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 sho