REGENERATION OF BANANA (Musa spp. AAA Group) cv. WILLIAMS via SECONDARY SOMATIC EMBRYOGENESIS AND CELL SUSPENSION FROM IMMATURE MALE FLOWER BUDS

Development of embryogenic cultures having high regeneration efficiency from important, commercial varieties of banana is a prerequisite for genetic manipulation and for in vitro propagation. In the present study, we have studied the induction of somatic embryogenesis from young immature male inflorescences of the banana cultivar "Williams" (Musa spp. AAA group) via secondary somatic embryogenesis and cell suspension. Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l alfa  naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline (MM1). Secondary somatic embryogenesis (SE2) was developed when primary embryos were subculture on SK4 medium (MS medium supplemented with 10 ml of coconut milk). Embryos differentiated when sub-cultured on SK8 medium (MS medium supplemented with 5 mg/l BA and the embryos germinated when subculture on MS free hormone medium. Suspension cultures were initiated from SE2 embryogenic tissues from when placed in liquid medium supplemented with 2,4-D (1mg/l), biotin (1 mg/l), L-glutamate (100 mg/l), malt extract (100 mg/l), and sucrose (45 g/l), pH of the medium was adjusted to 5.3. The packed cell volume (PCV) of the suspension increased 2-5 fold with each monthly cycle. The somatic embryos were developed when suspension culture were aspirated on MS medium supplemented with biotin (1 mg/l), malt extract (100 mg/l), Glutamine (100mg/l), NAA (1mg/l), Kinetin (0.5 mg/l) Zeatin (0.2 mg/l), sucrose (45 g/l), and phytagel (2.6 g/l) (SK13). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l 6-benzylaminopurine (BA) for development of mature somatic embryos, which were isolated and cultured on hormone-free MS medium for germination and development into plantlets. Approximately 55% of the somatic embryos germinated and developed into plantlets. Somatic embryogenesis via SE2 and cell suspension might be an excellent technique for mass propagation, developing a breeding strategy and genetic transformation of banana.


S. M. KHALIL
Agricultural Genetic Engineering Research Institute (AGERI), ARC, 12619, Giza, Egypt anana (Musa sp. L.) is the most important of the cultivated tropical fruits and is produced in the tropical and subtropical regions. Banana is grown in more than 130 countries throughout the world on an area 8.25 million ha with a production of 97.38 million tons (Singh, 2007). Banana and plantain in many countries represent major fruit exports and are essential sources of income for national economies, (Anonymous, 2001). The production is limited primarily by black Sigatoka (Mycosphaerella fugiensis), Fusarium wilt (Fusarium oxysporum var. cubense), viruses, and nematodes (Radopholus similes) (Sasson, 1997). Banana bunchy top disease (BBTD) caused by banana bunchy top nanovirus (BBTV) and banana CMV disease caused by banana-cucumber mosaic cucumovirus (Banana-CMV), have enormous negative impacts on banana productivity. Average costs for control of this fungal disease alone ranges between $ 0.30 and $1.0 per plant in banana plantations in order to avoid yield losses of 30-50% (Sagi et al., 1995). Secondary somatic embryogenesis (SE 2 ) is a phenomenon whereby new somatic embryos are initiated from other somatic embryos. Some cultures are able to retain their competence for secondary SE 2 for many years, and thus provide useful material for various studies, as described for Vitis rupestris (Martinelli et al., 2001), cork oak (Quercus suber) (Hernández et al., 2003) banana (Khalil et al., 2002). Recently, the development of different methods for somatic embryogenesis has considerably assisted in vitro technologies for these species. Diverse procedures have been originally described for somatic embryo induction in banana and differ depending on the type of explants employed, to include zygotic embryos, foliar bases and corm slices (Novak et al., 1989), in vitro cultured meristems (Dhed'a et al., 1991), and immature male (Escalant et al., 1994) and female (Grapin et al., 2000) flowers. Despite the many options, the most widely applicable methodologies for the establishment of regenerable embryogenic cell suspensions from valuable seedless cultivars have been restricted to the use of either in vitro multiple meristems (Xu et al., 2005;Kulkarni et al., 2006;Strosse et al., 2006) or immature flowers (Côte et al., 1996;Grapin et al., 1996;Ganapathi et al., 2001;Khalil et al., 2002;Suprasanna et al., 2006) as starting B material.
Nevertheless, somatic embryogenesis in banana is still far from being considered a routine technology and has not even been successfully applied to some cultivars.
The present study reports an efficient and reproducible protocol with high regeneration frequency for secondary somatic embryogenesis and cell suspension culture for mass propagation of banana plantlets to improve Musa spp. AAA group cv. Williams.

Plant material and culture initiation
The initial plant material used was the cultivar cv. "Williams" belong to the AAA genotype group. One month-old male flower buds about 30 cm in length were collected from the field. Cultures were initiated within two days after removing the male flowers from the mother plants. Tissues were sterilized by washing with 1% (v/v) detergent solution for 5 min and the outer bracts were removed until inflorescence was 5 cm long, surface sterilized by 100% Clorox® (5.25% sodium hypochlorite) for 15 min and finally rinsed 3 times with sterile water. In a sterile Petri dish, the outer bracts were peeled with forceps until flower axis was 1.5 cm in length. The remaining flower bud was cut into 4 segments longitudinally (orthogonally) through the shoot tip, into 2 mm segments (transverse section). A total of 16-20 such segments can easily be obtained from a single inflorescence male flower bud.

Establishment of primary somatic embryogenic cultures
Immature male flower explants were isolated and cultured on solid M1 medium ( Table 1). The cultures were incubated in the dark at 28C for two weeks.
The flower bud segments became swollen two weeks after cultures were initiated. Flower buds could be seen as whitish tissue protruding from the inflorescence tissues. These were carefully excised from the mother tissue, transferred to fresh M1 medium and kept in darkness. Primary somatic embryos were produced after tissues were transferred to modified MM1 medium which was composed of (M1 medium with 200 mg/l casein hydrozylate and 2 mg/l proline) for 2 months. Compact white calli and friable embryogenic tissues with globular structures containing primary somatic embryos were formed.

Induction of secondary somatic embryogenesis
After the production of primary embryogenesis in the first three months, the compact calli were transferred to seven modified different media (SK1 to SK7) illustrated in Table (1) to developed secondary somatic embryogenesis (SE 2 ). These cultures, and all subsequent transfers, were incubated at 28C under a 16/8 h (light/dark) photoperiod with light supplied by 40 W cool-white fluorescent lamps. At this stage, SE 2 were produced on SK4 medium and then sub-cultured on both SK8 and GTR media (Table 1) for embryo development. The response of SE 2 to several different concentrations of the cytokinin 6-benzylaminopurine (BA) in the differentiation media SK8 was investigated. Differentiation media were prepared with 0.0, 2.5, 5.0, 7.5 or 10.0 mg/l BA, and the production of mature embryos and plantlets from each medium was recorded. After two weeks, cultures were transferred from SK8 to MS (Murashige and Skoog, 1962) free hormone medium for further differentiation into mature embryos with leaf primordial.

Production of cell suspension cultures from SE 2
Suspension cultures were established from friable embryogenic calli containing SE 2 embryos. One gram of callus with SE 2 was transferred to 10 ml of liquid medium designated M2 (Table 1). pH of the medium was adjusted to 5.3. The suspensions were cultured in 250-ml Erlenmeyer flasks on a reciprocal shaker at 100 rpm in darkness for two weeks until suspension cultures start to multiply. Culture medium was changed weekly during the first month of culture and subsequently biweekly. Regular maintenance of suspension cultures was done by pipetting 1 ml of mother stock to 20 ml of fresh M2 medium. The medium was changed every 7-10 days. Cultures were maintained at 28C under a photoperiod of 16 h/8h (light/dark).

Proliferation of embryonic cells
Cell suspension cultures after four subcultures were collected through sieve (180 l) to avoid any cell aggregates. The 0.5 ml packed cell volume (PCV) of suspension culture was aspirated on sterile filter paper on the four different media, SK4, SK12, SK13 and SK14 to determine the suitable medium for embryo development. The compositions of four media were illustrated in (

Embryo germination and plantlet formation
Germination of embryos was performed on a medium designated SK10 medium which consisted of MS medium with MS vitamins and 30 g/l sucrose without growth regulators. The embryos were germinated in Petri dishes (diameter 9 cm). Embryos were maintained on SK10 medium for 15 days at 28C with a photoperiod of 16-h light and 8 h dark. The plantlets were then transferred to SK11 medium (Table 1) for elongation at 28C with a photoperiod of 16 h light and 8 h dark. Mature plantlets 5-6 cm long were obtained after 2-3 weeks on SK11 and were ready for acclimatization.

Callus formation with embryogenic structures in cv.Williams
Callus formation with embryogenic structures was characterized explants type immature male flowers using as starting material in the embryogenic process.
When cultured explants into M1 media and incubated in the dark for one month and re-subculture in the same for another one month. The white tissues were excised from the mother explants and subculture into MM1 medium (Table 1) for developmental callus, non-friable embryogenic tissue with early stages of primary embryos were formed (Fig. 1A). About (62.5%) explants producing embryogenic callus primary embryos. When added proline and casein hydrolysate into the MM1 medium to increase callus proliferation and production of primary embryos and the embryogenic potential in other banana cultivars. The similar results were obtained when used the M1 and MM1 media for developmental primary embryos from immature male flower explants in banana (Musa AAB cv. Dwarf Brazilian) (Khalil et al., 2002).

Induction of secondary somatic embryos (SE 2 )
After the production of primary embryogenesis in the first six months, the compact calli (Fig. 1A) were transferred to seven modified different media (SK1 to SK7) to develop secondary somatic embryogenesis (SE 2 ). After 28 days from subculture, results in Table (2) showed that significant percentages of SE 2 were produced on SK4 and SK5 media, 55% and 44.12%, respectively, while the lowest percentage of SE 2 (15.71%) was produced on SK7 medium. Similar results were reported by Khalil et al. (2002) on banana (Musa spp. AAB Group) cv. Dwarf Brazilian. Secondary somatic embryos typically entered the globular stage directly from primary somatic embryos However, in other embryogenic systems, the early developmental embryo stages (e.g. globular and heart stages) showed greater potential for secondary embryogenesis compared with later stages (Neves et al., 1999;Nair and Gupta, 2006).

Secondary somatic embryogenesis stages and development of mature embryos
The all stages of developmental SE 2 were demonstrated in Fig. (2). Secondary somatic embryogenesis typically entered the globular stage directly from primary somatic embryos and occurred in clusters ( Fig. 2A), initially appearing as small, on the surface of the clusters. The embryos passed through a recognizable heart-shaped stage, a primary torpedo stage (Fig. 2B), a later torpedo stage (Fig.  2C) and finally green plumules emerged from these embryos (Fig. 2D). The embryos passed through different developmental stages and finally green plumules emerged from these embryos. Concomitant with morphological development there was a change in opacity from hyaline to translucent to opaque with a simultaneous change in color from pale white to yellow to green. The green-plumule stage developed to germination stag with root initiation (Fig. 2E) after two weeks of subculture on SK8 medium with elongation of root and leaf primordial initiation (Fig. 2F). Khalil et al. (2002) and Pérez and Rosell (2008) reported the same trend in development of SE 2 from banana AAB group cv. Dwarf Bazilian.

Embryo differentiation of secondary somatic embryogenesis
Differentiation, maturation of embryos and development of plantlets were compared using two types of media SK8 medium and GTR medium. Results in Table (3) showed that SK8 medium was superior to GTR medium as it produced a higher number of globular. Vuylsteke (1989), reported that high BA concentrations induced shoot formation; however, our results showed a lower formation of mature embryos and plantlets at high (>5 mg/l) BA concentrations. The same results were reported by Khalil et al. (2002) on Dwarf Brazilian cultivars (AAB Group). The transfer of embryos to MS free hormone medium resulted in a threefold in-crease in the number of SE 2 that developed, irrespective of whether they were originally cultured on SK8 media. However, embryonic tissue originally cultured on SK8 produced three times more mature SE 2 when transferred to SK10 medium than calli originally cultured on GTR medium. Furthermore, transfer from SK8 to MS free hormone medium dramatically increased the number of germinated embryos and plantlets when compared to those that were formed on SK8 medium directly.

Plant regeneration via cell suspension culture of banana Musa spp. cv. Williams.
Suspension culture was established from the friable embryogenic callus with secondary somatic embryogenesis by transferring one gram from such callus of secondary somatic embryogenesis calli to 10 ml of M2 liquid medium according to Côte et al. (1996). The suspensions were cultured in 250-ml Erlenmeyer flasks on a reciprocal shaker at 100 rpm in darkness for three weeks until suspension cultures start to multiply. Culture medium was changed weekly during the first month of culture and subsequently biweekly with 20 ml of liquid M2 medium. Pipetting 1 ml of mother stock to 20 ml of fresh M2 medium and changing medium every 7-10 days did regular maintenance of suspension cultures. Cultures were maintained at 28C under a photoperiod of 16/8) h (light/dark). Kosky et al. (2002) developed embryogenic cell suspension (ECS) for large scale growth in temporary im-mersion systems and showed that different cell densities gave the best results of somatic embryo development with an initial inoculums of 100 mg and embryo diameter from 0.5 to 1.2 mm. Ma (1991), Escalant et al. (1994) and Jalil et al. (2003) obtained ECS cultures and plant regeneration of Musa acuminata by culture of male flowers. Data in Table (4) shows that SK13 medium has the highest number of clones and the highest number of embryos (Globular, torpedo and mature) under dark condition at 28C (Fig.  3C). Somatic embryos initially appeared small and rapidly enlarged into distinct globular structure, which passing through recognizable torpedo structure previously illustrated in (Fig. 2). Somatic embryos initially globular and torpedo were observed within 23-28 days after aspiration on SK13 regeneration medium. On the other hand, the SK12, SK14 and SK4 media have the lowest percentage of mature embryo in dark condition. The results in Table (4) indicated that the aspiration of embryogenic cell suspension on SK13 medium and incubation in dark at 28C the best media compensation and incubation for developed embryogenic cell suspension. Pinto et al. (2008) reported the same conditions were used for developed embryogenic calli.

Maturation of embryos
Embryo development was achieved by culturing the embryonic suspension culture on SK4, SK12, SK13 and SK14 medium then transferred two media SK8 (Khalil et al., 2002) and MM4 medium (Côte et al., 1996), to determine the best medium for embryo maturation and development regenerated banana plants. The cultures were incubated at 28C with a photoperiod of 16/8 h (light/dark) for four weeks. The data in Table (5) reported that the embryos were sub-cultured on the SK8 medium with the highest number of globular stage, later of torpedo, greenplumula stages and the total number of germination, embryos. Date in Fig. (3) indicated that the embryos initially on SK13 medium and subculture on SK8 medium development the highest number of regenerated banana plants. Data in Table (5) illustrate that the somatic embryos, which developed on SK13 medium and transferred to differentiation SK8 medium produced the highest number of germinated embryos compared with MM4 medium. Considering the effect of light on repetitive ECS, in general, and until the transition to mature stage, dark had a positive or neutral effect on embryo proliferation and total number of embryos in torpedo on stage. These quantitative and qualitative results strongly suggest that, in this species, cell suspension must be kept in the dark until they reach the globular stage, and should then be transferred to light on differentiation medium, the same data was reported by Pinto et al. (2008).
Mature somatic embryos, which differentiated on SK8 and MM4 media were separated from the culture mass and placed directly on MS free hormones medium (SK10 , Table 1). Somatic embryos gave rise to small plantlets (shoots and roots) within 10 days. The small plantlets were sub-cultured onto SK11 for elongation and development of new leaves. Figure (3) shows that the somatic embryos developed on SK13 and transferred to differentiation medium SK8 produced a higher number of germinated embryos (91%), but decreased the percentage to 30.5% when sub-cultured on MM4 medium. On the other hand, the percentagegerminated embryos were decreased when sub-cultured on SK12, SK4 and SK14 medium. However, embryonic suspension originally cultured on SK13 produced 96% embryos developed to shoots when transferred to SK8 differentiation medium. The date recorded on the regenerated banana plantlets from those of Escalant et al. (1994), who reported 60-70% germination using a temporary immersion system, Kosky et al. (2002), who reported an 89.3% regeneration using and cell suspension using a bioreactor.

Embryo germination and plantlet formation
To develop the mature true to type of regeneration cv. Williams banana germination of embryos was performed on a medium designated as SK10. Embryos germinated were maintained on SK10 medium for 15 days at 28C with a photoperiod of 16/8h (light/dark). The small plantlets were then transferred to SK11 medium for elongation. Mature plantlets (5-6 cm long) were obtained after 2-3 weeks on SK11 and were ready for acclimatization (Khalil et al., 2002) The stages of regeneration via cell suspension were demonstrated in Fig. (4) and production of plantlets from immature male flower buds in Musa cv. Williams, primary somatic embryogenesis from explants on modified M1 medium (Fig. 4A), suspension culture was developed on liquid M2 medium for multiply of embryonic cells (Fig. 4B), embryogenic cell suspension (ECS) aspiration and embryos induction on SK13 after four weeks from plating on culture medium (Fig. 4C), differentiation of embryos and maturation on SK8 medium (Fig. 4D), germination of embryos and production of small plantlets on SK10 medium two weeks post cultured on germination medium (Fig. 4E). Plantlets obtained were transferred to hormone-free MS medium and placed in light condition for formation of chloroplasts and further growth of the shoots and root (Fig. 4F). The successfully regenerated plantlets of banana cv. Williams acclimatized with suitable soil mixture were illustrated in Fig. (4 G and H).
Finally, the established protocol in this work for regeneration takes 12.5 months from initiation of suspension culture. We have successfully regenerated plants using the system described in this report via secondary somatic embryogenesis and cell suspension culture.

Plantlet formation and acclimatization of banana plants via SE 2 and cell suspension.
Mature embryos from SE 2 and cell suspension were sub-cultured on SK10 medium for germination, while the elongation was achieved by transferring the small plantlets to SK11 medium were obtained. After 2-3 weeks mature plantlets (5 to 6 cm in length) were obtained. Plants (10 plantlets/Fitakone or Jar) suitable for acclimatization were placed in a greenhouse maintained at 28C for 3-5 days. Medium was rinsed from the plantlets, and the latter were transplanted to 5-cmdiameter pots containing peat-moss, sand and vermiculite (1:1:1; v:v). The plantlets were acclimatized for three weeks in a green-house at 28°C, shown this stage in Fig. (4 G and H). To date, over 120 plants have been maintained in the greenhouse for more than two months. No abnormal morphological variations have been observed.

CONCLUSION
The development of an in vitro regeneration system is essential for genetic manipulation of banana. This is the first report using secondary somatic embryogenesis and cell suspension to produce a high percentage of germinated embryos and a high number of regenerated banana plants. The culture system described in this paper is a high regeneration capacity system and demonstrates the potential of using primary somatic embryos derived from immature male flower bud explants to raise highly proliferate secondary somatic embryo cultures of banana cultivar 'Williams', and this is the first report of tissue culture of this variety in Egypt. The use of secondary somatic embryos could provide an efficient solution to the problems limiting plant regeneration in other banana cultivars, and may result in a sys-tem for production of transgenic plants engineered for disease resistance or other purposes.

SUMMARY
Development of embryogenic cultures having high regeneration efficiency from important, commercial varieties of banana is a prerequisite for genetic manipulation and for in vitro propagation. In the present study, we have studied the induction of somatic embryogenesis from young immature male inflorescences of the banana cultivar "Williams" (Musa spp. AAA group) via secondary somatic embryogenesis and cell suspension. Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline (MM1). Secondary somatic embryogenesis (SE 2 ) was developed when primary embryos were subculture on SK4 medium (MS medium supplemented with 10 ml of coconut milk). Embryos differentiated when sub-cultured on SK8 medium (MS medium supplemented with 5 mg/l BA and the embryos germinated when subculture on MS free hormone medium. Suspension cultures were initiated from SE 2 embryogenic tissues from when placed in liquid medium supplemented with 2,4-D (1mg/l), biotin (1 mg/l), L-glutamate (100 mg/l), malt extract (100 mg/l), and sucrose (45 g/l), pH of the medium was adjusted to 5.3. The packed cell volume (PCV) of the suspension increased 2-5 fold with each monthly cycle. The somatic embryos were developed when suspension culture were aspirated on MS medium supplemented with biotin (1 mg/l), malt extract (100 mg/l), Glutamine (100mg/l), NAA (1mg/l), Kinetin (0.5 mg/l) Zeatin (0.2 mg/l), sucrose (45 g/l), and phytagel (2.6 g/l) (SK13). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l 6-benzylaminopurine (BA) for development of mature somatic embryos, which were isolated and cultured on hormone-free MS medium for germination and development into plantlets. Approximately 55% of the somatic embryos germinated and developed into plantlets. Somatic embryogenesis via SE 2 and cell suspension might be an excellent technique for mass propagation, developing a breeding strategy and genetic transformation of banana.
a All media used in this study contained MS salts, myo-inositol (100 mg l -1 ), nicotinic acid (1 mg l -1 ), thiamine HCl (1 mg l -1 ) and pyridoxine HCl (10 mg l -1 ), solidified with Phytagel (2.6 g l -1 ).   Fig. (1): Production of primary embryo on M2 medium (A) and development of secondary somatic Embryogenesis on SK4 medium using immature male flower buds explants. (B), development of secondary somatic embryogenesis from primary embryos after four weeks on SK4 medium.   ); B, suspension culture was developed on M2 medium (Bar 0.5 cm); C, embryos induction on SK13 medium four weeks after plating on culture medium; D, differentiation of embryos and maturation on SK8 medium (bar 0.5 cm); E, germination of embryos and production of small plantlets on SK10 medium two weeks post cultured on germination medium (Bar 0.5 cm); F, elongation and development of mature regenerated plantlet of banana on SK11 three weeks after subcultured on elongation medium (Bar 0.5 cm G., acclimatization of banana plantlets after 10 days of plantation with suitable soil mixture. H., regenerated banana plant with normal morphological growth and leaves, the all regenerated banana true to type.