Somatic embryogenesis and plant regeneration from male flower buds in banana

T. R. Ganapathi*, P. Suprasanna, V. A. Bapat, V. M. Kulkarni and P. S. Rao

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

Embryogenic cultures were established using young male flowers in five cultivars of banana (Musa spp.). Embryogenic callus continued to rapidly proliferate on MS medium supplemented with 0.22 m M BA and 1.14 m M indole-3-acetic acid (IAA) and somatic embryos developed during subcultures on the same medium. Green plumule emerged from the embryos followed by the development of roots within a span of 6 to 8 weeks upon transfer to ½ strength Murashige and Skoog medium (MS) + 0.5 g/l malt extract (ME) and 0.1% activated charcoal (AC) and subsequently developed into complete plantlets. Normal plant development was noticed following transplantation into paper cups in the greenhouse.

BANANAS and plantains are one of the world’s major fruit crops and staple food for millions of people. In India, banana ranks fourth in terms of gross value after paddy, wheat and milk and is the largest fruit crop, in terms of production. Majority of the edible bananas are triploid and are propagated vegetatively by suckers. In addition to this, sterility and polyploidy often hamper the breeding programmes for the development of superior banana varieties.

Plant cell and tissue culture techniques have helped in the rapid multiplication of elite varieties employing shoot tips or floral apices1. Somatic embryogenesis has been reported from leaf sheath or rhizome fragments from in vitro plants2, thin sections from proliferating buds3, immature zygotic embryos4 and male flower bud cultures5,6. Somatic embryogenesis leading to embryo production and plant regeneration offers advantages for mass propagation of elite cultivars and also provides a useful system for genetic manipulation7,8. In the present communication, we report on the initiation and maintenance of highly proliferating embryogenic callus and plant regeneration in some elite Indian banana cultivars.

Materials and methods

Experimental plant material included cvs. Rasthali (AAB), Basrai (AAA), Shreemanti (AAA), Lokhandi (AAA ?) and Trikoni (AAA ?). Male inflorescences

*For correspondence. (email: biotech@magnum.barc.ernet.in)

collected from the field-grown plants, were surface cleaned with absolute alcohol. Under a sterile laminar air flow, the outer enveloping bracts were removed and the inner part (2–3 cm in length) containing male flower primordia (from 0 to 15, 0 being the meristem) was isolated. Individual male flower primordia were cultured on Murashige and Skoog9 medium (MS) or White’s10 medium supplemented with 18.10 m M 2,4-dichlorophenoxy acetic acid (2,4-D), 5.37 m M napthalene acetic acid (NAA), 5.71 m M indole 3-acetic acid (IAA) and 4.09 m M d-biotin, 3% sucrose and 0.2% gelrite for callus induction. For the proliferation of callus and somatic embryo induction, a medium supplemented with 0.22 m M benzylaminopurine (BA) and 1.14 m M IAA, 3% sucrose and vitamins11 was used. Callus was also cultured on media supplemented with mannitol (3%), to study its effect on embryogenic callus development. The pH of all the media was adjusted to 5.7 prior to autoclaving and all the cultures were maintained in an incubation room at 25 ±  2° C and 12 h photo-period with 1000 lux.

For conversion of somatic embryos into plantlets, ½ strength MS medium supplemented with 0.5 g/l malt extract (ME) and 0.1% activated charcoal (AC), Morel’s vitamins, 3% sucrose and 0.2% gelrite were employed. Fully grown plantlets with good shoot and root system were then transplanted into paper cups with soilrite and then into polybags in the greenhouse.

Results and discussion

Male flower buds cultured on MS or White’s medium supplemented with 18.10 m M 2,4-D, 5.37 m M NAA, 5.71 m M IAA and 4.09 m M d-biotin, showed enlargement of the floral primordia and the development of whitish embryogenic callus, after 2 to 3 months of inoculation (Figure 1 a). MS medium was found to be better than the White’s medium (41.6%) for embryogenic callus (66.5%). The embryogenic callus showed several developed embryos on the surface and the smear preparations of the callus tissue showed early stages of embryo development (Figure 1 b). The embryogenic tissues showed repeated proliferation during subcultures on MS + 0.22 m M BA + 1.14 m M IAA, resulting in an increase by 8.07-fold callus fresh weight for each

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Figure 1 a–e.  Somatic embryogenesis and plant regeneration from male flower buds of banana cv. Rasthali. a, Embryogenic callus cultures from young male flower buds on MS medium with 0.22 m M BA and 1.14 m M IAA; b, Squash preparation of embryogenic callus showing globular stage embryo; c, Germination of somatic embryos on 1/2 MS medium + 0.5 g/l ME and 0.1% AC; d, A somatic embryo derived plantlet with good root system; and e, Regenerated plants in paper cups with soilrite in the greenhouse.

passage of four weeks. Embryogenic callus cultured on MS medium + 0.22 m M BA + 1.14 m M IAA with either 3% sucrose or 3% mannitol showed differences in callus morphology. The medium with 3% sucrose induced rapid proliferation of semi-friable embryogenic callus (90–95%), while with mannitol, the callus became nodular and compact (60–65%).

Five cultivars tested for embryogenic callus formation showed differential culture response (Figure 2). Rasthali was found to be highly responsive (50%) followed by 30% in Shreemanti. The other cultivars Basrai, Lokhandi and Trikoni were less responsive (8–15%). Embryogenic cultures of cv. Rasthali were maintained for a period of over 2 years without any decline in the capacity for proliferation. For the conversion of somatic embryos, the MS medium supplemented with 0.1% yeast extract, 1 g/l caesin hydrolysate (CH) and 1 g/l maltose was tested, either with agar or gelrite as the gelling agent. While the medium gelled with gelrite (0.2%) induced the formation of compact callus and globular embryos, the medium with agar (0.8%) showed > 80% browning of cultures.

Somatic embryos developed into plantlets (5–8%) on ½ strength MS basal medium without any growth regulators supplemented with 0.5% ME + 0.1% AC and gelled with 0.2% gelrite. Green plumule emerged from the embryos followed by the development of roots (Figures 1 c, d) within a span of 6 to 8 weeks. Plantlets developed normally when transplanted into paper cups with soilrite (Figure 1 e) in the greenhouse and 24 well-grown plants were transferred to the field.

Young floral tissues are known to be conducive to in vitro culture with good embryogenic potential12. In the present study, young male flower primordia of banana were used for the induction of embryogenic callus and somatic embryos which required different hormonal treatments. Inductive treatments included three auxins (IAA, NAA and 2,4-D) while a cytokinin with auxin (BA + IAA) was necessary for embryogenic callus
proliferation and somatic embryo development. Navarro et al.6 also noted that three auxins were essentially
required for induction. Among the cultivars with different genomic backgrounds, Rasthali (AAB) showed
good embryogenic response over Basrai (AAA) and Shreemanti (AAA) and others with AAA genome. A similar genotypic difference in culture response was observed in cultivars with AAA, AAB and ABB genomic status5.

In the present study, embryogenic callus exhibited rapid proliferation on a medium with BA + IAA, with embryos developing on the callus surface. Several embryogenic cell clumps and globular stage embryos were observed while some developed into somatic embryos. Escalant et al.5 also observed white, translucent callus and somatic embryos in cultures of young male flowers of cv. Grande Naine (AAA). Multiplication and mainte-

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Figure 2.  Frequency of embryogenic callus formation from young male flower buds in different cultivars of banana.

 

nance of embryogenic cultures was achieved by cultur-ing somatic embryos in a temporary immersion system, while Novak et al.2 and Dheda et al.3 described a liquid phase for the development of somatic embryos. In our studies, however, a liquid phase for somatic embryogenesis was not essential. Inclusion of mannitol (in addition to sucrose) in the medium is known to induce osmotic stress and embryogenic cultures were obtained in rice13. However, in this study, with the inclusion of mannitol, primary callus became nodular and more compact than on the sucrose medium.

The development of an in vitro regeneration system is essential for studies related to genetic manipulation of banana. The embryogenic culture system described in this paper demonstrates the potentiality of the male flower bud explants for use in raising highly proliferative, embryogenic cultures for banana cultivars. Cultivar Rasthali, responding better in vitro, has fruits with sweet taste and thin peel but is highly susceptible to Fusarium wilt. Developing protocols for in vitro plant regeneration may aid efforts in the selection of disease-resistant clones. Separate media manipulations for embryo maturation were not required as the embryos developed into plantlets on the same medium making the culture system much simpler. Although, the plant conversion rates are low, we believe that further refinement in the media would lead to enhanced embryo to plant conversion frequency. Further exploitation of the system of somatic embryogenesis for mass propagation, artificial seeds and for generating transgenic plants may provide a significant step in the improvement of banana.

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Received 28 January 1999; revised accepted 3 March 1999