Strategies for in vitro Propagation and Synthetic Seeds in Banana

T.R. Ganapathi, P.Suprasanna, V.M. Kulkarni, V.A. Bapat and P.S. Rao
Nuclear Agriculture and Biotechnology Division Bhabha Atomic Research Centre

Abstract
Bananas and plantains are one of the worlds major fruit crops and staple food crops for hundreds of millions of people. Although more than 300 kinds of bananas exist, only a few are commercially important. Improving plant productivity is a necessity as several biotic and abiotic constraints impede its cultivation and production. In our laboratory, protocols have been established for the in vitro propagation of twelve commercially important cultivars of banana. Tissue culture derived plants have been field planted and the performance of these plants has been encouraging with increase in yield, early maturity and quality fruits. In the seed propagated Enset (Ensete superbum), protocols for in vitro propagation have been established using male inflorescences. Low cost procedures for in vitro propagation have been developed using commercial grade sugar and simplified nutrient media. Shoot tips have been encapsulated to prepare synthetic seeds that offer as novel delivery system for tissue cultured plants. Future prospects for the improvement of banana using biotechnological tools are also described.

Introduction
The word banana is derived from the Arabic word for finger. There are more than 300 kinds of banana but only a few are commercially important. Banana is the man's oldest and most valued fruit crop. It is prized for its nutritive value with high carbohydrates (22.2%), fibre (0.84%) and protein (1.1%) with less fat (0.2%) and water (75.7%). The World production of banana is about 95 million tons and most of the production is consumed locally. In India, it is the most important fruit crop and is grown in 4.3 million hectare with a total production of 13.9 million tonnes. Several cultivars of banana are cultivated in the country among which, Dwarf Cavendish and Robusta are predominantly grown because of higher yields, resistance to strong winds and short cropping duration besides a good profit margin (Singh 1990). In addition to these, cultivars such as Poovan, Rasthali, Lalkela, Safed velchi and Karibale monthan are also grown. Banana is severely affected by viral (bunchy top virus, cucumber mosaic virus, banana streak virus), bacterial (moko or bacterial wilt, bacterial soft rot), fungal (black sigatoka, Fusarium wilt) agents besides insects/pests and nematodes.

Banana is a long duration crop of one and a half years and is propagated vegetatively by suckers. The production of suckers varies in different genotypes ranging from 5-10 per plant per year. Crop productivity and maturity is dependent on the size and age of suckers and uneven maturity extends the duration by 3-4 months. Suckers also carry soil nematodes, disease causing organisms such as bunchy top virus, leaf spot etc., thereby affecting the crop production considerably. In this regard, biotechnological approaches such as cell and tissue culture, protoplast fusion and gene transfer offer as useful tools (Novak et al 1993, Ganapathi et al.,. 2002). In vitro propagation of banana through shoot tip cultures is useful in the rapid multiplication of desirable disease free plantlets. In addition careful selection and updating of mother plants results in improved crop yield (Vuylsteke, 1989). Enset [Ensete superbum] is the Indian wild banana, propagated by means of seeds and does not produce vegetative side suckers naturally. The Ensets are the staple food of almost 10 million people in Ethiopia. These have capacity to withstand severe draught, and have also been used to help ward off famine in Ethiopia. The wild counterparts are

known for their broad genetic base and carry several desirable genes and thus the Ensets could be utilized for improvement of cultivated bananas by employing various in vitro techniques (Vuylsteke and Swennen, 1993; Novak et al., 1992). Conventional propagation of Enset is time consuming and hence there is a need for optimization of tissue culture techniques for the rapid propagation of enset. We describe here our studies on in vitro propagation of banana and enset, low cost strategies for in vitro propagation and synthetic seeds in banana.

et al., (1994). Shoot tips were grown on medium with CYE ( 10, 20, 40, 80 %) and also CYE (100%) alone, along with growth regulators. Experiments were conducted with commercial grade sugar (CGS) and tap water (TW) in place of sucrose and distilled water, respectively. Multiplication of shoots and rooting of shoots was tested on medium with CGS (3% ) and TW. Shoots were also encapsulated in sodium alginate (3%) and their conversion into plants was examined in the above media combinations (Ganapathi et al., 1995). For each treatment 24 shoots were used and all the experiments were conducted under controlled conditions of light (1000 lux), temperature (25+2o C) and relative humidity of 65%. Rooted plantlets were transferred to poly bags containing good horticultural soil and farm yard manure (1:1) in the green house at high humidity. About 8 week old hardened plants were used for field planting at Experimental Field Facility, Trombay, BARC and Gujarat State Fertilizers Company (GSFC) Ltd., Vadodara, Gujarat.

Materials and Methods

Suckers of ten varieties viz., Basrai, Shrimanthi, Rasthali, Lalkela, Poovan, Ardhapuri, Karibale monthan, Safed Velchi, Mutheli and Hazari were collected and established in the departmental green house. Cultivars, Williams and Grande Naine were obtained in the form of sterile cultures from INIBAPs Germplasm Transit Centre, Belgium. Male inflorescences of enset were collected from the wild plants growing on the steep hill slopes of Sinhagad hill fort of the Western Ghats, at an altitude of 800 m. Shoot tips were isolated from suckers by removing the sheathing leaf bases and were established in liquid MS medium (Murashige and Skoog 1962) supplemented with 5 mg/l benzyl adenine (BA). After 3 weeks these were transferred to semi-solid medium comprising of 2 mg/l BA and 30 mg/l adenine sulphate (AS) and 3% sucrose. Each shoot tip produced 3-5 shoots within a span of 3-4 weeks. Multiplication of shoots was carried out by isolating individual shoots and subculturing them on the same medium till a sufficient number of shoots were obtained. The individual shoots isolated from these multiple shoot cultures were used for various experiments. Individual shoots were transferred to medium with napthaleneacetic acid (NAA) for root induction. Investigations on the effect of cyanobacterial extracts (CYE) were conducted with cyanobacterial strain (Plectonema boryanum UT x 594) grown on BG11 medium. The detailed procedures are given in Ganapathi

Results and Discussion

In vitro propagation of banana Initially shoot tip cultures were established in vitro for the induction of multiple shoots (Fig.1A). The multiple shoots showed elongation and produced roots in liquid media (Fig.1B). Individual shoots transferred to rooting media formed complete plantlets within 4 weeks (Fig.1C). After another period of 4 weeks, plantlets transferred to polybags in the green house exhibited good growth by the emergence of new leaves (Fig.1D). More than 15,000 plants were regenerated in vitro and around 4,500 plants were planted at multilocations in the states of Maharashtra and Gujarat for field trials. A field trial was conducted at R&D farm Gujarat State Fertilizers Company, Baroda (Fig. 1E) and the data suggested an early maturity by about 6 weeks and an increase in yield of 33% in the tissue cultured plants of cv. Basrai as compared to control. The tissue culture raised

Fig. 1 : A. B. C. D. E. F. G. H. Isolated shoot tip used to initiate in vitro shoot cultures Multiple shoots developed in liquid medium. Rooted shoots Hardened plants in the greenhouse. Tissue cultured plants undergoing field trial at GSFC experimental field, Vadodara. Tissue culture derived banana plant bearing a fruit bunch. Encapsulated shoot tips of banana (synthetic seeds). Plantlet development from synthetic seeds of banana.

plants grown at the Experimental Field Facility at BARC, Trombay, showed more vigorous growth, early maturity and increase in bunch weight with good quality fruits (Fig.1F). In vitro propagation of Enset Male inflorescences were cultured on MS medium supplemented with BA (2 mg/l) and gibberellic acid (GA3 1 mg/l). The extreme apical region of male inflorescences necrosed, but the surrounding floral primordia showed growth and produced profuse leafy structures initially. After 2 subcultures, the proportion of leafy structures decreased substantially (<10 %) and a mixture of corm tissue and multiple shoots became predominant (>90%). Longer shoots (2.9 cm) were induced on MS + BA (2 mg/l) + AS (30 mg/l) + GA3 compared to shoots of 1.7 and 1.8 cm length on MS + BA (2 mg/l) + AS (30 mg/l) with and without caesin hydrolysate (500 mg/l) respectively (Kulkarni et al., 1997a). Efficacy of various media tested for root induction showed that MS medium alone was ineffective, whereas MS medium with NAA (1mg/l) gave highest number of roots followed by MS + indole-3-acetic acid (IAA) or indole-3- butyric acid (IBA). Addition of charcoal (0.1%) to these media did not improve rooting efficiency. Rooted plantlets exhibited normal growth upon transfer to polybags in the greenhouse and in the field. There have been very few reports concerning micropropagation of Ensets. Afza et al., (1996) reported micropropagation of E. ventricosum using shoot-tips from vegetative corms. Mathew and Philip (1997) reported regeneration using vegetative shoot apices in Ensete superbum. In the this study, cultured floral apices were employed as an alternative method for the in vitro propagation of enset. In the case of triploid banana cultivars, floral apices have successfully been used for the propagation of Chandrabale, Rasthali, Robusta (Doreswamy and Shahijram, 1989) and cvs. Monthan and Robusta (Balakrishnamurthy and Sree Rangasamy, 1988). The protocol for the regeneration E. superbum using male

inflorescences will be useful for rapid in vitro propagation. Low cost strategies propagation for in vitro

The main objective of this investigation was to develop a low cost in vitro technique for the micropropagation of banana by substituting laboratory grade sucrose and distilled water with commercial grade sugar and tap water, respectively, for minimizing the use of the expensive components of the nutrient media. The shoot tips grown on media prepared in distilled water containing lab grade sucrose or commercial grade sugar showed the formation of multiple shoots (4-5 shoots/ hoot tip) in four weeks. There was no significant change in the frequency of multiple shoot formation in both the media and almost 100 % of the shoots responded. In an another experiment when tap water was used instead of distilled water, more time was required for the induction of multiple shoots and proliferation and the time of the subculture passage had to be extended to 6 weeks. As presented in the Table 1, the excised shoots from multiple shoot cultures were transferred on various media for plantlet regeneration. The shoots cultured on MS medium prepared in distilled water and containing either laboratory grade sucrose or commercial grade sugar showed a good shoot growth having 2-4 roots with laterals in three weeks. The hoots showed comparatively slow growth on similar medium prepared in tap water. The frequency of plantlet formation was almost 95% and there was no change in the number of roots and their laterals. Plantlets regeneration was also observed on Knop's salts with MS minor, iron and vitamins with lab grade sucrose or commercial grade sugar, in medium prepared either with distilled water or tap water. The response was comparatively better in medium prepared in distilled water, as has been shown in Table 1. The frequency

Table 1 : Effect of different media on plantlet formation from shoot tips of banana Media + carbon source (3%) + NAA (1 mg/l) A. MS basal salts + I) sucrose/commercial grade sugar in distilled water II) sucrose/commercial grade sugar in tap water B. Knop's salts +MS minor, iron & vitamins+ III) sucrose/commercial grade sugar in distilled water IV) sucrose/commercial grade sugar in tap water C. Knop's salts + V) sucrose/commercial grade sugar in distilled water VI) sucrose/commercial grade sugar in tap water Data obtained after 4 weeks; 24 explants per of plantlet formation was around 75-80% on Knop's salt alone with lab grade sucrose or commercial grade sugar. The rooted plantlets (7-9 cm in length) thus produced were transplanted into ploybags for hardening in the green house for about two months before field planting. In the present study with banana tissue cultures, laboratory grade sucrose was totally eliminated by using 3 % commercial grade sugar and for the preparation of media clean tap water of good quality was employed instead of distilled water. Though all the components of MS medium were necessary for shoot multiplication, rooting and regeneration of plantlets was achieved only on Knop's salts (KNO3, Ca(NO3)2.4H2O, MgSO4.7H2O, KH2PO4) with NAA and commercial grade sugar. These alterations in the medium would be useful in lowering the total cost of tissue cultured bananas without affecting the rate of multiplication and quality of plants. Effect of cyanobacterial extract (CYE) The effect of cyanobacterial extracts (Plectonema boryanum UT x 594) was tested % cultures forming plantlets 100 95 85 85 75 75 treatment. No. of roots/ plantlet 2-4 2-3 2-3 2-3 2-3 2-3 Plantlet height after 4 weeks (cm) 7-9 7-8 6-8 6-8 6-7 6-7

on growth and multiplication of shoots from shoot tips as well as encapsulated shoot tips of banana. Shoot tips reared on MS medium containing different concentrations of CYE (10, 20, 40 and 80% v/v), showed induction of multiple shoot formation (2-4). Higher concentrations (40 or 80%) exhibited increased frequency of multiple shoot formation (Fig.2) and shoot growth compared to other concentrations. In one treatment, with complete elimination of MS basal medium, shoot tips were cultured on CYE supplemented with 3% sucrose, 5 mg/l BA and 0.8% agar. Although multiple shoot formation was noted in all the concentrations with varying frequencies, the shoots exhibited slow growth. Liquid cultures were also initiated with different concentrations of CYE (10-100% v/v) to minimize the use of nutrients and growth regulators. Among these, 10 and 20 % CYE induced 2-4 shoots while 100% CYE gave as many as 8 shoots and the shoots elongated with 2-3 pairs of leaves. To regenerate complete plantlets, the elongated shoots were cultured on MS medium with CYE (10, 20, 40 and 80% v/v) and plants were regenerated on all the four concentrations with varying frequencies

with 1 mg/l NAA or 5 mg/l BA and also on different substrates like sterile absorbent cotton ( Fig. 1H), soil, filter paper and soilrite (all moistened with 1/4th MS salt solution) for germination. Among these, White's medium gave high frequency (100%) plantlet development within a week compared to other substrates and MS media. Encapsulated shoot tips directly sown in petri plates Fig. 2 : Effect of Cyanobacterial Extract (CYE) on multiple shoot cultures and planet containing autoclaved formation in banana soil showed the emergence of shoots (10%) but failed to form (Fig.2). Similar to the controls (shoots on MS complete plantlets. medium with 1 mg/l NAA), 100% of shoots cultured on MS medium with 80% CYE The results revealed that the encapsulated developed into plantlets within 4 weeks and shoot tips can be handled like a seed and all the plants were transplantable. could be useful in minimizing the cost of Cyanobacterial extracts are known to production as 1 ml of medium is sufficient for stimulate somatic embryogenesis and the encapsulation of a single shoot tip compared conversion of synthetic seeds in carrot to 15-20 ml for conversion of shoot tips into (Wake et al., 1991, 1992) and sandalwood plantlets. By directly sowing the encapsulated (Bapat et al., 1996). Although the exact shoot tips in soil, the two stage process such factors that are involved in such a response as rooting and hardening can be eliminated. are unknown, it has been postulated that As compared to suckers, encapsulated shoot cyanobacterial extracts may synthesize a tips present as inexpensive, easier and safer wide variety of compounds including plant material for germplasm exchange, growth regulating substances (Metting and maintenance and transportation (Rao et al., Pyne 1986). In the present study, CYE has 1993). shown promotive effects on shoot multiplication and plantlet development Prospects for the improvement of suggesting that CYE can be employed in the banana in vitro propagation of banana. Banana has assumed an important status among fruit crops owing to the significant Synthetic seeds research inputs in the areas of cellular and Shoot tips excised from the shoot cultures of molecular biology. Globally research is being bananas cv. Basrai were encapsulated to focussed on aspects relevant to the needs of prepare synthetic seeds, in 3% sodium the developing countries. Mutation induction alginate solution prepared either in distilled using physical as well as chemical mutagens water or MS medium with 0.1% activated can be useful for the induction of genetic charcoal (Fig.1G) and an antibiotic mixture variability. Towards this goal, we have (Ganapathi et al., 1992). Encapsulated shoot conducted research as part of the tips were placed on MS or White's media Coordinated Research Project (CRP) of

Table 2 : Biotechnological approaches for the improvement and increasing productivity in banana Productivity Germplasm storage & Transportation Fungal diseases Viral diseases Nematodes Post harvest life Cloning of high yielding varieties Cryopreservation, Synthetic seeds Antifungal proteins, Antimicrobial compounds, Toxins Coat protein genes, Replicase Bt genes, Chitinases Anti polygalacturonase ( PG), Anti-ethylene biosynthetic Synthase, ACC Oxidase) genes (ACC

International Atomic Energy Agency (IAEA), Vienna, on the Mutation breeding and related biotechnologies for banana improvement (Kulkarni et al., 1997b, Rao et al., 1999). Somatic embryogenesis offers as an ideal system for the production somatic embryos on a large scale for use in the preparation of synthetic seeds, propagation and genetic transformation. In this direction, somatic embryogenesis has already been established in our laboratory from immature male flowers and proliferating shoot tips (Ganapathi et al., 1999, 2001). Embryogenic cell suspension cultures developed from proliferating shoot tips have been useful as the best target tissue for genetic transformation using Agrobacterium tumefaciens (Ganapathi et al., 2001). Introduction of genes for fungal disease resistance may enable the development of new lines with disease resistance in banana. One of the approaches includes the expression of antimicrobial peptides. Improving fruit quality with options for delayed fruit ripening can have significance to boost banana exports from developing countries. Another dimension in the genetic engineering is the production of recombinant proteins or peptides of pharmaceutical importance in transgenic plants. Production of edible vaccines in an edible plant tissue, for example, in fruits of banana can be of great benefit to the developing countries to gain access to the much-needed health care. Studies in this direction have already shown some success towards obtaining transgenic

plants of banana incorporated with useful characters like disease resistance and for developing edible vaccines (Ganapathi et al., 2002 ).

Acknowledgements
Authors thank Dr. R.K. Iyer, Molecular Biology and Agriculture Division, BARC, for providing the cyanobacterial strain (Plectonema boryanum UT x 594).

References
1. Afza R., von Durren M. and Morpurgo R. 1996. Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds. Plant Cell Rep. 15:445-448. Balakrishnamurty G. and Sreerangaswamy S.R., 1988. Regeneration of banana plantlets from in vitro culture of floral apices. Curr. Sci. 57: 270-272. Bapat V.A., Iyer R.K. and Rao P.S., 1996. Effect of cyanobacterial extracts on somatic embryogenesis in tissue cultures of sandalwood (Santalum album L.). J. Med. & Aromatic Plant Sci., 18 : 10-14. Doreswamy R. and Shahjiram L. 1989. Micropropgation of banana from male floral apices cultured in vitro. Sci. Hort. 40:181-188. Ganapathi T.R. Suprasanna P, Bapat V.A. and Rao P.S. 1992. Propagation of banana through encapsulated shoot tips. Plant Cell Reports 11 : 571 - 575.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Ganapathi T.R., Suprasanna P., Bapat V.A., Kulkarni V.M. and Rao P.S. 1999. Somatic embryogenesis and plant regeneration from male flower buds in banana. Curr. Sci. 76 (9) : 1228-1231. Ganapathi T.R., Suprasanna P, Bapat V.A. and Rao PS, 1994. Stimulatory effect of cyanobacterial extracts on banana shoot tip cultures. Trop. Agri. 71: 299-302. Ganapathi T.R., Chakrabarti A., Suprasanna P and Bapat V.A. 2002. Genetic transformation in banana. In : Plant Genetic engineering. Vol. 6: Improvement of fruits. Ed. PK Jaiwal and RP Singh. Sci-Tech Publ. Houston Texas, USA. (In press) Ganapathi T.R., Higgs N.S., Balint-Kurti PJ, Arntzen C.J., May G.D. and Van Eck J.M. 2001. Agrobacterium -mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB). Plant Cell Reports 20:157-162. Ganapathi T.R., Mohan J.S.S., Suprasanna P., Bapat V.A. and Rao P.S., 1995. A low cost strategy for in vitro propagation of banana. Curr. Sci., 68 : 646-649. Kulkarni V.M., Ganapathi T.R., Suprasanna P., Bapat V.A. and Rao P.S. 1997b. Effect of gamma irradiation on in vitro multiple shoot cultures of banana. (Musa species). Jour. Nuclear Agri. Biol. 26(3) : 168-174. Kulkarni V.M., Ganapathi T.R., Suprasanna P., Bapat V.A. and Rao P.S. 1997a. In vitro propagation in Ensete superbum (Roxb.) Cheesman, a species closely related to Musa. Ind. Jour. Exptl. Biol. 35 : 96-98. Mathew M.M. and Phillips V.J. 1997. Clonal propagation of enset [Ensete superbum (Roxb.) Cheesman] through shoot tip culture. Plant Cell Rep. 16:232234. Metting B. and Pyne J., 1986. Biologically active compounds from microalgae. Enzyme Microbiol. and Technol., 8 : 386-394. Murashige T. and Skoog F., 1962. A revised medium for repid growth and

16.

17.

18.

19.

20.

21.

22.

23.

bioassays with tobacco tissue cultures. Physiol. Plant. 15 : 473-479. Novak F.J., Brunner H., Afza, R., Morpurgo, R., Upadhyay R.K., Van Duren M., Sacchi M., Hawz J.S., Khatri A., Kahl G., Kaemmer D., Ramser J. and Weising K., 1993. Improvement of Musa through biotechnology and mutation breeding. In: Biotechnology applications for banana and plantain improvement . Proc. Of the Workshop. INIBAP, pp 143158. Rao P.S., Ganapathi T.R., Suprasanna P. and Bapat V.A. 1993 Encapsulated shoot tips of banana : a new propagation and delivery system. InfoMusa 2(2):4-5. Rao P.S., Ganapathi T.R., Bapat V.A., Kulkarni VM and Suprasanna P. 1999. Improvement of banana through biotechnology and mutation breeding. IAEA Tecdoc 1047 on Use of Novel DNA fingerprinting techniques for the detection and characterization of genetic variation in vegetatively propagated crops. IAEA, Vienna. pp 107-118. Singh H.P, 1990. Country paper report on banana and plantain - India. In: Banana and plantain R & D in Asia and the Pacific. INIBAP, pp. 161-185. Vuylesteke D.R., 1989. Shoot tip culture for the propagation, conservation and exchang of Musa germplasm. Practical Manual for Handling Crop Germplasm in vitro 2, IBPGR, Rome, Italy. Vuylesteke D.R. and Swennen R. 1993. Biotechnology: enhancing research on tropical crops Africa. In: Ed. Thotapilly G, Monti LM, Mohanraj DR., Moore AW, IITA. Pp. 143. Wake H., Umetsu H., Ozeki Y, Shimamura K. and Matsunaga T. 1991. Extracts of marine cyanobacteria stimulated somatic embryogenesis of Daucus carota L. Plant Cell Rep. 9 : 655-658. Wake H., Akasaka A., Umetsu H., Ozeki Y, Shimamura K. and Matsunaga T. 1992. Enhanced germination of artificial seeds by marine cyanobacterial extract. Appl. Micro. Biotechn. 36 : 684 - 688.

This paper was presented the Best Poster award in micropropagation in the National Symposium on Plant Tissue Culture : Commercialization, Diversification of Agriculture and Preservation of Fragile Ecosystems, held during April 10-12, 1996, at G.B. Pant University of Agriculture and Technology, Pantnagar
About the authors
Dr T.R.Ganapathi did his Ph.D. from Karnatak University,Dharwad, and joined BARC in 1991. He is working in the Plant Cell Culture Technology Section of Nuclear Agriculture and Biotechnology Division. His specialization is in the field of Plant tissue culture. He has standardized micropropagation protocols in banana and the technique has been transferred to user agencies. He has also established methods for somatic embryogenesis and agrobacterium mediated transformation in banana using embryogenic cell cultures. Currently, he is working on Genetic Transformation for the incorporation of useful traits into crop plants. Dr P. Suprasanna joined BARC in 1991 after obtaining his Ph.D. degree in Genetics from Osmania University, Hyderabad. He is working in the Plant Cell Culture Technology Section of NA & BTD and is engaged in plant biotechnological research on rice, banana and sugarcane. His contributions have been in the areas of cell and tissue culture, somatic embryogenesis, in vitro selection and synthetic seeds. Dr Suprasanna has to his credit, more than 60 research publications in journals and books published by national and international publishers. His interests are in using plant cell cultures for understanding mechanism of differentiation as well as for genetic manipulation. Dr Vishvas M. Kulkarni joined BARC in 1993 and subsequently obtained Ph.D. (Biotechnology) degree from Mysore University. In the Plant Cell Culture Technology Section of NA & BTD, he has been working on the use of tissue culture, molecular biological approaches in Banana and Pearl Millet and on mutagenesis in vitro using gamma irradiation for the isolation of banana mutants. He has also been involved in collaborative research projects, and has guided graduate and postgraduate students for the completion of their research projects. He has published several research papers in National and International Journals/Symposia/Workshops. Dr V.A. Bapat is working in the area of plant tissue culture for the last 31 years. The main thrust area of his work is on Micropropagation and Genetic Transformation of Plants. He has effectively contributed for clonal propagation, suspension cultures, protoplasts, synthetic seeds and bioreactor production of somatic embryos in a precious forest tree - Sandalwood. He has also done extensive work on micropropagation of Banana and Mulberry. He has several publications in National and International journals to his credit. Currently he is heading the Plant Cell Culture Technology Section. Dr P. S. Rao, former Head, Nuclear Agriculture and Biotechnology Division, joined BARC in 1966. and subsequently obtained his Doctorate in Botany from Delhi University in 1967. As a Visiting Scientist, he did advanced research in plant morphogenesis at the CNRS labs in Gif-sur-Yvette, France and in protoplast culture (1981-82) at the Max-Planck Institute, Koln, Germany. He is a Fellow of the National Academy of Sciences of India, and National Academy of Agricultural Sciences. Dr. Rao has guided several students for M.Sc., M.Tech. and Ph.D. He has published more than 170 research papers/articles. He has been the member of advisory board of several national research centres, universities and government bodies such as Department of Biotechnology (DBT), Department of Science & Technology (DST). He is a member of several professional societies in India and abroad, and is serving on the editorial board of scientific journals. Currently, Dr.Rao is the Vice-President (Biotechnology) of Indo-American Hybrid Seeds (India) Pvt. Ltd., Bangalore.