pBI121and EHA101/pIBGUS) were used. Among the two explants maximum transformation events were obtained from decapitated mature embryo. Transformation efficiency was found best when optical density of bacterial suspension was 1.0 at 600 nm, incubated for 45 min and cocultivated for three days. Selection of putative transgenic shoots were done using various concentration of kanamycin in the regeneration medium. All control shoots died on 150 g/l kanamycin. Selection medium containing 200 mg/l kanamycin found to be effective for selecting the transformed shoots. The transgenic nature of the fully developed plantlets were confirmed through histochemical GUS assay. The frequency of GUS positive plantlet was highest in Barichhola-4 transformed with EHA 101 (plasmid pIBGUS and Binachhola-4 when it was transformed with bacterial strain LBA4404 (with plasmid pBI121).
22. Detection of some important potato viruses and viroid through multiplex-RT-PCR - M. Salim Khan and H.-P. Muehlbach1, Tissue Culture Section, BCSIR Laboratories, Dhaka, Dhaka-1205, Bangladesh. 1Department of Genetics, Institute of General Botany and Botanical Garden, University of Hamburg, Ohnhorststr. 18, D-22609, Germany.
Rreverse transcription polymerase chain reaction (RT-PCR) protocols have been developed to detect individual viruses from infected plants and crops. However, detection of several individual viruses separately by RT-PCR is expensive. A multiplex RT-PCR, which accommodates several pairs of primers in one reaction in contrast to several individual PCR reactions, is even less expensive and faster. The m-RT-PCR assays developed is a reliable, rapid, and sensitive method for the detection of plant viruses in one reaction. In this study we describe the development and optimization of a multiplex reverse transcription polymerase chain reaction (Multiplex-RT-PCR) system for the detection of potato virus A, X, Y, S, potato mop top virus and potato spindle tuber viroid (PSTVd).
23. Transient GUS Expression in Papaya (Carica papya L.) Explants after Infection by Agrobacterium tumefaciens - F. Afroz and K.M. Nassiruddin, Department of Biotech-nology, Bangladesh Agricultural University, Mymensingh, Bangladesh.
Papaya ring spot virus coat protein (PRSV0cp) gene encoding GUS gene and NPTII gene (Conferring kanamycin resistance) was used for insertion into papaya tissues through Agrobacterium tumefaciens infection. Shoot tip and axillary bud, leaf, adventitious root and mature zygotic embryo from in vitro culture were used as explants. Successful delivery of genes took place in all types of explants as was evident by GUS assay. The rate of delivery of genes was highest in mature zygotic embryo. The gene was delivered best into the cut surface of the meristematic zone when infection time was three minutes and co-cultivation period was three days.
24. In vitro regeneration and Agrobacterium-mediated transformation of Kenaf varieties : two efficient protocols for plant development - Md. Mahmudul Hasan, A.N.M. Rubaiyath Bin Rahman and Asma Khatun1, Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh. 1Genetic Resource and Seed Division, Bangladesh Jute Research Institute, Dhaka Bangladesh.
Two efficient protocols of in vitro regeneration and Agrobacterium-mediated genetic transformation were developed for two kenaf (Hibiscus cannabinus L.) varieties (HC-2 and HC-95) from cotyledon with attached petiole of 8 days old seedling as explants. Both the varieties showed best response of callus induction and shoot regeneration on same MS supplemented with 3.0 mg/1 BAP and 0.5 mg/l IAA. The highest degree of callus induction and shoot regeneration was found to be 75 and 89%, respectively, for the variety HC-2 whereas 70 and 86% were for that of HC-95. The regenerated shoots were successfully rooted on basal MS without any growth regulators. In the next experiment, Agrobacterium-mediated genetic transformation was established by inoculating cotyledons with attached petioles and hypocotyle with Agrobacterium tumefaciens strains LBA 4404, harboring both salt and drought tolerant genes (CIPK and Gly1) along with the two marker genes ( GUS and NPTII). Explants were soaked to bacterial suspension for one minute before transferring to co-cultivation medium for 24 hours. Of the two varieties, HC-2 showed comparatively better response of GUS expression (90.0% positive) and regeneration potential. In both the varieties, the stable GUS gene integration was proved by PCR amplified DNA band using GUS specific primers set. The regenerated transformed plant was developed and successfully transferred to soil.
25. In vitro regeneration of Centella asiatica (L.) Urban - an important medicinal plant - Md. Jaheduzzaman, Md. Ahsan Habib1, Md. Salim Khan1 and A.N.M. Rubaiyath Bin Rahman, Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh. 1Plant tissue culture section, BCSIR Laboratory, Dhaka, Bangladesh
In vitro regeneration of Centella asiatica was obtained from the nodal and shoot tip explants using MS with different concentrations and combinations of growth regulators. Multiple shoot regeneration was observed from both the explants on MS containing either BAP or in combinations with IAA, NAA and Kn. However, of the two explants, nodal segment showed comparatively better response towards multiple shoot regeneration. The best response of adventitious shoot development was obtained on MS supplemented with 1.0 mg/l BAP and 0.2 mg/l NAA. In this concentration about 18 shoots were initiated from single explant. For root induction, the regenerated multiple shoots were cultured on both MS and half strength of MS with various concentrations of IBA, IAA and NAA. Profuse healthy roots were obtained on MS medium containing 0.2 mg/l IBA. The well rooted plantlets were successfully transferred to soil and their survival rate under natural condition was 90%.
26. Agrobacterium-mediated Transformation of PsCBL and PsCIPK Genes for Salt and Drought Tolerance in Sugarcane (Saccharum officinerum L.) - Md. Amzad Hossain1, Hossain Md. Faruquee, Nadira Islam1 and A.N.M. Rubaiyath Bin Rahman, Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh. 1Biotechnology Laboratory, Bangladesh Sugarcane Research Institute, Ishurdi, Pabna, Bangladesh
Agrobacterium-mediated genetic transformation of two gene constructs (PsCBL and PsCIPK) confers both salt and drought tolerance along with GUS gene to four sugarcane varieties (Isd 16, Isd 34, Isd 35 and Isd 36) has been studied. Meristematic spindle leaf sheath segments were used as explants for direct regeneration of transformed plants after infection for 60, 90 and 120 minutes. To improve the transformation efficiency, acetosyringone (30 mg/l) was used during infection. GUS assay was performed after 14 days of co-cultivation of infected explants. GUS positive putative blue colour was developed in 75 to 100% of explants depending on Agrobacterium strains, sugarcane varieties and infection times. GUS positive explants were cultured on shoot regeneration medium (MS + NAA 7.5 mg/l + Kn 0.5 mg/l) with kanamycin (50 mg/l). Shoot regeneration percentage ranged from 11.11 to 77.78% depending on two genes constructs, four sugarcane varieties and three infection times. MS supplemented with NAA (5.0 mg/l) and Kanamycin (50 mg/l) was used for root development. Transgene integration into the sugarcane genome of Isd 35 and Isd 36 was confirmed by PCR amplified DNA bands using GUS primer set. Regenerated transformed plants were evaluated for salt and drought tolerance capacity. For salt tolerance analysis, MS supplemented with NaCl of different concentrations (50, 100 and 150 mM for 13.11, 17.10 and 21.66 dS/m, respectively) was used, whereas osmoticum PEG (2.5, 5.0 and 7.5%) was used for drought tolerance. Transformed plants were able to survive on supplemented media up to 150 mM (21.66dS/m) NaCl concentration and 7.5% of PEG concentration while controlled plants died. These results showed the genetic transformation of salt and drought tolerance genes in sugarcane and indicate the possibility of the development of transgenic sugarcane varieties to salt and drought stresses.
27. Low cost biotechnology for production and utilization of Stevia and Mushroom as functional food - N. Islam, M.A. Hossain and M.A.S. Miah, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh.
Micropropagation protocol and hardening method are optimized for stevia and mushroom. Consequently easy utilization and production technology are developed. Transformation of bagasse-hemicellulose to functional food through mushroom culture has been optimized. Utilization of sugarcane bagasse as substrate for mushroom production has been proved as efficient and easy means of by product utilization. For the mushroom tissue culture high tech autoclave and laminar flow cabinet has been bypassed and successful low cost production system has been developed for poor farmers of the country.
28. Genetic diversity in Bangladeshi and exotic sugarcane varieties based on microsatellite markers - M.A. Hossain, N. Islam and M.A.S. Miah, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh
To enhance sugarcane variety improvement and sustainable productivity in Southeast Asia and Pacific 30 sugarcane varieties from Bangladesh, Indonesia, Malaysia, Philippines and Thailand were evaluated using microsatellite markers. Four microsatellite or SSR (Simple Sequence Repeat) primer pairs such as SMC119CG, SMC334BS, SMC336BS and SMC687CS were used to detect the relationship among the varieties. Genetic distance and cluster analysis were made using computer software NTSYS. A total 22 alleles (bands) were identified for the four microsatellite markers. All the 22 alleles were polymorphic. Based on the 4 SSR markers, the varieties formed two clusters. The first cluster consisted of 7 varieties from breeding programme of Bangladesh Sugarcane Research Institute and 1 variety from Thailand. The second major cluster consisted of Indonesian, Malaysian, Philippino and Thai varieties. Bangladeshi varieties are distantly related from the Indonesian, Malaysian, Philippino and Thai varieties based on genetic distance estimates and cluster analysis. In making cross combinations, genetically diverse parents should be used to obtain transgressive segregants or hybrids performing better than their parents. Cross should be made using parental clones coming from different clusters because they are genetic distantly related. The results indicate the scope of gene inclusion from exotic germplasm in the breeding programme for variety development in Bangaladesh.
29. Karyotype Analysis in Three Morphological Forms of Lasia spinos (L.) Thwaites (Araceae) - Sharmin Sultana and Sheikh Shamimul Alam, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh
Three morphological forms based on the leaf character of Lasia spinosa (L.) Thwaites viz. (i) sagittate, (ii) lamina dissected and (iii) a mixed of sagittate and lamina dissected were cytogenetically investigated. The sagittate form had 2n = 27. A minute subtelocentric chromosome was found in the sagittate form. Both lamina dissected and mixed form possessed 2n=26 chromosomes. The centromeric formula of sagittate form was 19m + 7sm + 1st. It was 9m + 15sm + 2st in the lamina dissected form and 14m + 11sm + 1t in the mixed form. The total length of 2n chromosome complement was 102.23 mm in the sagittate form, 57.78 mm in the lamina dissected form and 74.46 mm in the mixed form. In orcein-staining, one satellite was found in sagittate form, two in the lamina dissected form and five in the mixed form. In CMA-staining, two big CMA- positive satellites were found in the sagittate form. Two small CMA-positive satellites were found in the lamina dissected form. The mixed form had one big and one small satellite. Two CMA-positive bands were found in sagittate, five in lamina dissected and three in mixed form. The percentage of GC-rich region was 2.88 in the sagittate, 11.23 in the lamina dissected and 6.79 in the mixed form. The above features indicated that the mixed form might be a natural hybrid between the sagittate and the lamina dissected form. Each form possessed specific karyotype which could be applied to elucidate the taxonomic rank of these three forms in Lasia spinosa.
30. In vitro Propagation of Asparagus (Asparagus officinalis) - M.N. Huda, M. Zakaria, M.M. Hossain and M.A. Haque2, Department of Horticulture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh. 1Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh.
Two experiments were conducted for in vitro propagation of asparagus (Asparagus officinalis). In first experiment, five levels of NAA (0.0, 0.1, 0.5, 1.0 and 1.5 mg/l) and five levels of BAP (0.0, 0.1, 0.5, 1.0 and 2.0 mg/l) were used for shoot induction. In second experiment, five levels of NAA (0.0, 0.1, 0.2, 0.5 and 1.0 mg/l) and six levels of IBA (0.0, 0.5, 1.0, 2.0, 3.0 and 5.0 mg/l) were evaluated for root induction. Spear segments with one node and in vitro shoots were used as explants in first and second experiment, respectively. The treatments effects were found significant in most of the characters. The highest number of shoots (6.84) and maximum percentage of shoot inducing explants (91.13) were found with 1.0 mg/l NAA. The quickest shoot initiation (4.71 days) was observed with 1.5 mg/l NAA while the longest shoot (4.71 cm) was with 0.1 mg/l NAA. The concentration of 0.5 mg/l BAP performed best regarding the number of shoots (9.41) and percentage of shoot inducing explants (91.40). Shoot initiation was the earliest (4.9 days) with 2.0 mg/l BAP while the longest shoot (5.36 cm) was observed with 1.0 mg/l BAP. The combination of 0.5 mg/l NAA and 1.0 mg/l BAP was found to be the best for number of shoots per explants (15.50) at 60 days and percentage of shoot inducing explants (95.5) whereas the earliest (4.16 days) and longest shoot (10.10 cm) were found with the combination of 0.1 mg/l NAA and 0.5 mg/l BAP. The highest number of roots per shoot (5.12) and longest root (5.57 cm) were found with 0.5 mg/l NAA while 1.0 mg/l NAA produced the earliest rooting (16.10 days) and highest percentage of root inducing shoots (72.89). The concentration of 2.0 mg/l IBA was proved to be the best regarding the number of roots/shoot (5.12), percentage of root inducing shoots (87.83), earliest rooting (19.94 days) and longest root (6.94 cm). The combination of 0.5 mg/l NAA and 2.0 mg/l IBA performed best regarding the number of roots per shoot (7.88) at 60 days, percentage of root inducing shoots (91.67), earliest rooting (14.93 days) and longest root (9.85 cm).
31. Elucidation of the structure of Chlorella galactan-protein - Md. Ashraful Haque, Toshihisa Kotake1 and Yoichi Tsumuraya1, Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh. 2Department of Biochemistry and Molecular Biology, Faculty of Science, 255 Shimo-Okubo, Sakuraku, Saitama University, Japan
Galactan was purified from Chlorella cells by disruption of the cells, followed by hot-water extraction and several chromatographic operations with DEAE-cellulose, Sephadex G-100, and Sepharose 6B columns. The galactan (Mr 41,000) consists of a large amount of sugar (60%, w/w) and a proteinous component (4.7% of Kjeldahl nitrogen), suggesting a proteoglycan nature of the complex like AGPs in higher plants. The carbohydrate moiety of the complex is rich in Gal, accounting for 76% of total neutral sugars, together with other sugars as Rha, Ara, Xyl, Man, and 3-0-methyl hexose. The galactan-protein seems to have a fundamental framework composed of (1®3)-linked b-galactan backbones attached with (1®6)-liked b-galactan side chains. The carbohydrate moiety was digested with endo-b-(1®6)-galactanase, resulting in liberation of a large amount (62%, based on total sugar) of low-Mr products together with a high-Mr fraction (38%) resistant to the enzyme attack. The low-Mr products were tentatively identified as Gal, b-(1®6)-Gal2, and -Gal3 together with their derivatives substituted with methyl groups at 0-3. On the other hand, the sugar chains were slightly hydrolyzed by exo-b-(1®3)-galactanase with lesser extent (yield of low-Mr products, 23%). Based on these results, Chlorella galactan-protein appears to have an essential carbohydrate architecture similar to that of AGPs in higher plants.
32. Action of b-glucuronidase from Aspergillus niger on arabinogalactan-protein (AGP) - M.A. Haque, T. Kotake and Y. Tsumuraya1, Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur, Bangladesh. 1Department of Biochemistry and Molecular Biology, Faculty of Science, 255 Shimo-Okubo, Sakuraku, Saitama University, Japan
A b-glucuronidase purified from a commercial pectolytic enzyme preparation of Aspergillus niger hydrolyzed about half of the 4-0-methyl-glucuronic acid (4-Me-GlcA) residues located at the nonreducing terminals of (1®6)-linked b-galactosyl side chains of the carbohydrate portion of a radish arabinogalactan-protein (AGP) modified by treatment with fungal a-L-arabinosidase. Digestion of the a-L-arabinosidase-treated AGP with exo-b-(1®3)-galactanase released, by exo-fission of b-(1®3)-galactosidic bonds in the backbone chains of the AGP, neutral b-(1®6)-galactooligosaccharides with various chain lengths and their acidic derivatives substituted at their nonreducing terminals with 4-Me-b-GlcA groups. In contrast, successive digestion of the a-L-arabinosidase-treated AGP with b-glucuronidase followed by exo-b-(1®3)-galactanase liberated much higher amounts of b-(1®6)-galactooligomers together with a small portion of short acidic oligomers, mainly 4-Me-b-GlcA-(1®6)-Gal and 4-Me-b-GlcA-(1®6)-b-Gal-(1®6)-Gal. These results indicate that the b-glucuronidase acts upon 4-Me-b-GlcA residues in long (1®6)-linked b-galactosyl side chains of the AGP, whereas short acidic side chains survive the attack of the enzyme.
33. Improvement of Eggplant (Solanum melongena L.) through Agrobacterium- mediated transformation - R.H. Sarker, Sabina Yesmin and M.I. Hoque, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.
Agrobacterium-mediated transformation system was developed for two local varieties of eggplant (Solanum melongena L.), namely Singhnath and Baribegun-4 (kazla). Transformation experiments were conducted using Agrobacterium tumefaciens strain LBA 4404 harbouring binary plasmid pBI121, containing the GUS reporter gene and nptII gene conferring resistance to kanamycin. Transformation ability of different explants like cotyledonary leaves, hypocotyls, shoot tip and roots were tested. Among the four explants cotyledonary leaves explant showed highest percentage of GUS positive by transient assays. Cotyledonary leaves explants were also found to be more effective in formation of multiple shoots on MS supplemented with 1.0 mg/l BAP, 1.0 mg/l Kn and 0.1 mg/GA3 following Ageobacterium infections and selection. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin up to 200 mg/l. The selected kanamycin resistant shoots were rooted on MS without selection pressure. Transformed rooted plantlets (T0) were successfully transferred to soil where two of these plants produced fruits and set viable seeds. The stable expression of the GUS gene was observed in different parts of the transformed shoots such as leaf, stem, root, anther, corolla, stigma and pollen grains. The seedlings (T1) developed from these two plantlets (T0) were morphologically normal and also exhibited the expression of GUS gene.
34. In vitro regeneration of two varieties of potato (Solanum tuberosum L.) - Sabina Yesmin and Shamima Nasrin, Plant Division, NIB, Savar, Dhaka-1349, Bangladesh.
Two varieties of potato namely, Diamant and Granula were used in the present investigation to develop a suitable in vitro regeneration protocol using nodal and internodal segments. Maximum shoot regeneration was observed on MS supplemented with 1.0 mg/l BAP + 0.5 mg/l Kn + 0.1 mg/GA3 in both the varieties. MS without any hormonal supplement was sufficient for root induction from excised shoots. The in vitro regenerated plantlets were successfully transplanted to the soil and set tuber in green house condition.
35. Comparative Karyotype Analysis of Typhonium trilobatum and its two Morphological Forms - Shohana Huq, Hosne Ara, Md. Abul Hassan and Sheikh Shamimul Alam, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh
Typhonium trilobatum ( Coarse form) and its two morphological forms viz. tall and slender were cytogenetically investigated. Typhonium trilobatum and its tall form were found to possess 2n=18 chromosomes whereas the slender one has 2n=19 chromosomes. The extra chromosome of the slender form was a regular member of pair IX. The centromeric formula of the tall form was 12m + 6sm. It was 16m + 2sm and 17m + 2sm in T. trilobatum and the slender form, respectively. The orcein-stained interphase nuclei and prophase chromosomes revealed that the tall form had much less heterochromatins than T. trilobatum and the slender form. The Tall form possessed a pair of satellites which were found in orcein and CMA-staining. In DAPI staining, satellites showed negative banding which indicated its fully GC-rich nature. Satellite was not found in other two forms. A few chromosomes of each form fluoresced entirely with CMA. It indicated that these forms might have derived those chromosomes from a common ancestor. Due to the unique nature, those chromosomes could be used as marker. DAPI banded karyotypes showed similarities between T. trilobatum (Coarse form) and the slender form. It was much different in the tall form. The overall karyotypic features indicated that the tall form is quite different from the other two and thus it may be placed in different taxonomic rank. However, the Slender form may be considered as a trisomic variety of T. trilobatum (Coarse form).
36. Identification of a Leucine-rich repeat transmembrane protein Kinase gene in Jute - Zinnatun Nabi, Muhammad Shoyaib1, Shakhinur Islam Mondal2, Md. Maksudul Alam2, Md. Shahidul Islam2, Sazia Sharmin Sejuti2, Sarmah Bin Nayeem and Haseena Khan2, Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh. 1Institute of Information Technology, University of Dhaka, Dhaka-1000, Bangladesh. 2Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh.
Leucine rich repeat protein plays an important role on morphogenesis, embryogenesis, meristematic growth, pollen self-incompatibility and stress related signal in plant. We have identified a leucine-rich repeat transmembrane protein kinase (LRTP) using several gene finding software based on DNA sequences obtained from a jute genomic library constructed for designing jute specific SSR markers. Sequence analyses revealed that concurrence sequences of the leucine-rich repeat motifs are highly conserved among these proteins. Multiple alignments of the sequences revealed an overall nucleotide sequence similarity with leucine-rich repeat transmembrane protein kinase gene of Arabidopsis thaliana. We have finally confirmed the presence of this gene is jute genome by RT-PCR and DNA sequencing. Our future work is now based on using the degenerate primers (based on Arabidopsis thaliana leucine rich repeat sequences) to get the full length coding sequence of this gene.
37. Tissue Culture Independent Transformation Protocol for Jute - Abu Ashfaqur Sajib1, Md. Shahidul Islam2,3, Md. Shamim Reza2, Arpita Bhowmik2, Layla Fatema2 and Haseena Khan2, 1Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh. 2Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh. 3Bangladesh Jute Research Institute, Sher-e-Banglanagar, Dhaka, Bangladesh.
In vitro regeneration has been quite difficult with the Corchorus species through tissue culture techniques and several transformation attempts based on tissue culture have failed. In this attempt three different Agrobacterium tumefaciens mediated transformation techniques, all devoid of tissue culture steps, were used for establishing easy and efficient transformation protocols for both of the cultivated species of jute, Corchorus capsularis and C. olitorius. Heritable transmission of the transgene (GUS) to progeny from genetically manipulated plants was confirmed by GUS gene expression study following RT-PCR, assaying for GUS activity and PCR amplification of stably transmitted GUS gene sequence in plant genome. The transformation rate is sufficiently high. This has now paved the way for future genetic manipulations of jute.
38. Bioinformatic Analysis of Genomic DNA of Jute - Md. Maksudul Alam, Rozalynne Samira, Mahdi Md. Mosa1, Md. Ashraful Islam Bhuuiya1 and Haseena Khan, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh. 1Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh.
Our laboratory has 137 jute genomic DNA sequences most of which was not annotated previously. In this in silico experiment our aim was to annotate these sequences. We have characterized these sequences by implementing several bioinformatic tools available online. We have used Fgenesh+, GenScan, EST BLAST, TSSP, find miRNA, Expasy translation tool, VecScreen and CpG finder for finding out gene, ESTs, regulatory regions, miRNA, transposable elements, screening vector sequence, CpG island and Simple Sequence Repeats from these sequences. We annotated the putative genes based on their homology with known genes of other species. We have found several transcription regulatory genes, stress-related genes, transmembrane kinase and some housekeeping genes. Few genes showed homology with bacterial genes that could be probably for horizontal transformation. We believe such work for understanding molecular biology of jute will help us to find genes of agronomical importance as well as ESTs and STSs that will contribute to Jute Genome Project to be undertaken soon.
39. Jute SSR markers : for MAS and genome mapping - Minhaz Uddin Pahloan, Shamima Islam Keka, Jesmine Akhter, Saaimatul Huq, Shams Zaman1 and Haseena Khan, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh. 1Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh.
Infection with red and yellow mite is a major biotic stress of popular jute varieties that causes huge loss each year by detriorating fiber quality. An advanced line O-7/95 developed by BJRI is tolerant to mite infection. An F2 population was developed following a cross between O7/95 (mite tolerant) and O-72 (mite sensitive popular variety) at BJRI. Under this study, specific SSR primers were used to establish markers linked to mite resistant trait for facilitating marker aided selection (MAS). A preliminary genetic linkage map was constructed using ten SSR markers with an F2 population of 50 individuals. This analysis identified five different linkage groups where three belonged to single linkage clusters. Several manipulation of the linkage group analysis confirmed a true linkage between two markers and finally, one of the SSR markers, HK-66, appeared to be linked to mite tolerance trait in jute.
40. From Markers to Genes : Identification and Characterization of an Expressed Sequence Linked to low Temperature Tolerant in Jute - Samiul Haque, Nadim Ashraf, Aleya Awal, Saaimatul Huq, Md. Maksudul Alam, Nazlee Sharmin, Md. Shamim Reza and Haseena Khan, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh.
Jute usually grows at a base temperature of 20ºC, however, four accessions have been identified from the seed bank, which are able to grow at a lower temperature of 16ºC. In order to be used in marker assisted selection, 50 RAPD and 12 ISSR primers were used to screen the four tolerant accessions and compared with two low temperature susceptible jute varieties. The polymorphic markers identified in the experiment were used to screen an F2 population raised from a cross between Acc. 1805 (cold tolerance genotype) and O-9897 (high yielding variety), showing differential tolerance to low temperature. One randomly amplified polymorphic DNA (RAPD) marker, OPG05, was found to be strongly linked to the trait. This was then further characterized to identify single nucleotide variations at the RAPD binding site and within the fragment. The polymorphic marker was also sequenced, which revealed a 153bp expressed region showing a strong homology with the translated product of the terminal exon of an Arabidopsis gene. RT-PCR confirmed the expression of the same. A 5' RACE was then developed to determine the coding sequence of an unknown jute gene, based on primers designed from this 153b sequence. The sequences found from 5'RACE showed very strong homology with LDLKP proteins with other plants. Based on this strong homology degenerate primers were designed. Two steps of amplification using different sets of degenerate primers have provided additional sequences of this putative LDLP gene. This has enabled us not only to identify a molecular marker but also to identify a jute expressed sequence closely linked with the low temperature tolerant trait.
41. Agrobacterium-mediated genetic transformation of white jute (Corchorus capsularis L.) - G.M. Al-Amin, M.I. Hoque and R.H. Sarker, Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka -1000, Bangladesh
Quality and productivity of white jute (Corchorus capsularis) are affected by various biotic and abiotic stresses. About 60% of yield is lost due to incidence of fungal diseases alone. So, it is very important to develop improved varieties of jute resistant to biotic and abiotic stresses. Genetic transformation has been developed for two varieties of white jute, namely, CVL-1 and CVE-3 using Agrobacterium tumefaciens strain LBA4404 containing binary plasmid pBI121 with GUS reporter gene and nptII genes conferring kanamycin resistance. Maximum transformation ability was obtained from petiole attached cotyledon explants of CVL-1 and CVE-3 with bacterial suspension having an optical density of 1.1 at 600 nm monitored through transient GUS histochemical assay. In case of mature embryo explants the highest efficiency of transformation was achieved when the explants were incubated in bacterial suspension having the optical density of 1.72 at 600 nm. Regeneration from the transformed petiole attached cotyledon explants was obtained on MS with 0.2 mg/l BAP and 1.0 mg/l IAA for CVL-1. On the other hand such regeneration of explants from CVE-3 was obtained on MS with 2.5 mg/l BAP and 0.5 mg/l NAA. Selection of transformed shoots was carried out by applying kanamycin. Selection medium containing 50 and 200 mg/l kanamycin were found to be effective in selecting transformed shoots developed from petiole attached cotyledon and mature embryo explants, respectively. The shoots those survived following selection process were subjected to rooting on MS with 0.3 mg/l IBA. The stable expression of the GUS gene was observed in the different tissues of shoot, stem and root of transformed plantlets. Histochemical GUS expression was mostly observed in the plantlets obtained from the mature embryo explants. Genomic DNA was isolated from these shoots and stable integration of GUS and nptII genes was confirmed by PCR analysis.
42. Validation of DNA Markers from the Saltol Region in Specific Breeding Populations - A. Ferdousi, M. S. Rahman1, R. Alam, S. Rahman, Dante Adorada2, G. Gregorio2 and Z. I. Seraj, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka 1000, Bangladesh. 1Plant Physiology Division, BRRI, Gazipur-1701, Bangladesh. 2Plant Breeding, Genetics and Biochemistry Division, IRRI, Los Banos, Philippines
Salt tolerance is a polygenic trait and is therefore difficult to select by classical techniques. The advent of marker aided selection permits rapid and efficient identification of the individuals that contain the genes of interest. A major locus for salinity tolerance trait of the rice landrace, Pokkali, called Saltol has been previously identified on chromosome 1. Thirteen markers from this locus were used to analyze the F6 population between salt tolerant IR60494 and salt sensitive BR29. IR60494 has Pokkali and Nona Bokra as the source of salt tolerance trait. 978 individuals were screened to identify and select 198 tolerant and sensitive extremes for marker analysis and selective genotyping. Out of 25 markers tested, 7 were polymorphic. RM493 at 12.2 Mbp was found to be significantly linked to salt tolerance traits at the 1% level. Linkage of RM 1287, CP06224, and RM10793 located at 10.9, 12.07 and 12.5 Mbp were significant at the 5% level. The P-values (probability values) of these markers from the analysis of variance were 0.006. 0.014, 0.053 and 0.039, respectively. Thus, these markers should prove to be useful in selection of the Saltol locus in breeding programs.
43. Identification of Salt Tolerance QTLs in the Rice Landrace, Boilam and their Introgression into Farmer Popular Varieties - H.B. Shozib, M.J. Thomson1, M.A. Salam2, A.M. Ismail1 and Z.I. Seraj, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh. 1Crop and Environment Science Division (CESD), IRRI, Philippines. 2Plant Breeding Division, BRRI, Gazipur-1701, Bangladesh
Bangladesh is affected by soil salinity in about a third of its total cultivable land. The landrace Boilam grows well in the saline zone in SE Bangladesh; however it gives poor yields. Boilam is also photoperiod insensitive and early maturing, unlike the common salt tolerance donor rice, Pokkali. Pokkali is also not very receptive to crossing. This investigation was designed to identify novel Quantitative Trait Loci (QTL) linked to salt tolerance traits of Boilam and introgress these into modern rice varieties. BC2F2 progenies from a cross between salt tolerant rice Boilam and farmer popular rice variety BR27 was developed for this experiment. Their phenotypic and genotypic data were determined by using a set of 108 polymorphic markers and tolerance to seedling salinity stress. The data was analyzed by the Q Gene mapping software. Four putative QTLs linked to salinity have been identified on different chromosomes of rice with the help of the polymorphic markers. Qtl1, R9M30 (LOD 6.16, R2 25.81%), qtl2, RM490, (LOD 4.88, R2 21.67%), qtl3, RM17 (LOD 4.72, R2 20.26%) and qtl4, RM28746 (LOD 4.54, R2 19.57%) were identified on chromosome 9 and 1 and two on chromosome 12 respectively. Salt tolerant BC2F3 having the background genotype of BR27 will be developed further for release or use as parents in breeding programs with the help of the markers-linked to the salt tolerance QTLs.
44. Introgression of ‘Saltol’ QTL into Bangladeshi mega rice variety BR11 and BRRI dhan28 through marker assisted backcrossing - M.S. Rahman, M.R. Islam1, S. Rahman2, A. Ferdousi2, R. Malo2, M. J. Thomson3, M. A. Salam1, A. M. Ismail2 and Z. I. Seraj2, Plant Physiology Division, BRRI, Gazipur-1701, Bangladesh. 1Plant Breeding Division, BRRI, Gazipur-1701, Bangladesh. 2Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh. 3Crop and Environment Science Division (CESD), IRRI, Philippines
Marker Assisted Backcrossing (MAB) approach has been shown to be a very successful breeding approach for getting desirable genetic gain even over complex traits in the shortest possible time. MAB is, therefore, being used for incorporating ‘Saltol’ into the Bangladeshi mega rice varieties BR11 and BRRI dhan28 for targeting the T. Aman and Boro seasons, respectively. ‘Saltol’ is a major Quantitative Trait Loci (QTL) in the short arm of chromosome 1 and was mapped and validated earlier at IRRI (11.2-12.79 mbp). It is linked to the Na/K ratio in seedlings under salt stress and accounted for 65% of the phenotypic variation with a LOD >8.0. For introgression of ‘Saltol’ into BR11 and BRRI dhan28, crossing with FL378 (a RIL developed at IRRI from cross of IR29 and the salt tolerant donor Pokkali B) and repeated backcrossing with the recurrent parents was started at BRRI in the year 2006. For the foreground selection three markers i.e. AP3206 (11.20 Mb), RM3412 (11.50 Mb) and RM493 (12.20 Mb) were used to confirm the presence of the QTL. For the recombinant selection two markers flanking the QTL on either side, RM3627 (10.31 Mb) and RM562 (14.60 Mb) were used. For the background selection a set of 48 polymorphic markers were used for recovery of the recurrent parent genome. Out of 28 BC1F1 plants selected with the QTL and recombination for BR11 flanking Saltol, 5 were shown to have a background recovery in the range of 15.22-54.35%. BC2F1 progenies are now being analyzed by the three-step selection process. To obtain the desired salt tolerant BR11 with >90% RPG, would mean selfing of the selected progeny to BC2F2. Less than 90% RPG would necessitate another round of backcrossing and the three-step selection.
45. Agrobacterium-mediated transformation of OsNHX1 and PgNHX1 in Binnatoa under constitutive Actin1D and CaMV promoters - R.S. Tammi*, S.M. Touhidul Islam1, L.A. Lisa, S.L. Singla-Pareek2 and Z.I. Seraj, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh. 1Department of Genetic Engineering and Biotechnology, University of Dhaka, Bangladesh. 2International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. *Present address: Department of Biochemistry and Molecular Biology, Jahangir Nagar University, Savar, Bangladesh
Among the various stresses that impair crop productivity, salinity is a major threat to agriculture. A high salt concentration in the vicinity of a plant disrupts the ability of the roots for efficient water uptake, thereby leading to perturbation of crucial metabolic reactions inside the cell. Mechanisms that confer salt tolerance vary with the plant species; however the basic strategy works towards the maintenance of Na+ homeostasis in the cytosol. One of these is the sequestration of excess sodium into the vacuoles via vacuolar Na+/H+ antiporter (NHX). Na+/H+ antiporters, which catalyze the exchange of Na+ for H+ across membranes, contribute to the regulation of internal pH, cell volume and the sodium level in the cytoplasm. The antiporters are widespread membrane proteins found in animals, yeasts, bacteria and plants. In particular, we have investigated over-expression of vacuolar Na+/H+ antiporters in Oryza sativa cv Binnatoa to provide increased salt tolerance. In saline environments, an active vacuolar antiporter utilizes the proton motive force generated by vacuolar ATPases and pyrophosphatases (Pipases) to sequester excess Na+ into the vacuole, thereby reducing the toxic effects of Na+ inside the cytosol and utilizing these ions for maintenance of turgor in the vacuole for cell expansion and growth. Orthologs of Na+/H+ antiporter genes have been isolated from both glycophytes and halophytes. We have developed and functionally characterized Agrobacterium-mediated transformed Binnatoa with OsNHX1 (from rice) and PgNHX1 (from pearl millet, gift from ICGEB) under the constitutive Actin1D and CaMV35S promoter respectively. Our data demonstrate the potential of OsNHX1 and PgNHX1 for imparting enhanced salt tolerance capabilities to rice varieties for growing in saline areas.
46. Rice Transformation for Salt Stress Tolerance - R. Malo, L.A. Lisa, S. Jahan, L. Parvin, F. Naznin, M. Amin, R.S. Tammi*, N.M. Rasul, M. Nasiruddin, S. L. Singla-Pareek1, N. Tuteja1, R.H. Sarker2 and Z. I. Seraj, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh. 1International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. 2Department of Botany, University of Dhaka, Dhaka 1000, Bangladesh. *Present address: Department of Biochemistry and Molecular Biology, Jahangir Nagar University, Savar, Bangladesh
Salinity is one of the major problems encountered during crop production in Bangladesh. Transgenic approaches for solution to this problem have been reported, but success with rice has been limited. Several genes were used for producing transgenic rice and shown to confer improved salt tolerance. We have transformed the tissue culture-responsive landrace Binnatoa (BA), with the OsNHX1 construct containing the 5’ UTR and ORF and are also using the complete transcript. Transformed plants at the T3 were checked for integration by Southern and showed improved performance during seedling salinity screening and expression using semi-quantitative RT-PCR. The transgene has been backcrossed into farmer-popular dry-season cultivars, BR28 and BR45 and have shown expression in their new genetic background. Another gene, the Pea DNA helicase (PDH45), obtained from ICGEB, New Delhi, was also used for transformation. PDH45-transformed T1 BA rice having single gene insertion showed very good morphology as well as dramatically improved salt stress tolerance at the seedling stage. The glyoxalase pathway was also reported to be important in improving salt tolerance. Therefore, genes for the two enzymes of this pathway obtained from ICGEB, New Delhi, are also being used for transformation of BA rice. To express all these genes, a good promoter is essential. Commercial binary vectors contain the CaMV 35S promoter. It has been reported that CaMV 35S is a poor promoter for gene expression in rice. Therefore, we are isolating and characterizing promoters reported to be salt stress-inducible. The endogenous promoters of the NHX1 gene from salt tolerant Pokkali and salt sensitive IR64 fused with the GUS gene, PKN and IRN respectively, were shown to be 25-fold more responsive than CaMV 35S in rice calli. In regenerated transformed BA rice plants, PKN showed differential expression pattern in leaves under salt stress. Other stress related promoters- Asr, CCOMT, Apx and HKT have also been isolated and are being characterized.
47. Identification and Confirmation of Jute Genes of Agronomical Importance - Salim Ahmed, Zinnatun Nabi, Muhammad Shoyaib1, Shakhinur Islam Mondal2, Md. Maksudul Alam and Haseena Khan2, Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka-1000, Bangladesh. 1Institurte of Information Technology, University of Dhaka, Dhaka-1000, Bangladesh. 2Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh.
In molecular biology gene identification is a foremost and essential step not only for understanding a genome but also for its manipulations requiring incorporation of novel characteristics. But prediction of a gene and its confirmation is not an easy task. Although a few gene finding tools are available online but acceptance of their accuracy is not always beyond question. A combination of these online tools like GeneMarkhmm and Genscan with gene/exon finding programmes and BLAST searches along with the west lab techniques of molecular biology such as RT-PCR and DNA sequencing can satisfy the query of gene prediction with optimum firmness. In order to find genes of jute, the golden fibre of Bangladesh, techniques mentioned above were sequentially applied to clones of a genomic library of jute enriched for simple sequence repeats. Though in this study several sequences were established as the gene sequences of which a few were found interesting in terms of their agronomical properties.
48. In vitro regeneration and Agrobacterium-mediated genetic transformation of white jute (Corchorus capsularis L.) – Bivas Kumar Sarkar, M.I. Hoque and R.H. Sarker, Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka -1000, Bangladesh.
An in vitro regeneration protocol has been developed for the three local varieties of white jute (Corchorus capsularis L.) namely, CVL-1, BJC-7370 and BJC-83. The regeneration system is based upon direct organogenesis from cotyledon with petiole, cotyledonary node, and mature embryo explants. High frequency of shoot regeneration was achieved from cotyledon with petiole and cotyledonary node explants through the use of MS supplemented with 0.2 mg/l BAP and 1.0 mg/l IAA for CVL-1. In case of BJC-7370 best response for shoot initiation was obtained in 0.5mg/l BAP and 1.0 mg/l IAA. For BJC-83 high frequency of shoot initiation was obtained on 1.25 mg/l BAP and 0.25 mg/l NAA using the same explants. Multiple shoot formation and elongation of shoots was achieved on MS supplemented with 0.2 mg/l BAP for all the three varieties. Regenerated excised shoots developed effective in vitro root system in half strength of MS supplemented with 0.3 mg/l IBA for all the varieties. Plantlets from the three jute varieties were successfully transplanted into soil. Flowers and fruits were found to be developed on the in vitro raised plantlets identical to those observed in the original plants. Agrobacterium-mediated genetic transformation system was also developed for the production of transgenic white jute. For genetic transformation, genetically engineered Agrobacterium tumefaciens stain (LBA4404 with pBI121) possessing GUS and nptII gene within its left and right border of T-DNA was used. Among all the explants, cotyledon with petiole and mature embryo explants showed the best response towards transformation. A medium containing 50 and 150 mg/l kanamycin were found to be effective in selection of transformed shoots developed from cotyledon with petiole and mature embryo explants, respectively. Thus the plantlets survived in presence of selection pressure of kanamycin were primarily regarded as transformed plants. Histochemical GUS assay was used to detect the expression of GUS gene of the fully developed plantlets. Gus expression was mostly observed in the plantlets obtained from the mature embryo explants.
49. Preliminary studies on the development of fungal disease resistant peanut (Arachis hypogaea L.) through genetic transformation - Md. Niamul Kabir, M.I. Hoque and R.H. Sarkar, Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.
Leaflet explants of two varieties of peanut, namely BARI badam-6 and Dhaka-1 were collected from six - eight days old in vitro grown seedlings. MS medium with different combinations of BAP and Kn was used for shoot regeneration. Best direct multiple shoot regeneration was achieved when explants were cultured on MS medium containing 5.0 mg/l BAP + 0.5 mg/l Kn in case of BARI badam-6. For shoot elongation 0.1 mg/l GA3 was added in the regenerating medium. Addition of GA3 showed good response toward shoot elongation in case of BARI badam-6. MS containing 5.0 mg/l BAP + 0.5 mg/l Kn + 0.1 mg/l GA3 showed best response towards multiple shoot regeneration in case of BARI badam-6. However, Dhaka-1 showed best multiple shoot regeneration on MS containing 2.5 mg/l BAP + 0.1 mg/l Kn. Regenerated shoots were cultured on half strength of MS supplemented with IAA and NAA for root induction. Best root induction and subsequent development was observed on 0.2 mg/l IAA for both the varieties. Transformation experiments were performed using leaflet explants from Dhaka-1 and BARI badam-6. Three strains of Agrobacterium, namely LBA4404 (strain I) harbouring the binary plasmid pBI121 conferring GUS and nptII genes, Agrobacterium strain LBA44042301 (strain II) contained plasmid pICGEB-Ca-AFP1 conferring GUS, nptII and antifungal gene Ca-AFP1. The third strain was EHA105-pGII-pSOUP-VST-CHIT containing antifungal and nptII genes. Since strains I and II contained GUS gene, transformation experiments were monitored through GUS histochemical assay. About 87% of the explants showed GUS expression following transformation with LBA4404 harbouring the binary plasmid pBI121. Maximum transformation of leaflets was obtained with bacterial suspension having an optical density of 1.2 at 600 nm. Moreover, 60 min of incubation followed by 72 hours of co-cultivation was found to be most effective towards transformation. It was possible to recover transformed shoots using the bacterial strain LBA4404 containing GUS and nptII genes. Shoot regeneration was also observed following transformation with bacterial strain EHA105 containing antifungal chitinase, nptII and bar genes.
50. Development of fungal resistant Chickpea (Cicer arietinum) varieties - M. I. Hoque, R.H. Sarker and R. A. Sharmin, Plant Breeding and Biotechnology Lab., Department of Botany, University of Dhaka, Dhaka - 1000, Bangladesh.
The main objectives of the present investigation was to develop a suitable protocol for fungal resistant chickpea (Cicer arietinum) varieties through Agrobacterium-mediated genetic transfor-ation of locally grown chickpea varieties of Bangladesh. n order to develop a successful transformation protocol, a suitable and reproducible in vitro regeneration protocol was established using LS of embryo, cotyledon attach with embryo axis, decapitated embryo explants of chickpea varieties, namely Barichhola-2, Barichhola-4, Barichhola-5. Best response towards multiple shoot regeneration was obtained on MS supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn, 0.2 mg/l NAA along with double concentrations of CaCl2 and KNO3. Rooting of chickpea was very difficult. To solve this problem to LS of embryo explants was used on only MS medium. Here single shoots and roots were obtained on only MS medium. Following the formation of roots in vitro regenerated plantlets were successfully established in soil. For genetic transformation two strains of genetically engineered Agrobacterium tumefaciens EHA105/pG11 pSOUP Vst-N-Chit and LBA44042301/pICGEB-Ca-AFP1 were used. Three chickpea varieties, namely Barichhola-2, Barichhola-4, Barichhola-5 were used for this transformation. Three types of explants LS of embryo decapitated embryo and cotyledone with embryo axes were used. Among these explants maximum transformation events was obtained from decapitated embryo and LS of embryo. Most of the cases the transformation zones of these explants coincide with the regeneration points. Transformation efficiency was found best when density of overnight grown bacterial suspension was 1.0 of 600 nm and incubated for 45 minutes. Three days cocultivation period of the transformed explants was also found optimum for both the bacterial stains. Selections of putative transgenic shoots were done using various concentrations of kanamycin in the regenerating medium. Selection medium containing 200 mg/l kanamycin was found to be effective in selecting of transformed shoots. Shoots survived on the highest selection pressure for 15-20 days were considered putative transformed shoots. Stable expression of GUS gene was detected in the putatively transformed plantlets and shoot through GUS histochemical assay.
51. Development of fungal resistant Lentil (Lens culinaris Medik) varieties - M. I. Hoque, R.H. Sarker and Surupa Das, Plant Breeding and Biotechnology Lab., Department of Botany, University of Dhaka, Dhaka - 1000, Bangladesh.
The aim of the present investigation is to develop a suitable protocol for fungal resistant lentil varieties through Agrobacterium-mediated genetic transformation of locally grown lentil varieties of Bangladesh. In order to develop successful transformation protocol, a suitable and reproducible in vitro regeneration protocol was established using two local varieties of lentil (Lens culinaris Medik), namely Barimasur-2 and Barimasur-4. Three different explants of lentil such as cotyledonery node, cotyledone attached decapitated embryo, decapitated embryo were used for this purpose. As a integral part of transformation a series of experiments also conducted for the regeneration of in vitro plantlet using these explants. Best response towards shoot regeneration was obtained from cotyledon node on MS medium supplemented with 0.5 mg/l BAP + 0.5 mg/l kn + 0.1 mg/l GA3 + 5.5 mg/l tyrosine. But such shoots failed to produce effective root system. Under these circumstances to overcome the problem MS without any hormonal supplement is used. Here longitudinal section of embryo is used as explant. Highest frequency of roots were obtained on MS medium. Following the formation of roots in vitro regenerated plantlets were successfully established in soil. In vitro flowering and seed setting experiment were carried out in vitro raised shoots from cotyledonry node and cotyledon attached decapitated embryo. The best response regarding the development of in vitro flower was obtained on half the strength of MS containing 20 mg/l IBA, 0.50 mg/l NAA. For genetic transformation, two strains of genetically engineered Agrobacterium tumefactions EHA105/pG11 pSOUP Vst-N-Chit and LBA44042301/pICGEB-Ca-AFP1 were used. Agrobacterium infected cotyledon node and cotyledon attached decapitated explants MS supplemented with 0.5 mg/l BAP + 0.5 mg/l kn + 0.1 mg/l GA3 + 5.5 mg/l tyrosine. L.S. of embryo produced only on MS medium. Transformed shoots were selected using 200mg/l Kn. No transformed shoots was recovered using cotyledonery node as explants while a few transformed shoots were found to develop from cotyledone attached decapitated embryo following Agrobacterium infection and selection. Bacterial suspension having O.D. of 1(600 nm) in an incubation period of 60 min with three days cocultivation was found be optimum toward transformation of cotyledone attached decapitated embryo explants of lentil varieties. Shoot survived on the highest selection pressure for 15-20 days were considered putative transformed shoots. Stable expression of GUS gene was detected in putatively transformed plantlets and shoots through GUS histochemical assay.
52. Rapid Propagation of Watermelon (Citrullus lanatus Thumb.) From Field Grown Plants - M. Khalekuzzaman and M.H. Rashid1, Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh. 1Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh.
Maximum frequency (73%) of shoot apices of Citrullus lanatus survived and resumed growth when cultured on MS supplemented with 5 mg/l BA and 0.1 mg/l IAA. The cultures upon transfer to cytokinin enriched medium produced multiple shoots and 2.0 mg/l BA found to be optimum in this respect. Addition of GA3 in multiplication medium resulted better growth of shoots. Rooting rate was 65% when shoots from second subculture were cultured in medium with 1.0 mg/l IBA and the shoots were more prone to rooting by increasing subcultures. In vitro regenerated plants were successfully established in the soil.
53. Agrobacterium-mediated genetic transformation of Mungbean (Vigna radiata (L.) Wilczek) - Kazi Tariqul Islam, Md. Nurul. Islam, M.I. Hoque and R.H. Sarker, Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.
A reproducible in vitro direct regeneration protocol as well as Agrobacterium-mediated genetic transformation protocol was developed for locally grown mungbean [Vigna radiata (L.) Wilczeck] variety, BINA mug-5. Best response towards shoot regeneration was obtained from cotyledonary leaf explants on MS supplemented with 0.5 mg/l BAP while MS supplemented with 1.0 mg/l BAP was found to be most effective for cotyledon attached with embryonal axis (CAEA) explants. Half strength of MS supplemented with 0.5 mg/l IBA was found suitable for root induction from excised shoots. Transformation experiments using CAEA explants were conducted with Agrobacterium tumefaciens strain LBA4404 harbouring the binary plasmid pBI121 (conferring b-glucuronidase activity and resistant to kanamycin). Optimal conditions favouring transformation experiments were determined by transient GUS assays. Selection pressure was applied following shoot induction and putative transformed shoots were selected by increasing kanamycin concentrations up to 200 mg/l in regeneration medium. Shoots, survived during selection process, were subjected to rooting. Stable expression of GUS gene was detected in the putatively transformed plantlets through GUS histochemical assay.
54. In vitro flowering and seed formation from transgenic shoots of lentil (Lens culinaris Medik) following Agrobacterium-mediated genetic transformation - R.H. Sarker, Subroto Kumar Das, M.I. Hoque - Plant Breeding and Biotechnology Lab., Department of Botany, University of Dhaka.
Agrobacterium-mediated genetic transformation experiments were conducted with two local varieties of lentil (Lens culinaris Medik) namely, Barimasur-1 (BM-1) and Barimasur-4 (BM-4) using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing GUS (ß-glucoronidase) and nptII (neomycine phosphotransferase II) genes. Three different explants of lentil such as cotyledone attached decapitated embryo, decapitated embryo and cotyledonary node were used for this purpose. As an integral part of transformation a series of experiments were also conducted for the regeneration of in vitro plantlets using these explants. Regeneration of multiple shoots was achieved via organogenesis form these explants on MS medium supplement with 0.5 mg/l BAP + 0.5 mg/l Kn + 0.1 mg/l GA3 + 5.5 mg/l tyrosine, but such shoots were failed to produce effective root system. Under this circumstances to overcome the problem of rooting in vitro flowering and seed setting experiments were carried out using the in vitro raised shoots from cotyledonary node and cotyledone attached decapitated embryo explants. MS and half strength of MS containing various concentrations of auxins such as IAA, IBA and NAA were employed for this purpose. The best response regarding the development of in vitro flower was obtained on half strength of MS medium containing 20 mg/l IBA, 0.5 mg/l NAA. Agrobacterium infected cotyledonary node and cotyledone attached decapitated embryo explants produced shoots on MS medium supplement with 0.5 mg/l BAP + 0.5 mg/l Kn + 0.1 mg/l GA3 + 5.5 mg/l tyrosine. Transformed shoots were selected using 200 mg/l kanamycin. No transformed shoot was recovered using cotyledonary node explants while a few of transformed shoots were found to develop from cotyledone attached decapitated embryo following Agrobacterium infection and selection. Bacterial suspension having an OD of 1.0 (at 600 nm) in an incubation period of 60 minutes with 3 days of co-cultivation period was found to be optimum towards transformation of cotyledone attached decapitated embryo explants of lentil. The selected kanamycin resistant shoots developed in vitro flowers following their subculture on half strength of MS medium containing 20 mg/l IBA, 0.5 mg/l NAA and 50 mg/l kanamycin. After 12-15 days most of these flowers produced fertile seeds on the same medium. Seedling germinated from these in vitro raised seeds was successfully transplanted to soil for the development of further progenies. The transgenic nature of the fully developed plantlets was confirmed by histochemical GUS assay. Such expression of GUS gene was observed in various parts of transformed plantlets. This technique of in vitro flowering and seed formation can be exploited to develop transformed seeds in lentil since in vitro root formation appears to be a major constraint in obtaining complete plantlet under in vitro condition.
55. Molecular Characterization of 48 Cucumber Germplasm Using RAPD - D. Joyanti and M.G. Rabbani, Department of Horticulture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. Email: grabbani3@yahoo.com
RAPD markers were used to characterize 48 cucumber germplasm each 24 long (Sosha) and short (Khira) types. Out of 33 primers screened, 5 were selected which gave 51 clear markers in cucumber. Of the 51 bands, 49 (95.65%) were polymorphic and the rest were monomorphic. The estimate of Nei’s (1972) genetic diversity index was 0.036 and Shannon’s information index was 0.54. The highest genetic distance (0.93) was observed between CSL80 and CSS21 pair and lowest genetic distance (0.00) was observed between germplasm CSL195 and CSL197 pair. The highest intervariety similarity index Sij (94.18%) was observed between CSL32 and CSL37 pair and the lowest (00.00) was between germplasm CSL43 and CSS77 pair. Dendogram based on unweighted pair group method of arithmetic means revealed the segregation of 48 germplasm in two major two clusters viz. A and B. Long type cucumber germplasm belongs to cluster A and short type cucumber in cluster B. The study revealed that long type cucumber (Sosha) and short type cucumber (Khira) were distantly related at molecular level.
56. Molecular characterization of 24 sponge gourd germplasm using rapd - M.M. Rashid and M.G. Rabbani, Department of Horticulture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. E-mail: grabbani3@yahoo.com
Genetic variation and relationship among 24 sponge gourd germplasms were analyzed using RAPD. Out of 16 primers screened, 6 were selected, which gave 50 clear and bright fragments and 39 fragments were considered as polymorphic. The proportion of polymorphic loci and gene diversity values across all loci were 78% and 0.2640, respectively. The highest inter-accessions similarity index (100%) was found between LC09 and LC06, between LC010 and LC06 and between LC10 and LC09. The lowest inter-accessions similarity index (48.00%) was found between LC011 and LC01. Dendrogram based on Nei’s (1972) genetic distance upweighted pair group method of arithmetic means (UPGMA) indicated the segregation of 24 sponge gourd germplasms into 2 main clusters. The lowest genetic distance was found (0.0000) between LC09 and LC06, between LC010 and LC06, between LC010 and LC09 and between LC040 and LC039. The highest genetic distance (0.8210) was found between accessions LC011 and LC04.
57. Studies on genetic diversity of okra using morphological, biochemical and molecular markers - M. Saifullah1 and M.G. Rabbani, Department of Horticulture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. 1Horticulture Research Centre, Bangladesh Agricultural Research Institute, Jamalpur. E-mail: grabbani3@yahoo.com
Genetic diversity of 121 genotypes of okra (Abelmoschus esculentus) was investigated at morphological, biochemical and molecular levels using highly heritable morphological characters, peroxidase isozymes and RAPD, respectively. Twenty two quantitative and 16 qualitative characters were studied for morphological characterization of the okra accessions using partial lattice design. For molecular characterization, 39 random decamer primers were screened to amplify DNA by the PCR. After screening of primers, five primers generated 32 distinct polymorphic RAPD fragments. When the similarity index data of morphological, biochemical and molecular characterization were, correlation coefficients of 0.61 between biochemical and morphological data and 0.54 between molecular and morphological data were recorded. These results showed that the quantitative descriptors, biochemical and molecular approaches have high potential in characterization of okra germplasm.
58. Recovery from Initial Albinism of Anther Culture Derived Plantlets of Indica Rice (Oryza sativa L.) - A.K.M. Mohiuddin1, Nilufer Hye Karim, Shahanaz Sultana and Zannatul Ferdous, Biotechnology Division, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh. 1Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh.
A simple method was developed to overcome albino plant regeneration from anther culture of Hobigonj Boro IV and VI, two aromatic indica rice varieties of Bangladesh. Three factors such as induction medium component of M10, specific callus size (range 0.2 - 0.4 cm long) and height of shoot primordia (range 2-3 mm) were found crucial in turning albino shoot primordia to green plantlets. On immediate transfer of shoot primordia of 2 - 3 mm from M10 medium to regeneration medium and incubation under continuous fluorescent light at 100-lux intensity at 25 ± 1ºC, triggered the change of albino shoot primordia to green in 2 - 3 days. Shoot primordia of callus size ranging from more or less 0.2 to 0.4 cm length had no effect in changing albino shoot primordia green. Callus derived from anthers cultured in KA, KB, KC, KD and KE media also did not help recovery of albino plantlets in both the varieties. Transfer of shorter or longer length of shoot primordia to regeneration medium other than 2 - 3 mm long remained albino. All the albino shoot primordia initiated from Hbj B VI in M10 medium turned green (100%). However, in case of Hbj B IV, 79% shoot primordia resulted in a change to green plantlets. Subsequent culture and subculture of green plantlets showed rapid formation of new plantlets in multiple numbers.
59. Expression of peroxisome proliferator-activated receptor gamma and the growth inhibitory effect of its synthetic ligands in human salivary gland cancer cell lines.- Nasima Begum Mila, Department of Therapeutic Regulation of Oral Tumor, Institute of Health Biosciences, Tokushima University, 3-18-15 Kuramoto cho, Tomushima 770-8504., Japan. Email:mila@basic.med.tokushima-u.ac.jp/nasima_mila@yahoo.com
Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. It is expressed in several tissue types, including adipose tissue in which it stimulates adipogenesis. Recent studies have demonstrated that PPARgamma ligands induce cellular differentiation and inhibit cell growth in carcinomas of various organs including the breast, prostate, lung, colon, stomach, bladder, and pancreas. However, whether PPARgamma is expressed in human salivary gland tumors and its function in this tissue is unknown. In the present study, we examined PPARgamma gene expression in human salivary gland cancer cells and tested its ligands for any antitumor effect. PPARgamma mRNA was detected by RT-PCR in both benign and malignant salivary gland tumor tissues. The effect of PPARgamma on cell growth was investigated using four human salivary gland cancer cell lines; HSG, AZA3, HSY and TYS, which were confirmed to express PPARgamma1 mRNA and protein. Retinoid X receptor alpha protein, which forms heterodimers with PPARgamma, was also detected in all the cells tested. Data obtained by luciferase assay indicated that the intrinsic PPARgamma protein was activated by the synthetic ligands, troglitazone and pioglitazone, but not by the natural ligand, 15-deoxy-delta12, 14-prostaglandin J2. The synthetic PPARgamma ligands, particularly troglitazone, caused significant dose-dependent inhibition of cancer cell growth. Furthermore, the overexpression of PPARgamma1 or PPARgamma2 in cancer cells also reduced significantly their growth rate. These results suggest that PPARgamma and its synthetic ligands suppress the growth of human salivary gland cancer cells and it may be a useful molecular target for cancer treatment.
60. Genetic transformation in white jute (Corchorus capsularis) - S. T. Islam1, M. E. Hoque2, M. S. Hossain3 and A. Khatun4, 1Department of Genetics and Plant Breeding, 2Dept. of Biotechnology, Sher-e-Bangla Agricultural University, 3Dept. of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University, 4Genetics Engineering Lab, Genetic Resources and Seed Division, Bangladesh Jute Research Institute (BJRI), Dhaka-1207, Bangladesh.
In vitro seed germination potentiality, plant regeneration capacity and genetic transformation ability of white jute (Corchrus capsularis) was carried out. Per cent of seed germination (88.89) is higher in cotton-supported MS compared to agar solidified MS medium (77.77). The growth rate and seedling health also higher in the same medium. More areation facilities may be the probable reason for better growth and development in cotton-supported MS medium. Highest shoot regeneration (83.33%) was noticed in MS medium supplemented with 2 mg/l BAP + 0.5 mg/l IAA. An efficient and reproducible genetic transformation protocol was established for the development of transgenic white jute using Agrobacterium-mediated gene transfer methodology. The in vitro regenerated cotyledonary petioles used as explant for inoculating Agrobacterium tumefaciens strain LBA4404 carrying binary vector PBI121. The vector contains selectable marker gene nptII which confirms resistance to kanamycin and GUS reporter gene. Cocultivation, selection and GUS histochemical assay revealed successful transformation and express of reporter gene in the regenerated tissue of white jute. Among the two genotypes, the cultivar CVE-3 showed better (90%) response to GUS assay.
61. Anther and Filament Culture in Oilseed Brassica Species - M.A. Alam, A.H.M. Kamal, N. Pervin, M.A. Prodhan and L. Hassan, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Anther and filament of five Brassica species, namely BARI sariaha-7, Tori-7, Agrani, Daulat and Safal were cultured in vitro to observe their regeneration potentiality. MS was supplemented with different concentrations and combinations of growth regulators. The range of callus induction from anther and filament were 12.50 - 87.50% and 12.50 - 93.75%, respectively. Maximum callus induction (90.63 % from filament and 75.00% from anther) was observed on MS + 4 mg/l 2, 4-D + 1.0 mg/l BAP. Among the genotypes, BARI Sarisha-7 showed the highest percentage of callus induction (81.25 and 60.42 from filament and anther explants respectively). Among the treatments, highest percentages of shoot regeneration (93.75 % from filament and 75.00% from anther) were observed on MS + 4 mg/l BAP + 1.0 mg/l NAA. BARI Sarisha-7 also showed the highest rate of plant regeneration (79.17 and 72.92 from filament and anther respectively). Root induction was highest (87.50 and 75%) on half strength MS supplemented with 1.0 mg/l IBA and 0.5 mg/l NAA. The plantlets with sufficient roots thus obtained were transferred successfully to plastic pots and subsequently to the field. BARI Sarisha-7 and Tori-7 survived easily in the pots as well as in the field but Safal was very poor in survivability both in the pots and in the field.
62. Haploid Plantlet Regeneration Through Anther Culture of Five Oilseed Brassica Species - M. A. Alam, M. A. Haque, M. R. Hossain and L. Hassan, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Anthers of five Brassica species, namely BARI sariaha-7, Tori-7, Agrani, Daulat and Safal were cultured in vitro to observe their regeneration potentiality. MS was supplemented with different concentrations and combinations of growth regulators. The range of callus induction was 12.50 -87.50%. Maximum callus induction 75.00% was observed on MS + 4 mg/l 2, 4-D + 1.0 mg/l BAP. Among the genotypes, BARI Sarisha-7 showed the highest percentage of callus induction (60.42%). Among the treatments, highest percentage of shoot regeneration (75.00) was observed on MS + 4 mg/l BAP + 1.0 mg/l NAA. BARI Sarisha-7 also showed the highest rate of plant regeneration (72.92%). Root induction was highest (75%) on half strength MS supplemented with 1.0 mg/l IBA and 0.5 mg/l NAA. The plantlets with sufficient roots thus obtained were transferred successfully to plastic pots and subsequently to the field. BARI Sarisha-7 and Tori-7 survived easily in the pots as well as in the field but Safal was very poor in survivability both in the pots and in the field.
63. Standerdization of High Frequency Regeneration of Existing Genotypes of Jute (Corchorus olitorius) in Bangladesh - N. Pervin, M.A. Alam, A.H.M. Kamal, M.A. Haque and L. Hassan, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Four varieties of Corchorus olitorius were used to observe their regeneration potentiality and to establish a suitable in vitro plantlet regeneration protocol of jute. Cotyledons were used as explants. MS supplemented with different phytohormone concentrations and combinations were used to observe the callus induction ability of the explants. The highest callus induction (88.56%) was observed in O-9897 with the medium supplemented with 3 mg/l BAP + 0.5 mg/l IAA. Calli were maintained to get sufficient number of regenerants. The highest percentage of regenerants were found from O-9897 (88.94%) followed by O-72 (85.83%) and OM-1 (72.61%). Among the phytohormone combinations, MS supplemented with 3 mg/l BAP + 0.5 mg/l IAA showed the highest shoot regeneration (97.78%). The root formation from regenerants was the best (69.91%) on half strength of MS supplemented with 0.6 mg/l IBA. The in vitro regenerated plantlets from the genotypes O-9897, O-72, OM-1 and O-4 could be established in the field successfully.
64. Standerdization of High Frequency Regeneration of Existing Genotypes of Jute (Corchorus capsularis) in Bangladesh - N. Pervin, M.A. Alam, A.H.M. Kamal, M. A. Haque and L. Hassan, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.
Six varieties of Corchorus capsularis were used to observe their regeneration potentiality and to establish a suitable in vitro plantlet regeneration protocol of jute. Cotyledons were used as explants. MS supplemented with different phytohormone concentrations and combinations were used to observe the callus induction ability of the explant. The highest callus induction (83.89%) was observed in CVE-3 with MS media supplemented with 2 mg/l BAP + 0.5 mg/l IAA. Calli were maintained to get sufficient number of regenerants. The highest percentage of regenerants were found from CVE-3 (86.87) followed by BJC 7370 (74.64). Among the phytohormone combinations, MS medium supplemented with 2 mg/l BAP + 0.5 mg/l IAA showed the highest shoot regeneration (84.13%). The root formation from regenerants was the best on half strength of MS supplemented with 0.6 mg/l IBA. The in vitro regenerated plantlets from the genotypes CVE-3, BJC 7370, CVL-1, C-83, D-154 and CC-45 could be established in the field successfully.
65. Optimization of Callus Induction and Plantlet Regeneration Through Filament Culture of Five Oilseed Brassica Species - M.A. Alam, M.R. Hossain, M.A. Haque and L. Hassan, Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Filament of five Brassica species, namely BARI sariaha-7, Tori-7, Agrani, Daulat and Safal were cultured in vitro to observe their regeneration potentiality. MS was supplemented with ifferent concentrations and combinations of growth regulators. The range of callus induction was 12.50 -93.75%. Maximum callus induction 90.63 % was observed on MS + 4 mg/l 2, 4-D + 1.0 mg/l BAP. Among the genotypes, BARI Sarisha-7 showed the highest percentage of callus induction (81.25). Among the treatments, highest percentage of shoot regeneration (93.75) was observed on MS + 4 mg/l BAP + 1.0 mg/l NAA. BARI Sarisha-7 also showed the highest rate of plant regeneration (79.17%). Root induction was highest (87.50) on half strength MS medium supplemented with 1.0 mg/l IBA and 0.5 mg/l NAA. The plantlets with sufficient roots thus obtained were transferred successfully to plastic pots and subsequently to the field. BARI Sarisha-7 and Tori-7 survived easily in the pots as well as in the field but Safal was very poor in survivability both in the pots and in the field.
66. In vitro Response of BRRI Released Rice Varieties on Different Culture Media - K. Hossain1, M. S. Ali3 and L. Hassan2, Department of Biotechnology, 2Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh, 3Bangladesh Rice Research Institute, Gazipur, Bangladesh.
The experiments were conducted to identify the rice varieties having good in vitro culture response in order to select for future transformation studies. Dehusked seeds of ten BRRI released rice varieties (BR1, BR2, BR3, BR4, BR5, BR6, BR7, BR8, BR9 and BR10) were placed on five different media (MS, LS, KA, N6 and M10) to evaluate the performance of germination and callus induction through in vitro seed culture. The highest percentage of seed germination was recorded from BR1 and BR9 with LS and N6 media and the lowest percentage of germination was recorded from BR7 in different media. M10 medium showed excellent performance for germination but it was not efficient for callus induction. The highest percentage of callus induction was recorded from BR1 and BR9 with MS medium and BR7 performed was poor for callus induction against different media. In culture media, maximum performance of callus induction was exhibited from MS and poor performance of callus induction was exhibited from KA medium against the varieties. After germination and callus induction, the calli were transferred to MS generation medium for plant regeneration. The highest percentage of regeneration was obtained from BR10 and the lowest percentage of regeneration was obtained from BR4. So it is evident that, BR1 and BR9 have good in vitro culture response for germination and callus induction and BR10 performed best for regeneration.
67. In vitro Regeneration Potentialities of Different Varieties of Cabbage and Cauliflower - S.B. Shahid1, M.A. Quddus2 and L. Hassan2, 1Department of Biotechnology, 2Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
The experiment was carried out to examine in vitro regeneration potentiality and to establish an efficient protocol for in vitro regeneration of two cabbage and two cauliflower varieties. Different concentrations and combinations of growth regulators were used in MS to induce callus from hypocotyl explants. The range of callus induction was 70 - 100%. The highest percentage of callus induction (97.5%) was observed in MS media supplemented with 2.5 mg/l BAP, 0.1 mg/l 2, 4-D and 2.0 mg/l AgNO3 (T2). Among the varieties used, Early Tropical induced the highest percentage (98.3) of callus followed by Summer Star (86.6). For maintenance of calli, MS + 3.0 mg/l BAP + 0.1 mg/l 2,4-D + 2.0 mg/l AgNO3 (T3) showed the best result. Among the treatments the highest percentage of shoot regeneration (67.5) was observed in MS + 3.0 mg/l BAP + 0.1 mg/l 2, 4-D + 2.0 mg/l AgNO3 (T3). Among the four varieties tested, a large variation in shoot regeneration frequency was observed. Early Tropical showed best result (73.3%) in shoot regeneration followed by Tangail Special Pauslali (43.3%). The highest percentage of root induction (75.0) was observed in half strength of MS + 0.5 mg/l NAA. Tangail Special Pauslali had the highest response to root induction (66.7%). The plantlets with sufficient roots thus obtained were transferred successfully in plastic pots. The variety Tangail Special Pauslali showed the highest survival rate (82.5%) while the Summer Star was the least (58.33%).
68. Genetic Transformation in Indica Rice - S. Basak1, M. S. Ali3 and L. Hassan2, 1Department of Biotechnology, 2Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh, 3PSO, Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh
Two different experiments were conducted for in vitro regeneration and genetic transformation ability of indica rice varieties. In the first experiment, embryogenic calli from mature seeds of five indica rice varieties viz. BRRI dhan-29, BRRI dhan-32, BINA dhan-6, BINA sail and Hari dhan were used to observe their regeneration potentially and also to establish a suitable in vitro plantlet regeneration protocol and in the second experiment, two indica rice varieties were selected to observe their transformation ability. In the first experiment, MS supplemented with different phytohormone combinations were used to observe the callus induction ability of the explant. The highest callus induction (69.4%) was observed in Hari dhan over all the varieties and T3 (MS supplemented with 2 mg/l 2, 4-D + 50 mg/l L-tryptophan) over all the treatments (65.0%). Among the phytohormone combinations, T4 (MS + 8 mg/l Kn + 0.5 mg/l NAA) showed the highest shoot regeneration (52%). Among the genotypes, BRRI dhan-29 was highly responsive to shoot regeneration (56%) whereas root formation from regenerants was the best in MS media supplemented with 0.5 mg/l IBA (84%) in BRRI dhan-29. BRRI dhan-32 showed highest number of roots (21.77) in MS supplemented with 0.5 mg/l IBA (15.96). The in vitro regenerated plantlets thus obtained from those genotypes were successfully established in the field. The survival rate of the plantlet in pot was highest (96%) in BRRI dhan-29 but BRRI dhan-32 showed highest survival rate (93.33%) in soil. In pot and soil BINA dhan-6 showed the lowest survival rate of the plantlet 70 and 71.42%, respectively. In the second experiment, transgenic rice plants were developed by inoculating mature embryos with Agrobacterium tumefaciens strain LBA4404 carrying a binary vector pBI121 with GUS (reporter gene) and nptII (Kanamycin resistance) gene and the transformation experiment was performed by optimizing two important parameters: infection times and cocultivation periods. Infection was most effective when explants were inoculated for 30 minutes (96.67% GUS positive) and cocultivated for five days (100% GUS positive). Among the varieties, BRRI dhan-29 showed the highest response to GUS assay (93.33% GUS positive). Putative transformed plantlets were also highest percentage in BRRI dhan-29 (11.67). Thirty minutes infection time and co-cultivation period of four days were effective for regeneration of shoots (31.67 and 30%, respectively). Among the varieties, BRRI dhan-29 produced the highest percentage (60) of rooted shoots.
69. Filament Culture of Oilseed Brassica in B5 Medium - R. Rahman1, M. A. Quddus2 and L. Hassan2, 1Department of Biotechnology, 2Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
The experiment was conducted to develop a protocol for efficient in vitro regeneration of Brassica species. Investigation was also made to find out the regeneration ability of six genotypes, namely Tori-7 and Agrani of Brassica rapa; Rai-5 of Brassica juncea; BINA sarisha-3, BINA sarisha-5 and BARI sarisha-8 of Brassica napus. Different concentrations and combinations of 2, 4-D, BAP, NAA and IBA were used in B5 medium to observe the callus induction ability from filaments, used as explant. The minimum days (7.5) were required for callus initiation in the genotype Tori-7 on B5 medium with 2 mg/l 2, 4-D + 1 mg/l NAA. The highest percentage of callus induction (87.50) was recorded in Tori-7 with B5 medium + 2 mg/l 2, 4-D + 1 mg/l NAA followed by BINA sarisha-3 (85.50) on the same medium. The lowest percentage of callus induction (35.50%) was found in the genotype Agrani on B5 medium + 1 mg/l 2, 4-D + 1 mg/l NAA. The interaction between media and genotype showed significant variation. Interaction between the genotype Tori-7 and B5 medium with 2 mg/l 2, 4-D + 1 mg/l NAA showed the best performance for days to callus induction and percentage of callus induction. Significant variation was observed among the genotypes and hormone compositions for shoot initiation and root formation. The genotype Rai-5 induced the highest percentage of shoot (89.50) in B5 medium + 2 mg/l BAP + 0.5 mg/l IBA while Tori-7 took the minimum number of days (20.25) for shoot initiation and induced (86.50) of shoot on the same medium. The hormone composition 2 mg/l BAP + 0.5 mg/l IBA showed the best performance on days to shoot initiation. Among the genotypes Tori-7 took minimum days (14.00) for root formation and also produced the highest percentage of root (87.75) followed by Rai-5 (77.50). The interaction between Tori-7 and 2 mg/l NAA + 0.5 mg/l IBA was found to be the best performer for days to root initiation and percent callus showing root. Performance of the B5 medium with 2 mg/l NAA and 0.5 mg/l IBA was recorded better all through for days to root initiation and per cent rooting than the other media compositions. Considering overall performance, both Tori-7 and Rai-5 were found superior to the other cultivars.
70. Optimization of Regeneration Protocol of Ginger (Zingiber officinale Rosc.) through Meristem Culture - M.Z. Islam1, L. Rahman2 and L. Hassan2, 1Department of Biotechnology, 2Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
The study was undertaken to develop a suitable protocol for successful in vitro regeneration of ginger from meristem by the incorporation of plant growth regulators of optimum concentration. Two experiments were conducted during the present investigation. In the first experiment MS supplemented with a constant dose of IAA (1 mg/l) supplemented with different concentrations and combination of Kn (1, 2, and 3 mg/l) and BAP (3, 4 and 5 mg/l) was used to observe the regeneration performance using meristems of ginger as explant. The meristems of two varieties (Chinese and Local) of ginger were used. The shoot initiation percentage varied in different treatment combinations. The highest shoot initiation (93.8%) was recorded in 1 mg/l IAA + 2 mg/l Kn + 4 mg/l BAP in Chinese variety and in case of local variety, shoot initiation was 87.5% found in the same media composition. The days required for shoot initiation was influenced by different combinations of Kn and BAP. The minimum time (22.5 day) was required for Chinese variety, cultured in the combination of 1 mg/l IAA + 3 mg/l Kn + 5 mg/l BAP. Similar result was found in local variety in 1 mg/l IAA + 3 mg/l Kn + 4 mg/l BAP. The rate of shoot initiation also varied in different treatment combinations. The highest number of shoots/explant was obtained (6.50) in 1 mg/l IAA + 2 mg/l Kn + 4 mg/l BAP in Chinese variety. The highest number of leaves/shoot (4.50) was recorded in 1 mg/l IAA + 2 mg/l Kn + 3 mg/l BAP in Chinese variety. The longest shoot (6.35 cm) was found in 1 mg/l IAA + 2 mg/l Kn + 4 mg/l BAP. In the second experiment, regenerated shoot was transferred to half strength of MS containing different concentration of IBA (00, 0.5, 1.0 and 1.5 mg/l). The highest number of roots/plant (10.50 cm) was recorded in 1 mg/l IBA in both varieties and the longest root (5.25 cm) was also found in the similar treatment.
71. In vitro Regeneration of Garlic (Allium sativum L.) - M.S. Kabir1, M.A. Quddus2 and L. Hassan2, 1Department of Biotechnology, 2Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
The experiment was conducted to develop an efficient protocol for regeneration of garlic via callus culture from root tips and leaf base. This investigation was also done to find out the regeneration potentiality of two garlic varieties. Different concentrations and combinations of growth regulators were used in MS to observe the callus induction ability of different explants, namely leaf base and root tips. The ranges of callus induction from leaf base and root tips were 8 - 80% and 0 - 88% respectively. Maximum rate of callus induction was observed in root tip (88%) in MS + 2 µm 2, 4-D. Even though root tips induced higher rate of callus but proliferation rates were not satisfactory. Between the varieties used Indian variety showed slightly higher rate of callus induction ability than the local. Calli were maintained to obtain sufficient number of regenerants. MS media supplemented with 1.5 µm 2,4-D showed best performance in callus proliferation. BAP and NAA were able to regenerate shoots from these proliferated calli. Among different concentrations of BAP and NAA used highest percentage of regenerations (80 to 91). Indian variety had higher potentiality for shoot regenerations than the local variety and between the two explants used root tip showed better performance. Root initiation percentage and number of roots per plant were highest in MS supplemented with NAA. Maximum root formation was obtained from root tips when cultured on MS with 2 µm NAA. The regenerated plants thus obtained were successfully transferred to pots and subsequently to the field with satisfactory survival rate.
72. In vitro Clonal Propagation of Two Bamboo Species, Bambusa vulgaris and Dendrocalamus giganteus - Shyamal K. Roy and M. Azizur Rahman, Department of Botany, Jahangirnagar University, Savar, Dhaka, Bangladesh
Protocols for micropropagation of two bamboo species, Bambusa vulgaris and Dendrocalamus giganteus are described. Nodal segments of young lateral branches were used as explants. In vitro multiplication of shoots was achieved by culturing the explants on MS supplemented with 4.5 mg/l TDZ + 1.0 mg/l NAA for Bambusa vulgaris and 4.0 mg/l BA + 1.0 mg/l NAA for Dendrocalamus giganteus. In both the cases liquid medium was found to be efficient in induction of shoots and the subsequent subcultures were done on agar gelled medium of the same constituents. Shoots were multiplied up to 25 per culture during 5 passages. Addition of 10% coconut water to the medium enhanced the number of shoots per culture up to 2.5-fold. Ninety five per cent of the regenerated shoots rooted on half strength MS containing 3.5 mg/l IBA. Through hardening step, 85% regenerated plantlets were successfully transferred to soil.
73. In vitro Clonal Propagation of Rhynchostylis retusa from Nodal Segments of Mature Plant - Pinaki Sinha1, M. Lokman Hakim2 and M. Firoz Alam1, 1Department of Botany, Rajshahi University, Rajshahi, Bangladesh. 2Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Gonokbari. Savar, Dhaka, Bangladesh
A protocol for clonal propagation of Rhynchostylis retusa was developed from nodal segments derived protocorms through in vitro culture. Nodal segments from young shoot of a mature plant were cultured for 12 weeks on gelrite gelled half strength of MS supplemented with 1.5 mg/l BA, + 0.5 mg/l NAA + 3% (w/v) sucrose + 15% (v/v) coconut water + 2 g/l peptone + 0.5 g/l activated charcoal, which produced an average of 16 microshoots-PLBs. Subculture of microshoots-PLBs for eight weeks on the fresh medium of same nutrient composition enhanced the number of micro shoots-PLBs up to 100. The clump of microshoots-PLBs was cut into eight pieces and subcultured on gelrite gelled half strength of MS with 3% (w/v) sucrose + 15% (v/v) coconut water + 2.0 g/l peptone + 150 mg/l L-glutamine + 0.5 g/l activated charcoal, on which, during 8 weeks, microshoots elongated to shoots, PLBs proliferated and developed into shoots and new micro shoots-PLBs were induced from the base of the old ones. The regenerated shoots rooted within eight weeks on half strength of MS with 3% sucrose + 2 g/l peptone + 15% (v/v) CW + 0.5 g/l activated charcoal + 50 g/l banana pulp. Within the first 36 weeks after initiation of culture an average of 1500 plantlets as well as huge amount of microshoots-PLBs were achieved from a single explant. Regeneration of plantlets was continued by repeated subculture of microshoots-PLBs.
74. In vitro Micropropagation of Teak (Tectona grandis L.) from Nodal Explant - Md. Tabiur Rahman1, Md. Raihan Ali2 and Md. Abdul Matin3, 1FWT Discipline, Khulna University, Khulna-9208, Bangladesh. 2Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh. 3FWT Discipline, Khulna University, Khulna-9208, Bangladesh.
The experiment was conducted to establish an efficient and reproducible in vitro micropropagation technique for local strain of teak known as Jessore teak. Two to three weeks old seedlings were used as source of explant. Sterilized nodal segments were inoculated in WPM (Woody Plant Medium) and MS supplemented with different concentrations of BA ( 0.01 - 3.0 mg/l) with or without NAA (0.01 - 0.60 mg/l) for induction and proliferation of shoot. The WPM containing 1.0 m/l BA was found to the best (85.17%) followed by MS containing 1.5 mg/l BA (71.42%) for shoot initiation and proliferation. The WPM with 1.0 mg/l BA also produced the highest number of shoot (2.0 ) with 3.16 cm long shoot while the lowest number of shoots were produced in medium containing 0.10 mg/l BA in WPM medium or 3.0 mg/l BA in MS medium. It was observed that combination of 1.0 mg/l + 0.20 mg/l NAA (85.71%) did no improve the shoot formation efficiency. It was also observed that shoot induction as well as shoot number were gradually decreased with increasing concentration BA + NAA in WPM and MS media, respectively. The length of shoot was highest (4.10 cm) at 2.0 mg/l BA + 0.40 mg/l NAA in WPM medium after four weeks of inoculation. It was also observed that half strength of MS showed better performance than half strength of WPM. Hundred per cent shoots produced root in half strength of MS containing either 0.50 mg/l IBA + 0.25 mg/l NAA or 1.0 mg/l BA + 0.25 mg/l NAA while only 42.8%, or 57.14 % shoots produced roots in the WPM having same concentration of BA and NAA. The highest number of roots/shoot (9.00) also produced in MS containing 0.50 mg/l BA + 0.50 mg/l NAA but the percentage of rooting was also lower (71.42) in MS. The regenerated plantlets were successfully established into the pot after proper hardening.
75. Callus induction and shoot regeneration in Pointed gourd ( Trichosanthes dioca Roxb.) - Shafiuddin Ahmed, Md. Raihan Ali and S.M. Mahbubur Rahman, Plant Biotechnology Laboratory, Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.
Sterilized node, internode, leaf and petiole explants of pointed gourd (Trichosanthes dioca Roxb.) were cultured asceptically in MS supplemented with different concentrations (0.2 - 2.0 mg/l) of 2,4-D. The percentage of callus induction varies from 88-56 in different explants incubated at different concentrations of 2,4-D in MS. Eighty eight percentage of inter nodal segments produced the white and friable callus followed by nodal segments (84%) while the petiole showed the lowest (56%) performance. Hundred percent nodal and internodal explants induced to white and friable callus in MS containing 0.2mg/l 2,4-D followed by petiole (80%) in the same concentration. Petiole took shortest time (5.93 days) for callus induction while internodal segments took longest time (6.23 days) for the same. Different concentrations (0.2 - 4.0 mg/l) of BA, Kn, NAA and AgNO3 were added to MS to investigate their potentiality in organogenesis of pointed gourd. For regeneration of plantlet, the calli obtained from 0.2 mg/l treatment were sub-cultured in MS supplemented with different concentrations and combinations of BA, Kn, NAA and AgNO3 . Among different treatments used for shoot induction, BA @ 3.0 mg/l, 4.0 mg/l BA and 3.0 mg/l BA + 20 µM AgNO3 were only produced only shoots. All other treatments did not show any response. BA was found to be the best growth regulator for shoot induction in this experiment. Eighty per cent calli produced highest number of shoots/explant (2.13) in medium containing 3.0 mg/l BA followed by 4.0 mg/l BA (1.5). The longest shoot (3.02 cm) was produced in MS containing 4.0 mg/l BA followed by 2.6 cm in medium with 3.0 mg/l BA after two weeks of inoculation. Micro cuttings of regenerated shoots were inoculated in full strength of MS supplemented with different concentrations (0.2 - 2.0 mg/l) of IBA and IAA. MS supplemented with IBA showed better performance than IAA in root induction. The highest percentage (100) of micro-cuttings produced roots at 0.2 mg/l IBA with 13.8 roots/explant while no root was produced from the base of micro-cuttings but callus was formed at the base of micro-cuttings in the medium containing 2.0 mg/l IBA. The longest root (1.73 cm) was found at 0.5 mg/l BA followed by 1.54 cm in medium containing 0.2 mg/l. The in vitro regenerated plantlets were successfully established on potting mix composed of sand, soil and compost at 1:2:1. Survival of plantlets under ex vitro condition was about 70 per cent.
76. Introduction of Catalase Gene katE in Indica Rice Through Agrobacterium-mediated Transformation to Attain Salt Tolerant Rice - Shamsul H. Prodhan1,2, K. Nakao,2 K. Nagamiya2, T. Motohashi2, S. Jesmin3., A. Komamine4 and H. Morishima2, 1Department of Biotechnology, Shahjalal University of Science and Technology, Sylhet, Bangladesh. 2Laboratory of Genetics and Plant Breeding, Department of Agricultural Science, Tokyo University of Agriculture, Japan. 3Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan. 4The Research Institute of Evolutionary Biology. 2-4-28, Kamiyoga, Setagaya-ku,Tokyo, 158-0098, Japan.
In an attempt to improve the salt tolerance of rice, we introduced a catalase gene from E. coli, into the indica rice cultivar Kasalath by using Agrobacterium tumefaciens strain EHA101 harboring a binary vector pIES6/Hm/katE which contains genes for catalase katE, hygromycine resistance gene HPT and kanamycine resistance gene nptII in the T-DNA region. Presence of katE in transgenic plants were confirmed using PCR and Southern hybridization analysis. We checked the transgenic plants in three different growth stages. Four week aged plants in 100 mM formed immature inflorescence and in 250 mM survived up to 20 days. Six week aged plants were able to grow for more than 20 days in the presence of 250 mM sodium chloride and produced seeds for more than three months in the presence of 100 mM sodium chloride. On the contrary, non-transgenic rice plant could not survive for ten days even in the presence of 50 mM sodium chloride. Catalase activity was about two-fold higher in T1 plants than non-transgenics. Introduction of a single trait significantly improved the salt tolerance of indica rice plants which are reported so far. We hope to introduce multiple stress resistant genes (P5CS, katE, bet, P5CS etc) in our already transformed rice plant to obtain more stronger salt tolerant rice which would produce over 3 million metric tones of rice to fulfill our national and world food demand.
77. Molecular Targeting and Drug Discovery in the post-Genomics Era - Zaheed Husain, Institute of Biosciences, University Putra Malaysia, Immune Disease Institute, Harvard Medical School, Boston, MA
A fundamental paradigm shift - from traditional to genomics-based drug discovery - has occurred over the past few years. We have used three approaches to understand target identification and validation in diseases: (a) Conventional functional analysis of microarray-generated target gene. (b) RNAi-mediated gene silencing for proof-of-principle. (c) Bioinformatics-based analysis of gene expression profiles to determine regulatory networks. In studies of B cell lymphoma, we have identified a survival factor as a target for lymphoma therapy. A compound library screen using this gene as a target yielded a number of potential leads including a natural product-derived compound that can be used in selective therapy. In a separate system, study of NFkB knockout mice as a model for premature aging led to the identification of a novel target gene involved in the insulin signaling pathway. In vitro studies using RNAi, suggest possibility of targeting this gene in anti-diabetes research.
78. Mass propagation of Feronia limonia L. (Kothbel) through tissue culture - Most. Mosfeqa Zahan, Shahina Islam, Rezaul Karim Mondal, Miskat Ara Akther Jahan, Salim Khan, Ahashan Habib, Shahina Akter, Tanjina Akhtar, Plant Tissue Culture section, Biological Research Division, BCSIR, Dhaka-1205, Bangladesh.
Feronia limonia L. (Kothbel) is a familiar fruit in Bangladesh. It is a native fruit and the plant grows well in Bangladesh. Valuable medicinal compounds like umbelliferol, dictamnine, xanthotoxon, scoparone etc. found in this fruit. Large quantities of citric acids, mucilage and minerals found in its pulp, so the fruit could be good substrates for confectionary products such as jam, jelly, squash, pickle etc. Plantation of kothbel trees are decreasing due to ignorant utilization of the fruits and plants from seeds also reduce the quality due to cross-pollination. So, to solve the problem tissue culture is a dependable technique to develop good quality, large-number and disease free plants. In the present study shoot tips and nodal segments were excised as explants and different concentration of NAA, Kn, IAA and BAP were used singly or in combinations with MS to observe their effect on initiation and development of shoots. Finally MS supplemented with 0.2 mg/l BAP gave good number of multiple shoots with better plant growth. For a complete plantlet it is necessary to develop roots from in vitro growing shoots. Further experiments are going on to find out the suitable growth regulators for root induction.
79. Scope Of Agricultural Biotechnology After Green Revolution - Satyesh Chandra Roy, Department of Botany, University of Calcutta , Kolkata 700019, India. scroyind@yahoo.com
The first revolution in food production came through the emergence of Green Revolution . The important basic elements in the method of green revolution was double cropping method and using seeds of high yielding variety. Instead of one crop season per year based on the natural monsoon, another crop was made in the same year based on irrigation facilities through making Dams and using bore well facilities. The higher yield was made by using chemical fertilizers and pesticides in addition to the use of high yielding strains of crop plants. Thus many developing countries attained self-sufficiency in food production after the achievement of Green revolution. But a revolution of this magnitude created many problems now. It has created in environmental degradation particularly loss of soil fertility and hazards to human health due to excess use of fertilizers and pesticides. With the development of molecular biology and genetic engineering methods, the development and use of molecular markers have helped to identify the introgressive gene in the population and to select the desirable genotypes in the offspring. These are essentials for any improvement programme of crop plants. The results and scope of this method will be discussed.
80. Improvement of Pea against fungal diseases by Expression Family 19 Chitinase from Streptomyces olivaceoviridis ATCC 11238 - fathi hassan1,3; Heiko Kiesecker2 and Hans-Jorg Jacobsen1, 1Institute of Plant Biotechnology-Leibniz University of Hannover-Herrenhauser str.2, 30419 Hannover, Germany. 2German Collections of Microorganisms and Cell Cultures (DSMZ), Mascheroder Weg 1b, 38124 Braunschweig, Germany. 3General Commission for Agricultural Scientific Research (GCASR) Douma, P.O.Box 113 Damascus, Syria.
A chimeric bacterial chitinase gene Chit30 from the Gram-positive bacterium Streptomyces olivaceoviridis ATCC11238 was modified with an Arabidopsis leader peptides sequence under the control of either the constitutive 35S or an inducible plant vst promoter and was cloned into a pGreenII vector harboring bar gene for selection. Embryo axes excised from overnight imbibed mature pea seeds (Pisum sativum L., cv. Sponsor) were used as explants for Agrobacterium-mediated transformation following a modified protocol. Shoots were selected on MSB media containing up to 15 mg/l of PPT. Shoots were grafted onto decapitated seedlings to quickly recovering fertile plants producing the T1 progeny. Stable integration of the T-DNA was analyzed and confirmed by PCR. Copy number and integration pattern was studied in T0 and T1 generation using Southern blot analysis with different probes (chitinase, bar, and nptI) proving presence of a single copy in 17 clones and two copies in 4 clones of pea plants tested. No backbone sequence of the vector was detected using the nptI probe. Leaf paint analysis showed whether the level of PAT (the enzymatic product of the bar gene) was sufficient to confer resistance to the herbicide BASTA®. The accumulation and activity of chitinase in transgenic pea plants were examined by Western blot and immuno-staining analysis and in-gel-assays using glycol chitin as substrate, respectively. In-vitro bioassay using crude protein extract proved the successful inhibition of hyphal growth of Trichoderma harzianum, which could be observed 8 h and 24 hrs after treatment. This is the first report on the expression of a bacterial chit30 in transgenic pea.
81. Identification of Salt Tolerant Rice Lines in F3 Population of PNR-519× IR-29 Using SSR Markers - H. Sohrawardy, M.M. Islam2 and S.N. Begum2, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh. 2Bangladesh Institute of Nuclear Agriculture, P.O. Box 4, Mymensingh 2200, Bangladesh.
Twenty six F3 rice lines of PNR-519 ´ IR-29 were used to evaluate salinity at seedling stage and identify salt tolerant lines via SSR markers. A cross was made between salt tolerant rice mutant PNR-519 with high yielding salt susceptible IRRI variety IR-29. One thousand three hundred fifty F2 plants were grown and 37 plants were selected based on desired agronomic performances. In subsequent season, 37 F3 lines were grown and 26 F3 lines were selected for phenotyping and genotyping. Salinity screening was done at the seedling stage using hydrophonic system at glasshouse following IRRI standard protocol. Among these, line 05-28, 05-33 and 05-47 were found as tolerant, 4 lines were moderately tolerant and 11 lines were susceptible. Twenty six F3 lines along with parents (PNR-519 and IR-29) were genotyped with SSR markers for identification of salt tolerant rice lines. Parental polymorphism survey was done with eight SSR markers. Out of 8 markers, four polymorphic SSR markers viz., RM21, RM51, RM127 and RM510 were selected to evaluate F3 lines for salt tolerance. In respect of the marker RM21, 12 lines were identified as salt tolerant, 11 lines as susceptible and 3 lines as heterozygous in comparison with salt tolerant parent PNR-519 and salt susceptible parent IR-29. Marker RM51 identified 6 tolerant, 9 susceptible and 11 heterozygous rice lines. Five tolerant, 10 susceptible and 11 lines were identified when 26 F3 lines were genotyped with the marker RM127. The marker RM510 showed 7 lines were found as tolerant, 18 lines were susceptible and 1 line was heterozygous. Line 05-33 and 05-28 were observed tolerant as compared with parent PNR-519 when the marker RM21, RM51 and RM127 were tested. Line 05-37 and 05-16 were found susceptible compared with parent IR-29. Finally, line 05-28 and 05-33 showed tolerant compared with parent PNR-519 by using markers RM21, RM51 and RM127. Thus the tested markers could be efficiently used for tagging salt tolerant genes in marker-assisted breeding programme.
82. Plant Regeneration in Sweet Gourd - Jesmin Akter, M.S. Haque and M. Abdul Karim1, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. 1Department of Crop Botany, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.
Cotyledon, hypocotyl, root tip and shoot tip explants of in vitro germinated seedlings of sweet gourd (Cucurbita moschata) were cultured on medium supplemented with different concentrations and combinations of NAA (0.0, 0.2, 0.5 and 1.0 mg/l) and BAP (0.0, 0.5, 2.0 and 5.0 mg/l) to achieve direct and indirect regeneration. Among the explants, cotyledons were found to perform best in callus induction. The combination of 5.0 mg/l BAP and 0.2 mg/l NAA produced the highest callus frequency in cotyledon (90.0%). The calli derived from cotyledons, hypocotyls and root tips were cultured in MS medium supplemented with different concentrations of kinetin, BAP and/or NAA for shoot induction. Regeneration via callus was achieved only from cotyledon calli at a frequency of 65.0% on 2 mg/l BAP and 0.2 mg/l NAA. Shoot tips cultured for direct regeneration, in the same media containing 1.0 mg/L BAP or 1.0 mg/L BAP + 0.2 mg/l NAA resulted in 100.0% shoot differentiation. The regenerated shoots rooted on MS medium with or without 0.1 mg/l NAA (100%).
83. Genetic Diversity in Advanced Aromatic Rice Lines Using Ssr Markers - K. Kibria, S. N. Begum1 and M. M. Islam1, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh; 2Plant Breeding Div., BINA, Mymensingh, Bangladesh.
Molecular marker is useful tool for evaluating genetic diversity and determining cultivar identity. The purpose of this study was to assess the genetic diversity among 12 advanced aromatic rice lines along with their parents involving simple sequence repeat (SSR) markers. On the basis of phenotypic performances rice lines 43-28-5-3-1, 10-14-5-1-3 and 43-28-5-2-1 showed better yield and yield related traits than other lines studied. The lines were evaluated for polymorphisms after amplification with SSR primer pairs. A total of 3 SSR markers (RM223, RM342A and RM515) scored 46 bands among the lines, while the average number of effective allele ranged from 1.78 to 2.49. Marker RM223 showed highest polymorphism 66.67%. Based on Nei’s genetic distance, twelve lines were grouped into 2 clusters. The highest genetic diversity and genetic distance (2.306) were observed in line 43-28-5-3-1 and 10-14-5-1-3 indicating these lines derived from different origin and could be utilized in breeding programme for improving traits of interest.
84. Variability between Hori dhan and BR11: What is their molecular relationship? - Md. Mehedi Hasan1,2, Rokeya Begum1, Md. Sazzadur Rahman3 and Zeba I. Seraj1, 1Department of Biochemistry and molecular Biology, University of Dhaka,2Department of Genetic Engineering & Biotechnology, University of Dhaka,3Plant physiology Division, Bangladesh Rice Research Institute, Gazipur, Bangladesh.
Since ancient times, our farmers have developed enormous number of land races despite having no scientific training. Horipada Kapali is one of those farmers and responsible for developing his new rice “Hori dhan”. Hori dhan’s similarity to the most popular Bangladeshi rice variety BR11 (Mukta) resulted in a debate on its origin. So, a number of molecular studies were undertaken to show the relationship between Hori dhan and BR11. Around 125 SSRs distributed over the 12 rice chromosomes were used for characterization among which 24 SSRs showed polymorphism. Some of these polymorphic markers are located in the exon regions of genes; some are in the 5’ UTR (Untranslated Region), 3’ UTR and some in the intron regions. We are trying to predict the effect of the polymorphism loci in gene expression. Interestingly some small RNA signatures have also been found near the polymorphic loci. We have sequenced region from BR11 and Hori dhan amplified by RM180, RM229, RM210, RM21 and RM222 and we are trying to analyze variation at the nucleotide level. A comprehensive SSR map was built to pinpoint the overall variability. To test whether variability is restricted to specific loci or distributed generally along the genome, we are using 27 RAPD markers to test both varieties. So far RAPD markers did not show any significant variability. So it can be concluded that the phenotypic/morphological variations of Hori dhan and BR-11 occur due to some specific changes at the molecular level.
85. Chlorophyll fluorescence responses of two bean varieties during salt stress - Mouful Nahar and Bernhard Huchzermeyer, Institute of Botany, Gottfried Wilhelm Leibniz Universität Hannover, Germany.
In order to help solving the profound problem of soil salinity, it is essential to understand the consequences of damage occur in plants by salinity. In this study, chlorophyll fluorescence techniques have been effectively applied to asses the photosynthetic performance of two contrasting bean varieties (Phaseolus vulgaris L.) i.e., arroz tuscula (salt sensitive) and orfeo (salt tolerant) during the period of salt stress. In both varieties, chlorophyll fluorescence measurements (F0 and Fm value) revealed that no significant change occurred at the early stage of salt treatment. As a long term effect of salt stress, the maximal efficiency of PSII photochemistry i.e. the ratio of Fv/Fm was reduced in Tuscula compared to Orfeo. A decline of the photosynthetic efficiency of quantum yield (ΦPSII) was noticed in all plants irrespective of salt stress during flower initiation period. In addition to this, at flowering stage Tuscula showed a raise in minimal fluorescence (F0) and a decline of Fv/Fm ratio in presence of NaCl. At that particular time, Orfeo showed no major changes with respect to F0 and Fv/Fm values. Therefore, the increase of F0 yields during flower initiation in Tuscula can be correlated with the observed decrease of pod numbers under salt stress. Salt treatment increased sugar accumulation and reduced total chlorophyll and carotenoid content in both varieties. These fluorescence data together with results from pigment determination and sugar concentration measurement indicate that salt does not interfere with the primary reactions of photosynthesis but with the use of photosynthate (i.e. sugar export out of chloroplasts or from source leaves to sink organs).
86. Study of genetic diversity in some economically important members of Cucurbitaceae using isozyme, RAPD and ISSR markers - Biswanath Sikdar1, Moimee Bhattacharya2, Biplab Ghosh2, Ashutosh Mukherjee3, Anuradha Banerjee2, Enakshi Ghosh2, S.C. Roy2, 1Department of Genetics and Plant Breeding, University of Rajsahi, Bangladesh. 2Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata-700019, West Bengal, India. 3Department of Botany, Dinabandhu Mahavidyalaya, Bongaon-743235, 24 Parganas (N), West Bengal, India.
Understanding of the genetic diversity and phylogenetic relationship among plant species is vital for the successful breeding experiments, particularly in plants with high rate of cross pollination. In this study, biochemical and molecular markers have been used on eleven genera of Cucurbitaceae collected from lower Gangetic plains. 6 enzyme systems were selected. Among 40 primers examined, 14 RAPD and 10 ISSR primers were selected for the analysis. 100 RAPD and 100 ISSR fragments were generated. Level of variations was high among the species. Jaccard similarity indices were used for the evaluation of pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate interspecific diversity. Further, Principal Coordinate Analysis was performed to evaluate the resolving power of the three marker systems to differenciate among the species.
87. Mutation Breeding and Food Security: Achievements and Prospects - M.c. kharkwal, Indian Agricultural Research Institute, New Delhi-110 012, India. mckharkwal@gmail.com
The first and the longest period of history of mutation dates back to 300 BC to 1894 AD, when the phenomenon of spontaneous mutants were observed and documented. However, it was during the second brief period of 30 years only from 1895 to 1926 that the theory of induced mutations and mutation breeding concept was fully established. It was during this second period, more than a century ago, that the word "Mutation" was coined and introduced in the classical hypothesis by Hugo de Vries in 1901. Extensive experiments on induced mutations and crop improvement programmes through induced mutations were initiated about eight decades ago, immediately after the discovery of mutagenic effects of X-rays in Drosophila by Muller in 1927, and in barley and maize by Stadler (1928), during the third period running between 1927 to 1950. A great deal of work on basic aspects of induced mutation technique in understanding the mechanism of gene mutations, mode of action of physical and chemical mutagens etc. was done world over in the three decades that followed the pioneering work of Muller and Stadler to understand the nature of mutations, induction techniques and their role in evolution, genetics and plant breeding. These two discoveries resulted almost immediately in the practical recovery of some economically useful mutants in wheat and tobacco. Later, the induction of mildew resistance in barley by X-irradiation was reported. A major stimulus for much of later work in mutation breeding was the classic work in barley in which a very large number of mutations of especially for chlorophyll characters and stiffness of straw (erectoides) showing response to different external environments. The X-ray treatment of two-rowed barley in Sweden resulted to produce mutants having characters of dense ears and very stiff straw, which could withstand high doses of fertilizers and gave high yield. The Swedish programme covered several crop species and generated a lot of information on mutation breeding. The establishment of the International Atomic Energy Agency (IAEA) in 1957 proved to be a landmark in the history of applied mutation technique, induced mutagenesis and mutation breeding. Coordinated programmes on the use of mutation breeding technique were initiated by IAEA in a large number of crops in several countries of the world. Having understood the basics of mutation techniques as well as induced mutagenesis and the role of induced mutations and their practical utility as a tool in plant breeding, several countries including Canada, China, France, Germany, India, Italy, Japan the Netherlands, Pakistan, Russia, UK and USA took up the task of crop improvement through mutation breeding approaches. Over 3000 mutant varieties of crop plants including cereals, oilseeds, pulses, vegetables, fruits, fibers and ornamentals have been developed by the end of the year 2007. More than 60% of these mutant varieties were developed and released after 1985. While majority (>70 %) of the varieties were released as direct mutants, the rest has been released through cross breeding with mutants, a time tested methodology. Most of the mutant varieties (around 90 %) have been developed using physical mutagens (X-rays, gamma rays, thermal and fast neutrons), with gamma rays alone accounting for the development of >60 % of the mutant varieties. Later, mutagenic effects of mustard gas and several other chemicals were also demonstrated and it was established that alkylating agents are the most important group of chemical mutagens. A wide range of chemicals e.g., EMS, MMS, EI, NMU, SA and several others, have been successfully use as mutagens. A wide range of characters which have been improved through mutation breeding include plant architecture, yield, flowering and maturity duration, quality and tolerance to biotic and abiotic stresses. Mutation breeding has made a significant contribution to the national economy of the countries like China, India, Japan, Pakistan and USA. At the global level, the largest number of mutants have been released in cereals and millets (>1100), followed by ornamentals (> 575), legumes (>394) and fruits (>115), cash and industrial crops (>85), vegetables (>70) and oilseeds (>60). The numbers are constantly changing due to increasing number of mutants being identified/ released in one or the other country around the world.
With the release of more than 343 mutant cultivars belonging to >57 plant species, India has become a major recognized centre for work on induced mutations and also the second largest contributor of the mutant varieties in the world. Mutation breeding in India has been very successfully used in several crops as is evident from the number of mutant varieties released in each case of major crops such as rice (41), barley (13), finger millet (7), and pearl millet (5) in case of cereals; mungbean (15), cowpea (10), blackgram (9), chickpea (8) in case of pulse crops; groundnut (18), mustard (9), soybean (7), sesame (5) and castor bean (4) in case of oilseeds crops; cotton (9), sugarcane (9), jute (5) in cash crops; tomato (4) and turmeric (2) in vegetable crops. Besides these crops, a large number of mutant varieties have been released in case of ornamental crops such as chrysanthemum (49), rose (16), bougainvillea (13), dahlia (11) and portulaca (11). Mutant varieties have also been released in case of several medicinal plants, forage crops and fruit trees in India, although their number is limited.
The enormous achievements made in terms of such a large number of mutant varieties in more than 175 crop species in more than 60 countries of the world have made substantial contribution towards their national economies and have played a key role in solving their food security problems. An attempt will be made in this lecture to highlight the success stories of several important mutant varieties developed in various countries and their significant role in serving the alarming cause of national food security, particularly in the countries of the developing world.
In recent years interest has been rekindled in the use of mutation technique in several area of biological research, since induced mutagenesis is gaining importance in plant molecular biology as a tool to identify and isolate genes and to study their structure and function. These studies have an enormous potential for future crop improvement programmes. There is an urgent need to adopt molecular tools coupled with induced mutation techniques to redesign our crops by placing important traits on genetic maps and to equip them with the genes and attributes that could meet the enormous challenges in crop improvement. The accomplishments of mutation breeding in evolving superior crop varieties and its role not only in basic studies, but also in the preserving the food security of a country confer it an honorable niche in the ever-going crop improvement programmes in several countries.
88. Biotechnology in action: Performance of Sub1 lines in farmer’s fields - M. A. Mazid, M. A. Haque, H. U Ahmed1, M. A. Salam1, D. Mackill2 and A. M. Ismail2 -BRRI Regional Station, Rangpur, 1BRRI, Gazipur, Bangladesh. 3International Rice Research Institute, Los Banos, Philippines.
Flash flooding is a common constraint for high and stable rice productivity in flood prone areas of Bangaldesh, particularly if it occur at seedling or tillering stages. Farmers are compelled to use new seedlings from unknown source with the consequent low yields due to late sowing with older seedlings, or the land will remain fallow when seedlings are not available. Severity of losses depends on the duration and frequency of flooding and floodwater quality, as well as the extent of genetic tolerance of the varieties being used. Apparently, all modern rice varieties are severely damaged when flooded for even one week. Recent developments in biotechnology played an important role in developing submergence tolerant rice genotypes that can withstand submergence for up to two weeks. This is became possible after fine mapping and cloning of Sub1, a major QTL associated with tolerance to flash-flooding. This gene was subsequently introgressed into 6 popular mega varieties from different countries through marker assisted selection (MAS) breeding at IRRI. Swarna, a mega variety was the first to be used for introgression of Sub1 and BRRI received seeds of Swarna-Sub 1 for evaluation on-station at Ranpur as well as at BRRI Head Quarter in Gazipur. The variety was also tested in farmers’ fields at different locations of Bangladesh through BRRI-IRRI collaboration during 2005-07. Swarna-Sub1 could survive 10 to 17 days of submergence, and also showed quick recovery, particularly if managed properly, and this is reflected in higher yields (3.3-3.7 t/ha) over numerous seasons and locations. The BRRI Regional Station at Rangpur produced 82 kg of seeds of Swarna-Sub1 line by tiller separation technique from limited amount of seeds received earlier, and this was used for testing at farmer’s fields in 6 districts; Rangpur, Kurigram, Lalmonirhat, Nilphamari, Gaibandha and Sirajganj through BRRI-IRRI-NGO collaboration, during Aman season of 2007.Ten farmers’ fields grown to Swarna-Sub1 were submerged for 5-9 days, 12 farmers fields submerged twice for 2-14 days and 2 fields were submerged thrice for 3-10 days, but still produced high grain yield with an average of 3.88, 3.76 and 3.51 t/ha respectively. Even better grain yield of Swarna-Sub 1 was obtained with submerged for 8 days in one farmer’s plot at Lal monirhat (4.9 t/ha) where the yield of BR11 was very poor. Submergence delayed flowering of all genotypes including Swarna-Sub1 where it was delayed by 2-10 days in farmer’s fields. Farmers were excited to see the good performance of Swarna Sub1 in their fields and asked for seeds of submergence tolerant varieties for the next Aman 2008. BRRI Regional Station at Rangpur received small quantity of seeds of BR11-Sub1, but good amount of IR64-Sub1 and Sumba Mousuri-Sub1 seeds from IRRI through BRRI Head Quarter under BRRI-IRRI collaboration. The area used for seed production was increased by tiller separation techniques in the Boro season of 2008 to provide enough seeds for further evaluation of these submergence tolerant varieties in farmers fields during the next aman season. Tolerant varieties will further be tested along with local checks through farmer participatory trials (PVS) during 2008-2010 through BRRI-IRRI-GO-NGO collaboration. Development of submergence tolerant varieties is now feasible within less than 3 years using marker assisted breeding. This trait is expected to have substantial effect in increasing and stabilizing rice production in flash-flood prone areas of Bangladesh.
89. Mutation Breeding and Food Security: Achievements and Prospects - M.C. Kharkwal, Indian Agricultural Research Institute, New Delhi-110 012, India. mckharkwal@gmail.com
The first and the longest period of history of mutation dates back to 300 BC to 1894 AD, when the phenomenon of spontaneous mutants were observed and documented. However, it was during the second brief period of 30 years only from 1895 to 1926 that the theory of induced mutations and mutation breeding concept was fully established. It was during this second period, more than a century ago, that the word "Mutation" was coined and introduced in the classical hypothesis by Hugo de Vries in 1901. Extensive experiments on induced mutations and crop improvement programmes through induced mutations were initiated about eight decades ago, immediately after the discovery of mutagenic effects of X-rays in Drosophila by Muller in 1927, and in barley and maize by Stadler (1928), during the third period running between 1927 to 1950. A great deal of work on basic aspects of induced mutation technique in understanding the mechanism of gene mutations, mode of action of physical and chemical mutagens etc. was done world over in the three decades that followed the pioneering work of Muller and Stadler to understand the nature of mutations, induction techniques and their role in evolution, genetics and plant breeding. These two discoveries resulted almost immediately in the practical recovery of some economically useful mutants in wheat and tobacco. Later, the induction of mildew resistance in barley by X-irradiation was reported. A major stimulus for much of later work in mutation breeding was the classic work in barley in which a very large number of mutations of especially for chlorophyll characters and stiffness of straw (erectoides) showing response to different external environments. The X-ray treatment of two-rowed barley in Sweden resulted to produce mutants having characters of dense ears and very stiff straw, which could withstand high doses of fertilizers and gave high yield. The Swedish programme covered several crop species and generated a lot of information on mutation breeding. The establishment of the International Atomic Energy Agency (IAEA) in 1957 proved to be a landmark in the history of applied mutation technique, induced mutagenesis and mutation breeding. Coordinated programmes on the use of mutation breeding technique were initiated by IAEA in a large number of crops in several countries of the world. Having understood the basics of mutation techniques as well as induced mutagenesis and the role of induced mutations and their practical utility as a tool in plant breeding, several countries including Canada, China, France, Germany, India, Italy, Japan the Netherlands, Pakistan, Russia, UK and USA took up the task of crop improvement through mutation breeding approaches. Over 3000 mutant varieties of crop plants including cereals, oilseeds, pulses, vegetables, fruits, fibers and ornamentals have been developed by the end of the year 2007. More than 60% of these mutant varieties were developed and released after 1985. While majority (>70 %) of the varieties were released as direct mutants, the rest has been released through cross breeding with mutants, a time tested methodology. Most of the mutant varieties (around 90 %) have been developed using physical mutagens (X-rays, gamma rays, thermal and fast neutrons), with gamma rays alone accounting for the development of >60 % of the mutant varieties. Later, mutagenic effects of mustard gas and several other chemicals were also demonstrated and it was established that alkylating agents are the most important group of chemical mutagens. A wide range of chemicals e.g., EMS, MMS, EI, NMU, SA and several others, have been successfully use as mutagens. A wide range of characters which have been improved through mutation breeding include plant architecture, yield, flowering and maturity duration, quality and tolerance to biotic and abiotic stresses. Mutation breeding has made a significant contribution to the national economy of the countries like China, India, Japan, Pakistan and USA. At the global level, the largest number of mutants have been released in cereals and millets (>1100), followed by ornamentals (> 575), legumes (>394) and fruits (>115), cash and industrial crops (>85), vegetables (>70) and oilseeds (>60). The numbers are constantly changing due to increasing number of mutants being identified/ released in one or the other country around the world.
With the release of more than 343 mutant cultivars belonging to >57 plant species, India has become a major recognized centre for work on induced mutations and also the second largest contributor of the mutant varieties in the world. Mutation breeding in India has been very successfully used in several crops as is evident from the number of mutant varieties released in each case of major crops such as rice (41), barley (13), finger millet (7), and pearl millet (5) in case of cereals; mungbean (15), cowpea (10), blackgram (9), chickpea (8) in case of pulse crops; groundnut (18), mustard (9), soybean (7), sesame (5) and castor bean (4) in case of oilseeds crops; cotton (9), sugarcane (9), jute (5) in cash crops; tomato (4) and turmeric (2) in vegetable crops. Besides these crops, a large number of mutant varieties have been released in case of ornamental crops such as chrysanthemum (49), rose (16), bougainvillea (13), dahlia (11) and portulaca (11). Mutant varieties have also been released in case of several medicinal plants, forage crops and fruit trees in India, although their number is limited.
The enormous achievements made in terms of such a large number of mutant varieties in more than 175 crop species in more than 60 countries of the world have made substantial contribution towards their national economies and have played a key role in solving their food security problems. An attempt will be made in this lecture to highlight the success stories of several important mutant varieties developed in various countries and their significant role in serving the alarming cause of national food security, particularly in the countries of the developing world.
In recent years interest has been rekindled in the use of mutation technique in several area of biological research, since induced mutagenesis is gaining importance in plant molecular biology as a tool to identify and isolate genes and to study their structure and function. These studies have an enormous potential for future crop improvement programmes. There is an urgent need to adopt molecular tools coupled with induced mutation techniques to redesign our crops by placing important traits on genetic maps and to equip them with the genes and attributes that could meet the enormous challenges in crop improvement. The accomplishments of mutation breeding in evolving superior crop varieties and its role not only in basic studies, but also in the preserving the food security of a country confer it an honorable niche in the ever-going crop improvement programmes in several countries.
90. Designer plants for drug discovery and functional foods for human welfare - Swapan K Datta and Karabi Datta, Botany Department, University of Calcutta, 35 Ballygunge Circular Road, Kolkata-700 019, India, swpndatta@yahoo.com
Molecular tools provide the opportunity to design a plant or to target the production of secondary metabolites in plant cell factories. About 250,000 plant species provide greater diversity of bioactive compounds. More than 100,000 compounds have been isolated from plant sources and about 4000 new compounds are being discovered every year. Through centuries plants have been evolved and survive in response to environmental stresses (biotic and abiotic) and grown in diversified ecological conditions. Such conditions allow plants to produce a large number of active bio-molecules. With the co-evolution of nature and environment the biochemical profile of the plants, differ when collected from different places at different times. Hence, the need of a designer plant or a product based research of one or more secondary metabolites for the benefit of human health. The international market of herbal products estimated to be U$ 62 billion and is expected to be U$ 5 trillion by the year 2050. India’s share of this attractive market currently is about 5.5 U$ billion and is rapidly expanding. The Indian systems of Medicines have identified 1500 medicinal plants and >500 plants are mostly used in preparation of drugs. With the advancement of the new technologies, attention has also been shifted from traditional medicinal plants to model/cultivated plants to produce plant derived compounds such as vincristine, podophyllotoxin, diosgenin to functional foods such as pro-vitaminA (Golden rice), Lysine-maize, bio-engineered oil plants with improved fatty acids and high iron rice. I would elaborate and discuss the subject emphasizing genomics, cell and chemical differentiation in plants and a few examples of plant based Functional Foods.
91. Production of Jatropha cureas Plants through Somatic Eembryogenesis and Zygotic Embryo Culture - P. Mukherjee and T. B. Jha, Department of Botany, Presidency College, Kolkata-700 073, India.
Productions of biofuel from seeds of non-edible crops are gaining ground. Jatropha cureas is primarily targeted as one potential source of non-edible biofuel-producing energy crop in today's world. However, uses of J. curcas in herbal medicines and other purposes are well known. Seeds, constituting the primary source of non-edible oil, are genetically heterozygous and pose a problem to genetic fidelity in terms of oil content. Sustainable need of quality planting materials cannot be met with traditional breeding alone. Application of somatic embryogenesis and zygotic embryo culture, two important techniques of plant biotechnology as ideal system for future breeding programme and transgenic research is felt. In vitro methods of plant regeneration through somatic embryogenesis and excised zygotic embryo culture has been successfully achieved for the first time in this species. Plants raised through these methods are now growing in the field condition and bear flowers. Methods and well-documented results will be discussed.
92. In vitro Techniques for Biotechnological Improvement of Chlorophytum borivilianum Sant.et. Fern - S. Basu, A. Banerjee1 and T.B. Jha, Plant Biotechnology Laboratory, Department of Botany, Presidency College, Kolkata-700073, India. 1West Bengal State Council of Science & Technology, Kolkata, India.
In vitro culture techniques in recent years have become a powerful tool for studying and solving basic and applied problems in plant biotechnology. These techniques may offer an ideal answer to large-scale production of clonally uniform plants throughout the year and in vitro regeneration protocols are pre-requisite for application of the advanced biotechnological tools. The application of tissue culture techniques for mass propagation, long term conservation and improvement of medicinal plants holds great promise. Chlorophytum borivilianum Sant.et. fern (Liliaceae) is a rare, near-endemic, high value aphrodisiac herb which deserves in vitro biotechnological methods for mass propagation and conservation. The plant is native to the subcontinent and can grow well in different climatic zones. The present report highlights different modes of regeneration of C. borivilianum like: direct and indirect organogenesis, somatic embryogenesis, artificial seed production and in vitro tuberization using various explants (stem disc, shoot base, leaf and seedling derived axillary meristem). MS basal medium fortified with different growth regulators and various additives has been used throughout the study. The regeneration protocols developed are highly cost effective and may find application not only in mass propagation but also in commercial purposes and genetic transformation studies.