A rapid protocol for somatic embryogenesis mediated regeneration in banana (Musa spp.) cv. Nendran Lekshmi, R.S., *Soni, K.B., Swapna Alex, Deepa S. Nair, Lekha Sreekantan and B.R. Reghunath College of Agriculture, Vellayani, Thiruvananthapuram, Kerala - 695 522, India *E-mail : soni.kb@kau.in ABSTRACT A simple and rapid protocol for somatic embryogenesis in banana cv. Nendran (AAB) using immature male flowers (IMF) has been developed. The IMF produced pale- white to yellow, globular embryogenic callus on MS medium supplemented with BA (0.05 – 0.50mgL-1) and picloram (0.50 – 2.00mgL -1) with explant response of to 30 per cent. Addition of ascorbic acid (20mgL -1) and Gelrite© (0.45 per cent) to callus induction medium reduced interference from phenolic exudation. Embryogenesis was induced (33.3 to 60 per cent) on semisolid (0.30 per cent Gelrite©) MS medium supplemented with BA 2mgL-1 + IAA 0.5mgL-1. The somatic embryos showed 60-80 per cent germination on half- strength semisolid MS medium with BA 2mgL-1 + IAA 0.5mgL-1. Transfer of germinated embryos to semisolid MS medium supplemented with BA 2mgL-1 + NAA 1mgL-1 under 14 h light /8h dark photoperiod resulted in hundred percent conversion to plantlets. This protocol takes merely 6 months for producing plantlets from immature flower buds through somatic embryogenesis, without any intermediate liquid cultures. Key words: Immature male flowers, somatic embryogenesis, picloram, Musa spp., Nendran INTRODUCTION Bananas and plantains belonging to the genus Musa of Musaceae family, are perennial herbaceous monocots. Musa is a major crop in the tropical and sub-tropical regions of the world, with an approximate production of 70 million tonnes per annum. India is rich in genetically diverse varieties of banana, cultivated over an area of 802.6 (000ha), with production of 297.24 lakh tonnes, contributing 33.4 per cent of the global production share (Indian Horticulture Database, 2015, National Horticulture Board, Ministry of Agriculture, Govt. of India). Owing to female sterility, parthenocarpy and polyploidy, production of new banana cultivars is difficult and time-consuming. Genetic manipulation is a promising technique for introducing desired traits in banana. A rapid and effective in vitro regeneration protocol is a prerequisite for any such manipulation techniques. Single-cell origin of the somatic embryos and their potential to produce non-chimeric plants makes it a desirable system for genetic manipula tion. Somatic-embryogenesis- mediated regeneration is also a method for large-scale multiplication of banana. Immature male inflorescence has been used to initiate embryogenic cultures of several banana and plantain cultivars (Cronauer and Krikorian, 1983; Escalant et al, 1994; Cote et al,1996; Navarro et al, 1997). Compared to soil-grown suckers, IMFs show less contamination during micropropagation, and, selection of male buds with desirable characteristics such as high number of hands and fruits per bunch is also possible (Resmi and Nair, 2007). Nendran is a popular banana cultivar in South India where it is relished as a fruit and is used in making sever a l va lue-a dded products. T his var iety is characterized by its fruits having a distinct neck with a thick, green skin, which turns buff-yellow on ripening and has a pale yellow flesh. Fruits of Nendran are larger, silky, with a firm flesh and are sweet. Bananas offer a wonderful nutritious diet due to their high protein content, are easy to digest, especially in infants. Attempts made on somatic embryogenesis in Nendran J. Hortl. Sci. Vol. 11(2): 116-123, 2017 117 are scarce. Most reports are on Cavendish varieties, which involve lengthy procedures (taking 18-26 months for plantlet development). This study was undertaken to develop a rapid and simple protocol for somatic- embryogenesis-mediated regeneration system in Nendr an, suita ble for ge netic tr ans forma tion experiments. MATERIAL AND METHODS Preparation of explant for inoculation Male flower inflorescence was collected from field-grown Musa spp. cv. Nendran plants, after completion of bunch formation (i.e., 4 weeks after eme rge nc e of the infloresc enc e). Bra cts were removed from the inflorescence to reduce its size to 7-8cm length. These explants were then surface- sterilized with 90% ethanol, inside a laminar air- flow cabinet. Immature male flower (IMF) clusters from 0-15 position from inside were separated using a sterile bla de . T he tiny bra cts c overing ma le relative humidity. Callus initiated was subcultured onto the same parent medium at monthly intervals for multiplication. Induction of somatic embryos Calli developed on different treatments were transferred to media of four different compositions: (1) MS+ 2,4-D 2mgL-1 + Zeatin 0.2 mgL-1 + Gelrite 4% (2) MS+ 2,4-D 2mgL-1 + IAA 1mgL-1+NAA 1mgL1 + Biotin 1mgL-1+ Glutamine 100mgL-1 (3) MS+BA 2mgL-1 + IAA 0.5mgL-1 + Gelrite 0.3%, and (4) SH + MS vitamins + Glutamine 100mgL-1 + picloram 1mgL-1. Sucrose at 4.5% and Gelrite at 0.2% was used in all the media. Fifteen replications with three clumps each were maintained in this study. Cultures were incubated in the dark at 25±2ºC and at 50-60 per cent relative humidity. Maturation and germination of embryos Somatic embryos were transferred to half- stre ngth MS (ha lf-stre ngth ma cr o nutrie nts) supplemented with BA 2mg L-1 + IAA 0.5mg L-1. Cultures were incubated in the dark for 3 weeks for maturation and germination of somatic embryos. Conversion of somatic embryos into plantlets Germinated embryos were transferred to MS medium supplemented with BA 2mg L-1 + NAA 1mgL-1 for development into plantlets. Cultures were maintained at a light intensity of 31.4µmolm-2S-1 and 14h light/8h dark photoperiod. Plantlets at 3-4 leaf stage were transferred to coir pith compost in protrays and hardened in a mist chamber for a month. Plantlets were then transferred to polybags with soil and cow dung (1:1) mixture. RESULTS AND DISCUSSION In the present study, a protocol was developed for plantlet regeneration via somatic embryogenesis in banana cultivar Nendran. Somatic embryogenesis has been reported in many banana cultivars such as Bluggoe (ABB), Grand Naine (AAA) and Rasthali (AAB) (Novak et al, 1989; Escalant et al, 1994). However, few reports are available on this route of in vitro culture in the variety, Nendran (AAB group). Somatic embryos are ideal targets for genetic manipulation studies owing to their single cell origin. A rapid protocol for somatic embryogenesis can reduce time taken to develop transformants and their analysis. Somatic embryogenesis in banana (Musa spp.) cv. Nendran J. Hortl. Sci. Vol. 11(2): 116-123, 2017 Fig. 1. Imma ture ma le flow ers ino culate d o n ca llus induc t io n me d iu m flowers (hands) were removed without damaging the apical dome and were inoculated onto callus induction medium (Fig.1) Callus induction from IMFs The explants were cultured in 250ml culture bottles containing 40ml MS medium (Murashige and S koo g, 19 62 ) s up ple m e n te d w it h v a ri ou s combinations of 2,4-D (0.5 to 4mgL-1), IAA (0.5 – 1mg L-1 ), BA (0.5 – 4mg L-1 ) and picloram (0.5 – 10mg L -1) a long with a sc orbic ac id 20mg L -1 . Different combinations of growth regulators used for callus induction (Treatments A, B and C) are shown in Table I. Gelrite© at 0.4% was used as the solidifying agent. pH of the medium was adjusted to 5.7 using 1N NaOH or HCl and was autoclaved at 121°C, 1.06 Kg cm-2 pre ssure for 20 minutes. Culture s we re incubated in the dark at 25±2ºC and at 50-60 per cent 118 Immature male flowers were used as explants in this study for establishing an in vitro regeneration system via embryogenesis. IMFs have been used as explants for initiating cultures of several bananas and plantain cultivars (Becker et al, 2000; Khalil et al, 2002; Namanya et al, 2004; Houllou-Kido et al, 2005). As the explants were collected after formation of the complete bunch, it was possible to superior traits of mother plant assure selection of explants for the study, based on visual evaluation of bunch quality, number of hands, pest and disease resistance, etc. Sterilization and culture establishment from IMFs Contamination-free cultures were established with a simple surface-sterilization procedure. Dipping the flower bud in 90% ethanol for one minute, or, wiping it with ethanol soaked cotton-swab, was found to be effective. Immature flower buds are perfectly and tightly packed within the bracts of the inflorescence. This may be the reason for obtaining contamination- free cultures with this simple procedure. Endogenous bacterial contamination is very low in inflorescence meristems compared to that in the shoot meristem, favouring selection of this explant for in vitro culture. A serious problem encountered in establishing IMF culture s in c v. Nendran was prese nc e of polyphenols. The explant turned brown in 2 to 3 days after inoculation, and decayed subsequently. In this study, 20 mgL-1 of ascorbic acid was added to the medium in all the treatments, thus reducing the blackening of explants due to phenolic oxidation, and preventing decay of the explants. Use of antioxidants like ascorbic acid (Namanya et al, 2004) and citrate (Soubir et al, 2006) has been shown to avoid browning and improve callus formation in banana. Ascorbic acid is a reducing agent and can prevent oxidation of phenolics in the medium and it scavenges oxygen radicals produced during wounding of tissues. In this study, Gelrite© was used in the medium as a gelling agent, replacing agar. This too could overcome problems associated with phenolic exudation (Fig. 2). Spread of exudates was very restricted when Gelrite© was used. Similar findings have been reported by Khatri et al (2005) in callus induction studies using various banana cultivars. They also reported a positive correlation of light with browning, which may be due to the higher physiological activities under light than in the dark. In the present study too, cultures were incubated in the dark to overcome the ill effects of phenolics. Effect of growth regulators on callus induction in IMFs Callus induction was observed on IMFs in 45 to 50 days after inoculation on different media tested (Table1). Explants inoculated onto MS medium supplemented with 2,4-D (0.5 - 4 mgL-1) and IAA (0.5 – 1 mgL-1) turned brown within a week of inoculation (A1 to A16). However, after 4 weeks of inoculation, the explants exhibited a swelling; and, after another two weeks, callus initiation was observed in treatments A1 to A14. Among treatments with a combination of 2,4-D and IAA, callus induction was highest in treatment A9 (45.80 per cent), while treatments A1 & A13 produced the least (8.30 per cent). Higher concentration of 2,4-D, above 3.5mgL-1 (A15, A16), did not produce any callus. Calli induced in different treatments varied in morphology too (Fig. 3a-&-b). Ganapathy et al (1999) were a ble to produc e embryogenic callus from male flower buds of five bana na c ultivars on MS or White ’s medium supplemented with 2,4-D, NAA, IAA and biotin within 2 to 3 months of inoculation. According to Meenakshi et al (2011), IMF inoculation in 2,4-D supplemented MS medium could induce 77.70 per cent callus within a few weeks. Karintanyakit et al (2014) also reported the effect of 2,4-D on IMF in seven banana cultivars, a nd obs er ve d tha t ca llus induc tion wa s genome-specific. IMFs inoculated on picloram (0.5-10 mgL-1) supplemented medium (B1 to B11) turned brown at the base within 3 days of inoculation, but induced yellow coloured callus in treatments B4 to B11 (Fig. 3c), with the explant response ranging from 15 per cent to 60 per-cent in B4 and B8, respectively, in 45 days. Houllou- kido et al (2005) made a similar observation in their study where IMFs inoculated onto MS medium J. Hortl. Sci. Vol. 11(2): 116-123, 2017 Lekshmi et al Fi g. 2. Ef fect of soli di fyi ng agen ts on in te rferen ce from p h enol ics in I MF s on call u s in d u ct ion m e di u m : a) I MF s in ocu l ated on cal lu s i n d uc ti on m e- d iu m sol id i fi ed w it h agar, an d b ) M ed i um sol id i fi ed wi th Gelr it e on 10th d ay fr om i n ocu l ati on . 119 Table 1. Effect of growth hormones on callus induction in IMF of Musa spp. cv. Nendran Treatm 2,4-D BA IAA Picloram Morphology Explant ents (mgL-1) (mgL-1) (mgL-1) (mgL-1) of callus response % A1 0.5 - 0.5 - Pale white, hard 8.3 A2 0.5 - 1 - Pale white, hard 16.6 A3 1 - 0.5 - Pale white, hard 25.0 A4 1 - 1 - Pale white, hard 41.6 A 5 1.5 - 0.5 - Pale white, hard 33.3 A6 1.5 - 1 - Pale white, hard 29.1 A7 2 - 0.5 - Pale white, hard 33.3 A8 2 - 1 - Pale white, hard 33.3 A9 2.5 - 0.5 - Pale white, hard 45.8 A1 0 2.5 - 1 - Pale white, hard 25.0 A1 1 3 - 0.5 - Pale white, watery 20.8 A1 2 3 - 1 - Pale white, watery 20.8 A1 3 3.5 - 0.5 - Pale white, watery 8.3 A1 4 3.5 - 1 - Pale white, watery 25.0 A1 5 4 - 0.5 - No response 0 A1 6 4 - 1 - No response 0 B1 - - - 0.5 No response 0 B2 - - - 1 No response 0 B3 - - - 2 No response 0 B4 - - - 3 Yellow, watery 15.0 B5 - - - 4 Yellow, watery 35.0 B6 - - - 5 Yellow, watery 35.0 B7 - - - 6 Yellow, watery 50.0 B 8 - - - 7 Yellow, watery 60.0 B9 - - - 8 Yellow, watery 40.0 B10 - - - 9 Yellow, watery 30.0 B11 - - - 10 Yellow, watery 40.0 C1 - 0.05 - 0.5 Yellow, globular 8.3 C2 - 0.05 - 1 Yellow, globular 20.8 C3 - 0.05 - 2 Yellow, globular 16.6 C4 - 0.1 - 0.5 Pale white, globular 16.6 C5 - 0.1 - 1 Pale white, globular 30.0 C6 - 0.1 - 2 Pale white, globular 25.0 C7 - 0.2 - 0.5 Pale white, globular 8.3 C8 - 0.2 - 1 Pale white, globular 12.5 C9 - 0.2 - 2 Pale white, globular 8.3 C10 - 0.5 - 0.5 Pale white, globular 16.6 C11 - 0.5 - 1 Tiny, yellow, globular, friable 12.5 C12 - 0.5 - 2 Tiny, yellow, globular, friable 8.3 Note: Callus initiation occurred in 45-50 days on all the media tested J. Hortl. Sci. Vol. 11(2): 116-123, 2017 Somatic embryogenesis in banana (Musa spp.) cv. Nendran 120 supplemented with picloram developed necrosis in the first two weeks. However, after two months of inoculation, 80 per cent callus initiation was observed beneath the bracts. Smitha and Ashalatha (2011) reported the effect of picloram on callus induction from leaf-sheath explants. They obtained a brown, spongy callus with yellow globular structures using low concentrations of picloram in MS medium. At higher concentrations of picloram, a brown compact callus with white globular structures developed. In treatments with BA and picloram (C1 to C12), IMFs produced small-to medium-sized, yellow, globular friable callus with a dense yellow cytoplasm in C1,C2 and C3 (Fig. 3d), whereas, treatments C4 to C10 produced pale-white, globular callus with a dense cytoplasm (Fig. 3e), and, treatments C11 and C12 (Fig. 3f) developed yellow, watery callus. Among the treatments with BA and picloram combinations in MS medium, C5 produced the maximum (30 per cent) callusing rate. Callus initiation occurred in 45-55 days from inoculation in this combination. In a study by Smitha and Ashalatha (2011), embryogenic callus was obtaine d fr om le af-she a th e xpla nts on MS supplemented with picloram in Musa acuminata cv. Njalipoovan (AB). In the present study, among all the treatments tested for callus initiation, from phenolics interference was less in BA and picloram combination. Callus initiation occurred in 45-55 days of inoculation in this combination. Induction of somatic embryos from callus Embryogenic potential of the callus induced in various treatments mentioned above was studied in four different media (Table 2). But, embryo formation was obs er ve d only on se mis olid MS me dium supplemented with BA 2mgL-1, IAA 0.5 mgL-1 and 0.3 per cent Gelrite©. Only calli induced in treatments C8, C9 and C10 produced somatic embryos on this medium within 30 days of inoculation. Callus from C9 yielded 10 per cent embryo induction, which was the highest response in this study. These monocot embryos seemed elongated, with a glassy appearance (Fig. 4). Even though callus induction was high in treatments A and B, these failed to produce embryos. According to Arnold et al (2002), competence for somatic embryo induction may be the result of varying auxin sensitivity of cells. They observed that although callus initiation was comparatively high in treatments with 2,4-D and IAA, and, that with picloram alone, these culture failed to induce somatic embryos. Hence, it can be inferred that somatic embryogenesis may have been influenced by picloram and BA used for callus initiation. Rema kantha n e t al (2014) obta ine d direc t J. Hortl. Sci. Vol. 11(2): 116-123, 2017 Fig. 3. Morphology of calli induced on MS medium supple- mented wit h 2,4-D and IAA: A) Pale-white, hard callus formed in treatments A1 to A10, i.e., 2,4-D upto 2.5mgL-1 and IAA (0.5 or 1mgL-1) B) Pale-white watery callus formed in treatments A11 to A14 i.e. when 2, 4-D was more than 2.5 mg L-1 C) Yellow coloured callus formed on MS medium with picloram (Treatments B4 to B11) D) Yellow globular callus formed in treatments C1 to C3 (MS medium with BA 0.5mgL-1 and picloram 0.5 to 2mg L-1) E ) Pale-white globular callus formed d in treatments C4 to C10 ( MS with BA 0.05 to 0.5 mg L-1 and picloram 0.5 to 2mg L-1) F) Yellow callus formed in treatments C11 and C12 (MS with BA 0.5 and picloram 1or 2mg L-1) Fig. 4. Glassy, elo nga te d mo no co t embr yos developed on se mis o lid MS mediu m supplement ed with BA 2mg L-1 + IAA 0.5mg L-1 on 0.3 per cent Gelr ite Lekshmi et al 121 Table 2. Embryogenic response of callus initiated from various treatments Sl. Treatment for inducing somatic Response of callus induced on Per cent No. embryogenesis various treatments embryogenesis* A1 to B4 to C1 to A16 B11 C12 1 MS + 2,4-D 2 mgL-1, zeatin 0.2 mgL-1 + _ _ _ Gelrite 4% 2 MS+ 2,4-D 2 mgL-1 + IAA 1 mgL-1 + NAA 1 _ _ _ mgL1 biotin 1mgL-1 + glutamine 100 mgL-1 + Gelrite 0.3% 3 MS + BA 2 mgL-1 + IAA 0.5 mgL-1 + Gelrite _ _ Embryo C8 C9 C10 0.3% induction only in callus 33.33 60 50 from C8, C9, C10 4 SH+ MS vitamins + glutamine 100 mgL-1 + picloram 1 mgL-1+ sucrose 4.5% + Gelrite 0.2% *Per cent embryogenesis was calculated as the number of calli showing embryogenesis out of the number of callus clumps inoculated embryogenesis from split shoot tips of Grand Naine on MS medium supplemented with picloram and BA, used alone or in combination, obtaining the best embryogenic response when used in combination. When they used BA alone, explants produced only shoots. In almost all the protocols reported for somatic embryogenesis from callus, an intermediary liquid medium is involved. Maintenance of suspension cultures is a somewhat sophisticated process, even though rate of multiplication realized is high. In our study, we could replace the same with a simple semisolid medium. Germination of somatic embryos and plantlet formation Somatic embryos induced on semisolid MS with BA 2mgL-1 + IAA 0.5mgL-1 failed to germinate on the same medium; But, when half-strength MS with the same combination of growth regulators was used, 60- 80% germination was obtained in 3 weeks of inoculation in dark (Table 3). At this stage, shoots were pale-white to pale- green in colour (Fig. 5a).When germinated embryos were transferred onto MS medium with BA 2mgL-1 + NAA 1mgL-1 and incubated at a light intensity of 31.4µmol-2S-1 and 14 h light/8h dark photoperiod, these turned green and developed into complete plantlets (Fig. 5b). Meenakshi et al (2011) used BA and IAA to supplement MS medium with 0.2 per cent Gelrite© for conversion of somatic embryos into plantlets. Okole and Schulz (1996) and Lee et al (1997) reported that high BA concentrations induced only shoot formation. But Khalil et al (2002) reported lower rate of shoot formation at BA >5 mgL-1. In the present study, BA concentration maintained at 2mgL-1 on half-strength MS medium resulted in regeneration of soma tic embryos . Ganapathi et al (1999) also observed that development of embryos into plantlets wa s good in IAA a nd BA combina tion. Better Table 3. Germination of somatic embryos on semisolid half MS with BA 2mg L-1+IAA 0.5 mg L-1 Treatment for No. of embryo clusters No. of clusters Per cent germination callus induction inoculated showing germination C8 10 6 60.00 C9 18 13 72.22 C10 15 11 80.00 J. Hortl. Sci. Vol. 11(2): 116-123, 2017 Somatic embryogenesis in banana (Musa spp.) cv. Nendran 122 J. Hortl. Sci. Vol. 11(2): 116-123, 2017 Lekshmi et al Fig 5. Germination of embryo and plantlet development A) Germinating embryos in semisolid 1/2MS with BA 2 mgL-1+ IAA 0.5 mgL-1 B) Plantlet conversion in MS medium with BA 2 mgL-1 + NAA 1 mgL-1 c) Plantlet placed for primary hardening in coir-pith compost development of plantlets was observed when IAA was subs titute d with NAA 1mgL -1 . MS me dium supplemented with NAA and 0.2 per cent Gelrite© was used by Meenakshi et al (2011) for root development. The plantlets, after primary hardening in coir-pith compost (Fig. 5c) and secondary hardening for one month in soil and cow-dung (1:1) mixture in a mist chamber, showed 100% survival. CONCLUSION Embryogenic culture system described in this paper demonstrates the potential of immature male flowers as explants for raising highly proliferative, e mb ry oge n ic c ult ur e s wi th s im ple ho rm on e combinations. In most of the protocols reported, liquid me dium is use d for prolife ra tion of the e mb ry os . Ma i nt e na n c e o f e mb ryo ge n ic c e ll suspensions is la borious a nd time -consuming. Fre que nt subc ultur es ne ede d in the re por ted proc edures may le ad to soma clona l va riation, microbial contamination and, eventually, loss of morphogenetic potential (Kulkarni and Ganapathi, 2009). The protocol described in our present study does not require any intermediate cell suspension syste m, making it a more convenient and very effective method for small laboratories lacking facilities for continuous agitation. According to Georget et al (2000), a common feature of many ba na n a c e ll c u lt ur e p r ot oc o ls is th e s l ow de v e lo pme nt o f c e ll c l us t e rs , m a kin g pl a nt regeneration time-consuming (18-24 months). The current protocol requires just 6 months for producing plantlets from the explant. It is, thus, suitable for ge neti c tra ns forma tion e xpe rim ent s w her e by tra ns form ants c a n be e a sily r e ge ne ra te d a nd evaluated. Further refinement of the medium may improve conversion rate, thus making it suitable for mass propagation as well. ACKNOWLEDGEMENT We gratefully acknowledge funding provided for the study by Kerala Agricultural University. Authors’ contribution statement: Lekshmi, R.S. (lekshmirs84@gmail.com), Ph.D. student, carried out the work; Soni, K.B. is the principal investigator; Swapna, A., Deepa S. Nair, Lekha, S. and Reghunath, B.R. are Members, Advisory Committee of the student. REFERENCES Arnold, S.V., Sabala, I., Bozhkov, P., Dyachok, J. and Filonova, L. 2002. Developmental pathways of somatic embryogenesis. Plant Cell Tiss. Org. Cult. 69:233-249 doi:10.1023/A:10156732 00621 Becker, D.K., Dugdale, B., Smith, M.K., Harding, R.M. and Dale, J.L. 2000. Genetic transformation of Cavendish banana (Musa spp. AAA group) cv. Grande Naine via micro-projectile bombardment. Pl ant Cell Rep ., 19:2 29-2 34 do i:1 0.100 7/ s002990050004 Cot e, F.X., D omergn e, R., Monma rson , S., Schwendiman, J., Teisson, C. and Escalant, J.V. 1996. Embryogenic cell suspensions from male flower of Musa AAA cv. Grand Naine. Physiol. Pl an t, 97 :28 5-2 90 do i: 10 .103 4/ j.139 9- 3054.1996.970211.x Cronauer, S. and Krikorian, A.D. 1983. Somatic embryos from cultured tissues of triploid plantains (Musa ABB). Plant Cell Rep., 2:289291 doi: 10.1007/ BF00270183. Escalant, J.V., Teisso n, C. and Cote, F.X. 1994. Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivar (Musa spp.). In vitro Cell Dev. Biol., 30:181- 186 doi:10.1007/BF02823029 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 Georget, F., Domergue, R., Ferrière, N. and Côte, F.X. 2000. Morphohistological study of the different constituents of banana (Musa AAA, cv. Grand Nain) embryogenic cell suspension. Plant Cell Rep., 19:748-754 doi:10.1007/s002999900188 123 Houllou-Kido, L.M., Kido, E.A., Falco, F.C., Silva, M.C., Figueira, A.V.O., Nogueira, N.L., Rossi, M.L. and TulmannNeto, A. 2005. Somatic embryogenesis and the effect of particle bombardment on banana ‘Maçã’ regeneration.Pesquisa Agropecuária Brasileira., 40(11):1081-1086 http://dx.doi.org/ 10.1590/S0100-204X2005001100005 Indian Horticulture Database, 2015, http://nhb.gov.in/ area-pro/horst_galance_2016.pdf Karintanyakit, P., Suvittawat, K., Chinachit, W., Silayoi, B., and Saratultad, P. 2014. The impact of genome and 2,4-D on callus induction from immature male flowers of seven banana cultivars. Acta Hort. 10 24: 25 3-2 55 d oi: 1 0.1 766 0/ ActaHortic.2014.1024.33 Khalil, S.M., Cheah, K.T., Perez, E.A., Gaskill, D.A. and Hu, J.S. 2002. Regeneration of banana (Musa spp. AAB cv. ‘Dwarf Brazilian’) via secondary somat ic embr yogen esis. Pla nt Cel l Rep ., 20(12):1128-1134 DOI 10.1007/s00299-002- 0461-0 Khatri, A., Khan, I.A., Dahot, M.U., Nizamani, G.S., Siddiqui, M.A., Raza, S. and Naqvi, M.H. 2005. Study of callus induction in banana (Musa sp.) Pak. J. Biotechnol., 2(1-2):36-40 Kulkarni, V.M. and Ganapathi, T.R. 2009. A simple procedure for slow growth maintenance of banana (Musa spp.) embryogenic cell suspension cultures at low temperature. Curr.Sci., 96(10):1372-1377 http://www.ias.ac.in/currsci Lee, K.S., Zapata-Arias, F.J.B.H. and Afza, R. 1997. Histology of somatic embryo initiation and organogenesis from rhizome explants of Musa spp. Pl. Cell Tiss. Org. Cult., 51:1–8 doi: 10.1023/ A:1005985401756 Meenakshi, S., Shinde, B.N. and Suprasanna, P. 2011. Somatic embryogenesis from immature male flowers and molecular analysis of regenerated plants in banana ‘Lalkela’ (AAA). J. Fr. & Orn. Pl . Res., 19(2 ): 15 -30 htt p:/ /www.isad.p l/ jofaop.html Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cul tur es. Ph ysio l. Pla nt , 15 (3 ): 47 3-4 97 doi:10.1111/j.1399-3054 Namanya, P., Magambo, S.M., Mutumba, G.M. and Tushem erei rw e, W. 200 4. S omat ic emb ryogen esis fr om immat ur e m ale inflorescences of East African highland banana cv. ‘Nakyetengu’. African Crop Sci. J., 12(1):43- 49 doi: 10.1007/s11627-014-9598-0 Navarro, C., Escobedo, R.M. and Mayo, A. 1997. In vitro plant regener ation f rom embryogenic cultures of a diploid and triploid Cavendish banana. Pl. Cell Tiss. Org. Cult., 51:1725 doi:10.1023/ A:1005965030075 Novak, F.J., Afza, R., Van Duren, Perea, M., Dallos, B., Conger, V. and Xiaolang, T. 1989. Somatic embr yogen esis and pl ant regene rat ion i n suspension cultures of dessert (AA and AAA) and Cooking (ABB) bananas (Musa spp.). Biotechnol., 7:147158 doi:10.1038/nbt0289-154 Okole, B.N. and Schulz, F.A. 1996. Micro-cross sections of b anan a an d plan tai ns ( Mu sa sp p.) : morphogenesis and regeneration of callus and shoot buds. Pl. Sci., 116:185-95 doi.org/10.1016/ 0168-9452(96)04381-6 Remakanthan, A., Menon, T.G. and Soniya, E.V. 2014. So mati c emb ryogen esis in ban ana ( Mu sa acuminata AAA cv. Grand Naine): effect of explant and culture conditions. In Vitro Cell. Dev. Biol. (Plant), 50(1):127-136 doi:10.1007/s11627- 013-9546-4 Resmi, L. and Nair, A.S. 2007. Plantlet production from the male inflorescence tips of Musa acuminata cultivars from South India. Pl. Cell Tiss. Org. Cult., 88:333-338 doi: 10.1007/s11240-007- 9206-7 Smitha, P.D. and Ashalatha, S.N. 2011. Effect of picloram on somatic embryogenesis from leaf sheath explants in diploid Musa acuminata cv. Njalipoovan. Pl. Tiss. Cult. Biotechnol., 21(1):83- 87 doi:10.3329/ptcb.v21i1.9612 Soubir, T., Salil, K.B., Ajoy, M., Md. Sadrul, A. and Sarder, N.U. 2006. Control of phenolic compound secretion and effect of growth regulators for organ formation from Musa spp. cv. Kanthali floral bud explants. Amer. J. Biochem. Biotechnol.,2(3):97- 104 doi:10.3844/ajbbsp.2006.97.104 J. Hortl. Sci. Vol. 11(2): 116-123, 2017 Somatic embryogenesis in banana (Musa spp.) cv. Nendran (MS Received 20 March 2016, Revised 25 March 2016, Accepted 30 Decenber 2016)