Plant Cell Tiss Organ Cult (2009) 99:133–140 DOI 10.1007/s11240-009-9585-z ORIGINAL PAPER ABA enhances plant regeneration of somatic embryos derived from cell suspension cultures of plantain cv. Spambia (Musa sp.) Nasser J. Y. Sholi Æ Anjana Chaurasia Æ Anuradha Agrawal Æ Neera Bhalla Sarin Received: 4 November 2008 / Accepted: 10 August 2009 / Published online: 23 August 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Creamy friable calli were induced from meristems (scalps) of proliferating shoots of plantain (Musa sp.) cv. Spambia (genome AAB) incubated on a semi-solid modified Murashige and Skoog (MS) medium supplemented with 4.5 lM 2,4-dichlorophenoxyacetic acid (2,4D) and 1.0 lM zeatin. About 25% of shoot-tip explants formed scalps, and about 98% of scalps developed embryogenic calli. Small dense aggregates of cells, were obtained when these calli were transferred to liquid MS medium supplemented with 4.5 lM 2,4-D and 1.0 lM zeatin. Upon transfer to semi-solid MS medium of the same composition as described above, aggregates of cells formed somatic embryos. In the presence of 2.5 lM abscisic acid (ABA), maturation of somatic embryos was 2.6-fold higher than that of control (lacking ABA), and regardless of the type of cytokinin used in the medium. Upon transfer to MS medium supplemented with 1.25 lM 6-benzyladenine (BA), 80% of germinated embryos developed into plantlets. Keywords Banana
Plantain
Musa spp. embryogenic callus
Somatic embryogenesis
Cell suspension
Abscisic acid N. J. Y. Sholi
A. Chaurasia
N. B. Sarin (&) School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India e-mail: neerasarin@rediffmail.com A. Agrawal National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110 012, India Abbreviations BA 6-Benzyladenine 2,4-D 2,4-Dichlorophenoxyacetic acid ABA Abscisic acid IAA Indole-3-acetic acid MS Murashige and Skoog (1962) medium TDZ Thidiazuron Introduction Banana and plantain (Musa spp.) are nutritious and widely grown fruit crops with annual production of around 104 million tonnes (FAO 2004). Due to sterility and polyploidy, developing banana and plantain with resistance to various diseases and pests through conventional breeding has been limited (Sagi et al. 1997). Therefore, using the tools of biotechnology will overcome problems of genetic enhancement of these crops. Embryogenic cell suspensions (ECSs) of banana have been successfully initiated from proliferating meristems (Dhed’a et al. 1991; Schoofs 1997; Schoofs et al. 1998), immature male and female flowers (Escalant et al. 1994; Grapin et al. 1996, 1998; Jalil et al. 2003), immature zygotic embryos (Cronauer-Mitra and Krikorian 1988; Escalant and Teisson 1999; Marroquin et al. 1993), corm tissues and leaf bases (Novak et al. 1989), and protoplasts (Assani et al. 2001). However, embryogenic response is poor, as in East African Highland banana (Escalant et al. 1994; Panis et al. 1993), and plantlet regeneration remains low. To date, there are only a few published reports on somatic embryogenesis from plantain. Embryogenic suspension cultures have been developed from rhizomes in triploid cooking bananas Cardaba (ABB genome), SH-3362 (AA genome) and Bocadillo (AA 123 134 genome) (Novak et al. 1989). Young flowers isolated from male buds have been used as explants to induce embryogenic suspension cultures of East African highland bananas (Escalant et al. 1994). Somatic embryogenesis has been also induced from young male flowers of French Sombre (AAB genome) and female flowers of false horn plantain (AAB genome) (Grapin et al. 1996, 2000). There is only a single report on establishing of ECS cultures from shoot-tip meristems of proliferating shoot cultures, which has been reported in AAB group plantains Agbagba, ObinoI’Ewai and Orishele (Strosse et al. 2006). Somatic embryogenesis from shoot-tips is highly desirable for large scale plantain micropropagation as these tissues are not subjected to seasonal variation and irrespective of presence of female or male flowers (Strosse et al. 2004). In this study an efficient system of regeneration from cell suspension cultures from shoot-tips of proliferating shoots of plantain cv. Spambia (AAB) was established, and the influence of abscisic acid ABA on maturation of somatic embryos was investigated. Plant Cell Tiss Organ Cult (2009) 99:133–140 (EC). Scalps of varying sizes (2–8 mm) were transferred to embryogenesis induction medium, ZZss, consisting of modified MS medium (half-strength MS macroelements and iron, 0.4 mg l-1 thiamine HCl, 10 mg l-1 ascorbic acid, 4.52 lM 2,4-D, 1 lM zeatin and without myoinositol). The effect of different concentrations of 2,4-D (1.13, 2.26, 3.39, 4.52 and 9.05 lM) combined with zeatin (1 or 2 lM) on formation of EC, was investigated. For every treatment, 80 explants were used and the experiments were repeated three times. Calli were subcultured on the same medium for 3–4 cycles of 15 days each under dark conditions. Data on number of scalps developing EC were recorded. Initiation and maintenance of embryogenic cell suspension cultures In vitro-grown shoot cultures of plantain (Musa sp.) cv. Spambia (genomic group AAB) (EC 431405) were obtained from the In Vitro Genebank at the National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India. Cultures were maintained on Murashige and Skoog (MS) (1962) medium supplemented with 10 lM benzyladenine (BA), 1 lM indole-3-acetic acid (IAA), 3% sucrose, and solidified with 2.0 g l-1 gelrite. Cultures were incubated at 28°C under 16-h photoperiod provided by fluorescent white light 50 lmol m-2 s-1. Embryogenic calli were transferred to 20 ml ZZl medium (same as ZZss but without gelrite) in 150 ml Erlenmeyer flasks and kept on a rotary shaker at 90 rpm. A small aliquot of suspension culture was removed, placed on a glass slide and observed for form and quality of cell aggregates, using a light transmission microscope (Nikon microscope Model no. C-FID, Japan). After one week, suspension cultures were filtered through a sterilized metallic sieve (Sethi, India) of 500 lm pore size. The filtrate was transferred to a sterile graduated tube (50 ml) and allowed to settle down for 10 min. About 10 ml of the supernatant was pipetted, discarded and fresh medium added to maintain 2.5–3% of the settled cell volume (SCV). This was then transferred to a fresh 100 ml sterilized flask, and cultures were kept on a shaker at 90 rpm in darkness. This protocol was carried out once weekly for a period of one month, and then biweekly until embryogenic clusters were obtained. Multiple meristem cultures Induction of somatic embryos from ECS These were obtained when apical meristems were cultured on 25 ml of MS basal medium in 150 ml culture tubes (one explant/tube) supplemented with BA (22.2, 44.4 or 90 lM), IAA (1 lM) and 56.8 mM ascorbic acid, and solidified with 2.0 g l-1 gelrite and maintained in darkness. Cultures were routinely subcultured onto fresh medium of the same composition every 4 weeks for 8–12 months, until shoots converted into compact clumps of meristematic buds known as scalps (Dhed’a et al. 1991). These scalps were used as explants for inducing somatic embryogenesis. An aliquot of 200 ll of settled cells was placed on a double layer of Whatman filter paper no. 1, and kept in a 90 mm petri dish containing 25 ml ZZss medium. Filter papers were allowed to soak excess liquid medium from the aliquot of settled cells for 10 min, after which the lower filter paper was removed allowing cells settling on upper filter paper to be in direct contact with the medium. Cultures were maintained for 4 weeks, after which they were transferred to fresh ZZss medium for 2–3 months. Materials and methods Plant materials Somatic embryo maturation Induction of embryogenic calli Scalps were excised from the uppermost part of meristematic clumps and used for initiation of embryogenic calli 123 Torpedo shaped somatic embryos observed under a dissection microscope were individually picked up and transferred onto RD1 medium (semi-solid MS medium Plant Cell Tiss Organ Cult (2009) 99:133–140 containing half-strength major salts and iron, full-strength minor salts and vitamins and 100 mg/l myoinositol) supplemented with different concentrations of ABA (0, 1, 1.25, 5 or 10 lM) for 3 weeks for maturation. About 100 somatic embryos were cultured per petri dish. Germination of somatic embryos and hardening of plantlets in medium containing different cytokinins The effects of BA, zeatin and TDZ at concentrations ranging between 1 and 10 lM in semi-solid MS medium on somatic embryo germination was evaluated. Germinated somatic embryos were transferred to semi-solid MS medium supplemented with 8.89 lM BA. After 4–6 weeks, regenerated shoots were transferred to MS basal medium with 1% sucrose for rooting. Plantlets with well developed roots and shoots were hardened in pots containing agropeat (Prakruti Agrotech, Bangalore, India) and irrigated with Hoagland’s solution (Hoagland and Arnon 1950). Pots were transferred to the green house and grown at 25 ± 2°C with 16 h photoperiod and 70% relative humidity for 2 months. Plantlets were then transferred to soil and grown in the nursery. Results and discussion Scalp formation BA at 44.4 lM was optimal for meristem proliferation (Table 1). More than 69% of the shoot-tip explants developed scalps (cauliflower like-structures). Groups of closely packed meristems were observed at the base of explants within 2 months following culture of explants on MS medium supplemented with 44.4 lM BA and 1 lM IAA (Fig. 1a, b). Upon subculture, scalps were obtained within 8–12 months (Fig. 1c, d). Previously, Dhed’a et al. (1991) reported that 10 lM BA was necessary to induce scalp formation in plantain cv. Bluggoe (ABB). While, Strosse et al. (2003) reported that scalp formation requires about 7– 9 monthly subcultures of explants of Musa spp. (AAA) on a medium supplemented with high BA concentration (100 lM). These findings suggested that different Musa genotypes would require different levels of plant growth regulators to induce scalp formation in vitro. Induction of embryogenic calli Among scalps cultured on various combinations of 2,4-D and zeatin, those of 3–6 mm in diameter swelled within 2–3 days, and gave rise to cream coloured EC after 5–6 weeks (Fig. 2a). Highly EC were obtained after 3–4 passages, subcultured once a month onto fresh ZZss 135 Table 1 Effect of different concentrations of BA in the presence of 1 lM IAA on proliferation of meristem explants of plantain cv. Spambia BA (lM) Number of explants Mean number ± SD of scalps formed* 22.2 35 3 ± 1.52 b 44.4 36 25 ± 3.00 a 90.0 40 5 ± 2.00 b * Values followed by the same letter within columns are not statistically different by Duncan Multiple Range Test at P = 0.05 medium (Fig. 2b); whereas, non embryogenic nodular calli were yellowish in colour and detected within 6 weeks following culture (Fig. 2c, d). The frequency of embryogenic callus formation varied from 0 to 98%, depending on the medium (Table 2). Explants less than 0.3 mm in size turned necrotic; while, those C0.7 mm did not yield EC. Excluding IAA from the scalp formation medium resulted in lower frequency of scalp formation, 10%, and accompanied by formation of yellow and nodular structures. None of the scalp explants incubated on medium containing low concentration of 2,4-D (1.130 lM) formed embryogenic callus. Amongst all combinations of 2,4-D and zeatin tested, highest embryogenic callus response (98%) was obtained on medium supplemented with 4.52 lM 2,4-D and 1 lM zeatin. These results are in agreement with those of both Dhed’a et al. (1991) and Strosse et al. (2006). These frequencies are higher than those previously reported for some plantains and cooking bananas, 3%, (Schoofs et al. 1998; Cote et al. 1996) and for other Musa genotypes, 3–22%, (Strosse et al. 2004). Escalant et al. (1994) also reported a frequency of 0–7% embryogenic callus formation from responding male flowers of three genotypes of Musa AAA, AAB and ABB. Effect of medium composition on establishment of ECS culture Embryogenic cell suspension cultures were successfully established, *42%, when embryogenic callus was incubated in ZZl medium supplemented with 4.52 lM 2,4-D and 1 lM zeatin (Fig. 3a). Out of inoculated 55 flasks, efficient ECS formation was observed in 23 flasks; while, cultures in remaining turned slimy no ECS was observed. Following 1 week of initiation of ECS, embryogenic cells with prominent nuclei were observed, while number of vacuolated cells decreased. The embryogenic cells and embryogenic cell aggregate formation in ECS could be observed within 6 weeks following ECS initiation. The proportion of embryogenic cell aggregates increased while the number of single cells decreased in ECS in the first 2 months. These embryogenic masses varied in size from 123 136 Plant Cell Tiss Organ Cult (2009) 99:133–140 Fig. 1 Scalps of plantain cv. Spambia at different stages of development. These were grown in MS medium supplemented with 44.4 lM BA and 1 lM IAA. The bar scale represents 1 mm. a, b Groups of closely packed meristems after 1 month and 2 months of culture respectively. c Increased number of meristematic clumps (designated with an arrow) formed after 8 months. d Ideal scalp with large number of highly meristematic clumps (shown with an arrow) after 11 months of culture 80 to 300 lm, and finally resulted in cell suspensions of thick consistency after 2 months (Fig. 3b, c). ECS cultures were maintained up to 18 months and retained their embryogenic and regeneration competence. For those ECS cultures established in medium supplemented with either 3.39 lM 2,4-D and 1 lM zeatin or 9.05 lM 2,4-D and 2 lM zeatin, cells formed larger clumps composed of vacuolated cells with and without prominent nuclei after which they lost their regeneration capacity. This might be attributed to either low concentration of 2,4-D, in the first medium, or high concentration of zeatin in the second medium respectively. transfer of 200 ll of settled cell volume of ECS to ZZss medium, translucent somatic embryos were visible after 45 days (Fig 3e). Induction of somatic embryos Somatic embryo formation was observed after two months of initiation of ECS cultures (Fig. 3d). After 5 months of initiation, cell suspensions were mainly composed of embryogenic cell aggregates of 250 microns in size. Upon 123 Somatic embryo maturation Torpedo shaped embryos were observed after 3 weeks on RD1 medium supplemented with ABA. Maturing embryos grown on ABA supplemented media were creamy in color and slightly larger (1.5 mm) than embryos (1 mm) grown on control medium (without ABA) (Fig. 3f). It was also observed that presence of 2.5 lM ABA in RD1 medium subsequently enhanced somatic embryo regeneration frequency (Table 3). Effect of cytokinins on somatic embryo germination It was observed that MS medium supplemented with either TDZ, BA or zeatin enhanced somatic embryo germination Plant Cell Tiss Organ Cult (2009) 99:133–140 137 Fig. 2 Callus formation induced from scalps of plantain cv. Spambia. The bar scale represents 2 mm. a Embryogenic callus after 5 weeks of subculture on ZZss medium. b Highly embryogenic callus after monthly subculture of callus obtained from stage (a) on ZZss medium for 3–4 cycles at intervals of 1 month each. c, d Nodular callus formed on top of the scalp after 5–6 weeks of subculture on ZZss medium Table 2 Influence of plant growth regulator combinations on induction of EC of plantain cv. Spambia Media code Plant growth regulators No. of explantsa % Explants developing EC (mean ± SD)* 2,4-D (lM) Zeatin (lM) Z1 1.130 1 249 0.0 ± 0.0 f Z2 2.262 1 243 50.68 ± 8.03 c Z3 3.393 1 246 74.46 ± 8.23 b Z4 4.524 1 255 98.06 ± 0.6 a Z5 9.048 1 249 24.09 ± 2.1 e Z6 1.130 2 240 0.0 ± 0.0 f Z7 2.262 2 240 43.64 ± 10.92 cd Z8 3.393 2 240 36.22 ± 2.44 d Z9 4.524 2 240 77.70 ± 12.2 b Z10 9.048 2 245 18.32 ± 5.88 e Data collected after 6 weeks of culture in dark F = 61.486, at P \ 0.01, Mean square = 47.463 A total of three replicates of *80 explants each were used for each treatment combination a * Values followed by the same letter within columns are not statistically different by Duncan Multiple Range Test at P = 0.05 in comparison to the control MS basal medium (Table 3). Somatic embryos turned green in colour within 2 days, and shoots emerged after 1 week. For most germinating somatic embryos, shoot formation preceded root formation (Fig. 3g). The presence of 2.5 lM ABA in combination with 1.25 lM BA enhanced germination of somatic 123 138 Plant Cell Tiss Organ Cult (2009) 99:133–140 Fig. 3 Plant regeneration from somatic embryos of plantain cv. Spambia. The bar scale represents 1 cm in a, g, h and i; 100 lm in b, c and d and 1 mm in e and f. a ECS in ZZl medium. b, c Cell aggregates after 2 months. d Emerging SE after 2 months in ZZl medium. e Emerging SE after 45 days on ZZss medium. f Mature somatic embryos on RD1 media. g Germinating somatic embryos on MS medium supplemented with 1.25 lM BA after 3 weeks. h Rooted shoots grown on MS medium with 1% sucrose. i Potted plantlets. j Mature plants growing in the nursery embryos up to 85% compared to that of control (32%) (Table 3). Frequency of plantlet regeneration frequency in this study (80%) was higher than that of control treatment (15%) and for those reported previously by Dhed’a et al. (1991), 10–23% for cv. Bluggoe (ABB), and Novak et al. (1989), 10–40% for cv. French Somber, 5% for cv. Grand Naine and 1.5–12% for the diploid clone, Bocadillo. This frequency of regeneration in this study was also higher than those recently reported by Ganapathi et al. (1999) for cv. Rasthali (AAB), 5–9%, Marroquin et al. (1993), 20–36% for Musa acuminata and Strosse et al. 2006 75% for various Musa spp. Moreover, shoots obtained in this study continued to grow in MS medium supplemented with 8.89 lM BA and formed roots within 2 weeks when transferred to basal MS medium with 1% sucrose (Fig. 3h). These plantlets were acclimatized (Fig. 3i) with a survival frequency of 90%, when transplanted to the nursery (Fig. 3j). 123 Role of ABA in somatic embryo development Although ABA has been reported to induce development of somatic embryos in a various species including Brassica, Datura, Nicotiana (Sethi et al. 1990), Hordeum (Rengel and Jelaska 1986), Picea (Dunstan et al. 1988; Roberts et al. 1990; Attree et al. 1995), Pinus (Sen et al. 1989), Medicago (Fujii et al. 1990), Triticum (Brown et al. 1989; Qureshi et al. 1989), Apium (Nadel et al. 1990), Prunus (Lydia et al. 1999), Allium (Van-der-valk et al. 1992), and Asparagus (Li and Wolyn 1995), this is the first report on somatic embryo developed in Musa spp. (AAB). The role Plant Cell Tiss Organ Cult (2009) 99:133–140 139 Table 3 Effects of ABA in combination with either BA, zeatin, and thidiazuron on somatic embryo germination and conversion of plantain cv. Spambia Frequency of somatic embryo germination (%) Frequency of plantlet recovery (%) ABA concentrations (lM) used for maturation 0 0.1 1 2.5 5 0 0.1 1 2.5 5 BA conc. (lM) 1 10 bcd 16 bcdef 28 bcde 30 ab 28 abc 10 abcde 16 bcd 27 bcde 30 ab 24 abcd 1.25 32 a 40 a 49 ab 85 a 46 a 15 abc 15 bcde 30 bcde 80 a 33 a 2.5 20 ab 35 ab 64 a 60 ab 40 ab 15 abc 35 a 60 a 40 ab 27 ab 5 18 abc 33 abc 42 abc 55 ab 28 abc 18 ab 18 bc 40 ab 35 ab 27 ab 10 9 bcd 14 cdef 16 cde 17 b 12 bc 4 bd 12 cde 10 cde 10 b 9 bcd 1 10 bcd 22 abcde 23 bcde 32 ab 27 abc 8 bcde 15 bcde 22 bcde 28 ab 25 abcd 1.25 22 ab 30 abcd 39 abcd 55 ab 40 ab 14 abcd 16 bcd 30 bcde 45 ab 30 ab 2.5 23 ab 31 abcd 43 abc 57 ab 38 ab 20 a 29 ab 38 abc 52 ab 25 abcd 5 17 abc 23 abcde 35 abcd 53 ab 30 abc 15 abc 16 bcd 34 abcd 31 ab 26 abc 10 10 bcd 12 def 15 cde 27 ab 20 abc 7 cde 10 cde 10 cde 9b 9 abc 9 bcd 8 bcd 10 ef 8 ef 10 de 12 de 46 ab 63 ab 5c 16 abc 5 cde 1e 4 cde 2d e 6 de 7de 30 ab 45 ab 3 cd 14 abcd Zeatin (lM) TDZ (lM) 1 1.25 2.5 4 cd 4 ef 10 de 45 ab 33 abc 5 cde 2 de 8 de 28 ab 24 abcd 5 3 cd 2f 5e 36 ab 17 abc 2e 1e 3e 21 b 14 abcd 10 0d 1f 3e 5b 4c 0e 1e 2e 2b 2d About 100 embryos were subcultured on ‘ MS medium supplemented with different concentrations of ABA for 3 weeks for maturation after which they were transferred to MS medium with different concentrations of cytokinins for germination and plantlet recovery. Values followed by the same letter within columns are not statistically different by Duncan Multiple Range Test at P = 0.05 of ABA on somatic embryo maturation and subsequent regeneration still remains unclear, although ABA has been reported to induce expression of genes responsible for maturation as well as synchronization of maturation of wheat embryos (Morris et al. 1990). The role of ABA in suppressing abnormal embryo development, inhibiting precocious germination, conferring desiccation tolerance and promoting accumulation of storage lipids and proteins has been well documented in (Bornman 1993). Etienne et al. (1993) showed that slow desiccation or maturation of somatic embryos on medium containing sucrose (120 g l-1) and ABA (1 lM) strongly improved germination and conversion of somatic embryos of Hevea brasiliensis. Attree et al. (1995) demonstrated that in presence of ABA was important in maintaining somatic embryos of white spruce (Picea glauca) in a developing state. Sharma et al. (2004) reported that upon treating somatic embryos of Camellia sinensis (L) with 18.9 lM ABA for 14 days, starch, protein, and soluble sugar contents increased by several folds and resulting in improved germination of somatic embryos. Whether similar physiological and metabolic changes were instrumental in enhancing maturation and germination of somatic embryos of Musa sp. Spambia remains to be determined. Acknowledgements Sincere thanks to ICCR and Ministry of Palestinian Authority of Higher Education for providing scholarship and financial assistance to Nasser Sholi, to UGC, India for J. R. F to Anjana Chaurasia, to National Bureau of Plant Genetic Resources, New Delhi for providing plant materials, to Professor S. C. Maheshwari and Dr. Nirmala Maheshwari of ICGEB, Prof. P. S. Ganapathi from Delhi University, Dr. V. M. Kulkarni from BARC, and Dr. Mohan Jain from Helsinki University, Finland for their critical comments and suggestions. 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