Desiccation and ABA treatment improves conversion of somatic embryos to plantlets in banana (Musa spp.) cv. Rasthali (AAB)

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1 Indian Journal of Biotechnology Vol 5, October 2006, pp Desiccation and ABA treatment improves conversion of somatic embryos to plantlets in banana (Musa spp.) cv. Rasthali (AAB) L Srinivas, T R Ganapathi P Suprasanna and V A Bapat* Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay Mumbai , India Received 18 March 2005; revised 16 August 2005; accepted 25 October 2005 Somatic embryos of banana cv. Rasthali were subjected to 0-8 h desiccation and abscisic acid (ABA) treatment for improving plant conversion. Somatic embryos exposed to 10 μm ABA followed by 2 h desiccation exhibited an increase in plant conversion (66%) compared to control (56%). Embryos treated with ABA followed by 2 h desiccation survived up to 5 weeks with 56% plant conversion upon storage at 10 C. This study suggests that combined treatment of banana somatic embryos with ABA and desiccation can improve conversion frequency. Keywords: banana, somatic embryos, abscisic acid, desiccation IPC Code: Int. Cl. 8 A01H4/00 Introduction The regeneration of plants through somatic embryogenesis represents a remarkable feature of plant cells and this has led to the development of novel approaches for plant propagation and genetic manipulation. Such application often necessitates the production of somatic embryos in large numbers and high conversion of somatic embryos into plants. In many plant species, low conversion of embryos is related to poor quality of embryos and lack of maturation and desiccation tolerance 1,2. Low vigour and poor quality of somatic embryos results in poor development into plants, which is also attributed to reduced levels of storage reserves 3. Provision of an artificial endosperm to the encapsulated somatic embryo has been shown to increase vigour 4 but not with much success. Alternatively, enhancing the storage reserves in somatic embryos through the acquisition of desiccation tolerance during maturation process as a means of stabilization and hardening of somatic embryos offers a potential choice 5. Desiccation tolerance in developing somatic embryos may be induced by exogenous signals in the tissue culture medium such as addition of osmotically active substances like abscisic acid (ABA), poly ethylene glycol (PEG), sucrose and chilling stress *Author for correspondence: Tel: ; Fax: vabapat@magnum.barc.ernet.in These have been found to be effective for the maturation and stimulation of embryo-plant conversion in wheat 12, Hevea 13, sugarcane 14, white spruce 15 and wild cherry 16. Often embryos display signs of precocious germination, which is due to incomplete maturation phase between the stages of embryo development and or embryo germination 17. Treatment of somatic embryos with ABA to reduce water content and to allow preservation of synthetic seeds was demonstrated by Senaratna et al 18. Shiota et al 19 used a combined treatment of desiccation and ABA to examine their effect on somatic embryo viability during preservation of carrot somatic embryos at low or sub-zero temperatures. In banana, somatic embryogenesis leading to plant regeneration has been demonstrated from bases of leaf sheaths and corm tissue, thin sections from in vitro proliferating meristems, immature zygotic embryos, male and female flowers Although somatic embryos can be produced with higher frequency, maturation and conversion into plants are still critical, requiring optimization of nutritive and environmental conditions. Considering the importance of somatic embryogenic systems in banana for large-scale propagation, genetic manipulation and conservation, experiments on the improvement of conversion of somatic embryos into plants are underway in our laboratory. In an earlier paper, we reported stimulatory effects of cyanobacterial extracts on the conversion of somatic embryos into plants 23. In this report, our

2 522 INDIAN J BIOTECHNOL, OCTOBER 2006 results on the effects of ABA and desiccation on the conversion and storage of somatic embryos of banana cv. Rasthali (AAB) are described. Materials and Methods Establishment of Cultures Shoot tip cultures were established in vitro from field grown plants of cv. Rasthali (AAB genomic group) as per the method described earlier 24. The in vitro multiple shoot cultures were grown and maintained on medium containing MS 25 salts supplemented with benzylaminopurine (BAP, 4.5 μm), indole 3-acetic acid (IAA, 1.14 μm) and sucrose (87.7 mm). Thin ( μm) longitudinal sections were cut from 1 cm long shoot tips (4-6 whorls of leaves) under aseptic conditions. These were then transferred to MS medium supplemented with 9.05 μm 2,4- dichlorophenoxyacetic acid (2,4-D), 1 μm zeatin and 1 mg L -1 d-biotin for embryogenic callus development. Suspension Cultures and Somatic Embryo Development Suspension cultures were established from 6- month-old, whitish, friable embryogenic callus tissue as described by Ganapathi et al 26 in liquid M2 medium [(MS supplemented with 2,4-D (4.5 μm), d- biotin (1 mg L -1 ), L-glutamine (100 mg L -1 ), malt extract (100 mg L -1 ), sucrose (87.7 mm)]. These cell suspensions were plated onto half strength MS medium (RR) supplemented with zeatin (9.12 μm) and sucrose (87.7 mm) for embryo development. Globular embryos were formed in 2-3 weeks after plating the cells and fully developed embryos in 4-6 weeks. These were used for desiccation and/or ABA experiments. Treatment with ABA ABA (10 μm) was added aseptically to MS basal medium containing 87.7 mm sucrose. About 5 g somatic embryos were transferred to 200 ml MS medium with ABA in 500 ml Erlenmeyer flask and cultured on gyratory shaker (100 rpm) at 25 C in dark for 48 h. Following incubation, medium was decanted and embryos were washed with hormone-free MS medium and used for desiccation and calculation of water content. Determination of Water Content (WC) About 5 g of ABA treated and non-treated embryos were tested for measuring the WC. The embryos were kept on dried autoclaved filter paper and laid on 20 g silica gel in a desiccator. Initial weight of embryos before desiccation was noted and after every hour weights were taken and relative water content (RWC) was calculated using the following formula: Initial weight Final weight RWC= 100 Initial weight Desiccation of Embryos 5 g of ABA treated and non-treated embryos were used for desiccation using 20 g autoclaved silica gel in a sterile desiccator. Embryos were desiccated for 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 9 h and after each desiccation period, 500 embryos were taken from the desiccator and placed on dried filter paper in Petriplates and sealed with parafilm. Storage of Somatic Embryos ABA treated and non-treated, desiccated and nondesiccated embryos were subjected to storage at three different temperatures, 25, 10 and 20 C. Conversion of Somatic Embryos After incubation at a given temperature, desiccated and non-desiccated, with or without ABA treatment, embryos were placed every week on conversion medium containing MS salts supplemented with 1.62 μm BAP, 87.7 mm sucrose and gelled with 0.2% phytagel (Sigma, USA). In each experiment, about 50 embryos were checked for conversion. Conversion frequency was calculated as the somatic embryos that developed with good shoot and root system, and for each embryo, shoot length and root lengths were recorded. All the experiments were repeated twice with three replications and standard error was calculated for the results. Results Embryogenic cell suspension (ECS) cultures consisted of embryogenic cells and clumps (Fig. 1A). Translucent spheres characterized the highly embryogenic cultures and torpedo shaped embryos were observed after 4-6 weeks of transfer to RR medium (Fig. 1B). The embryos converted into tiny plantlets with the emergence of coleoptiles at a frequency of 56% (Fig. 1C). Well-developed embryos derived from these ECS were employed for ABA and/or desiccation treatments. Somatic embryos treated with or without ABA exhibited relative water contents in the range of % in the control (Fig. 2). The pattern of decrease

3 SRINIVAS et al: CONVERSION OF SOMATIC EMBRYOS TO PLANTLETS IN BANANA BY DESICCATION W*rm(%l Fig. 2-Changes in relative water content of ABA treated and non-treated somatic embryos of banana during desiccation for 0-8 h. Fig, 1--Conversion of somatic embryos of banana cv. Rasthali following ABA and desiccation treatment: A. Embryogenic cells as visible in the cell suspension culture (note the cells with dense cytoplasm studded with starch grains); B. 6 week-old somatic embryos developed on MS medium supplemented with zeatin; C. Conversion of somatic embryos (Control) on MS medium supplemented with BAP; D. Conversion of ABA treated somatic embryos on MS medium supplemented with BAP; E. Conversion of ABA treated and 2 h desiccated somatic embryos on MS medium supplemented with BAP. was similar for ABA treated and non-treated embryos during desiccation. The relative \~VC was 44.6% (without ABA) and 42.5% (with ABA) during desiccation for 2 h. The WC dropped slowly to % during desiccation for 7 h (Fig. 2). Compared to 56% conversion in the control, initial desiccation for 1-2 h showed 44 and 48% conversion and subsequent desiccation regimes showed a decreasing trend (Fig. 3). The conversion response of somatic embryos treated with ABA andlor desiccation, during storage at 25 C is presented in Fig. 4. Somatic embryos that were neither ABA treated nor desiccated survived up to 6 weeks at 25OC with a gradual decrease in the conversion response. Compared to 56% conversion in the initial period with maximum shoot length (2.03 cm), there was 46% in the first week followed by a drop up to 6 weeks (4%) with the gradual decrease in shoot length (Table 1) and thereafter the embryos did not survive. On the contrary, 20% of the ABA-treated embryos converted upon transfer to the conversion medium. The conversion frequency (58%) and shoot lengths increased in 2nd and 3rd weeks during storage and maximum shoot length (3.06 cm) was observed (Fig. ID, Table 1-1. Embryos that were desiccated for 2 h showed a reduction in conversion from 48 to 16% when stored for 5 weeks. An increase in shoot length was observed only up to 2 weeks (Table 1). Immediately after desiccation, conversion was 48% while up to 3 weeks the decrease was in the range of 36-44%. However, embryo conversion fell rapidly thereafter. In the case of treatment with ABA followed by desiccation for 2 h, somatic embryo conversion was observed till 6 weeks of storage; 66% somatic embryos converted in the l" week of storage (Fig. 1E) followed by 60% in the 2"d week, 42% in the 3rd week and 30% in the 4" week. Maximum shoot length of 2.2 cm was observed in embryos

4 524 INDIAN J BIOTECHNOL, OCTOBER 2006 Fig. 3 Effect of desiccation on conversion of control somatic embryos (without ABA treatment and storage). Conversion was noted after 4 weeks of culture. Fig. 5 Comparison of conversion of somatic embryos (2 h desiccated and ABA treated + 2 h desiccated) upon storage at 10 C. Table 1 Shoot length of somatic embryos following ABA, 2 h desiccation and ABA +2 h desiccation treatment. Data was taken after 4 weeks upon transfer to conversion medium Period of storage (week) Control ABA Desiccation (2 h) ABA+ desiccation (2 h) Fig. 4 Conversion of somatic embryos treated with ABA and/or 2 h desiccation during storage at 25 C. transferred to conversion medium after one week of storage. In contrast, none of the somatic embryos that had neither been treated with ABA nor desiccated survived when stored at low temperatures. Embryos treated with ABA and desiccated for 2 h exhibited better conversion during storage at 10 C after 5 th and 6 th weeks as compared to those desiccated but without ABA treatment (Fig. 5). Compared to low conversion rate (14%) during the first week, ABA treated and desiccated embryos showed conversion of 56% after storage for 5 weeks. It gradually dropped to 24% in the 6 th week, 12% in the 7 th week, and beyond this, all the embryos died with no conversion. Treatment with ABA followed by desiccation up to 4 h did not show any conversion while 6 h desiccation resulted in 2-4% conversion during storage at 20 C up to 5 weeks (data not shown) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Discussion In this study, an attempt was made to examine the role of ABA and desiccation on the conversion and storage of somatic embryos of banana. Results showed the significance of ABA treatment in conferring desiccation tolerance to somatic embryos. Without prior ABA treatment and desiccation, embryo conversion dropped from 56% in the first week to 4% in 6 th week when stored at 25 C. Somatic embryos either with ABA exposure or desiccation showed increased conversion during storage at 25 C in the initial 1-3 weeks and thereafter there was a gradual decline, however, the combined treatment (ABA+desiccation) resulted in higher conversion (66%) only during initial 1-2 weeks. Such a decrease in conversion might be due to the high metabolic activity of cells and lack of nutrition. Embryos that were stored at 10 C showed comparatively longer storability (up to 6 weeks) with 56% conversion (Fig. 5),

5 SRINIVAS et al: CONVERSION OF SOMATIC EMBRYOS TO PLANTLETS IN BANANA BY DESICCATION 525 suggesting that in such somatic embryos, metabolic activity might be very low and hence high germinability was noticed. Somatic embryos that were desiccated showed decline in conversion (< 10%) upon storage at 20 C. This could be due to freezing injury caused by the formation of ice crystals between intracellular spaces 27. The positive effects of desiccation on the conversion of somatic embryos to plantlets have been demonstrated in many plants. In horse chestnut, somatic embryos were subjected to different desiccation periods after a pretreatment with 80 μm ABA and/or 50 g L -1 PEG. Although heat and chilling stress can induce desiccation tolerance in somatic embryos, ABA plays a major role in conferring tolerance as reported for various species including Apium graveolens 28, Brassica oleracea 29 and Picea spp. 30. Although unclear, ABA by suppressing abnormal development, inhibiting precocious germination, conferring desiccation tolerance and promoting accumulation of storage lipids and proteins, induces a synchronized maturation that results, upon its withdrawal, in uniform high frequency germination of somatic embryos 31. ABA has been known to induce the expression of maturation genes in wheat embryos 32 and to inhibit precocious germination resulting in accumulation of storage proteins in interior spruce somatic embryos 33. ABA seems to play, at the structural level, a role in cell expansion; ABA treated embryos have small and densely cytoplasmic cells, whereas ABA untreated embryos have vacuolated and expanded cells 34. In addition to the known effects on embryo conversion, studies have also indicated that ABA treated and/or desiccated somatic embryos can avoid damage caused by the formation of ice crystals in intracellular spaces, when stored at sub-zero temperatures 19. The damage in the frozen state is generally due to the formation of ice crystals in the intracellular spaces in plant cells 35. Carrot callus grown on medium containing high sucrose or ABA reduced water content and could be preserved at 80 C for a year 36. The results in this study also indicate that desiccation preceded by exposure to ABA can avoid such freezing due to low water content in the somatic embryos. The 6-8 h desiccated embryos had less than 10% conversion and could be stored at 20 C. Shiota et al 19, demonstrated long-term preservation of ABA treated and desiccated embryos with viability for more than three years at 25 C. Our results showed that banana somatic embryos exposed to a combined treatment with ABA and desiccation could be preserved at low temperatures for 7-8 weeks. Preservation of plant tissue through cryopreservation is done by the addition of cryoprotectants and gradual freezing to ensure the growth of ice crystals in the extracellular spaces 37. Additionally, vitrification has also been used successfully for the preservation of plant cells. In this method, cells are exposed to a vitrification solution, cooled rapidly in liquid nitrogen and rewarmed rapidly in order to avoid the formation of ice crystals 38,39. Although these methods have successfully been employed for the preservation of plant cells and tissues, rapid freezing conditions or ultra temperatures or thawing steps generally used restrict their large-scale and routine application 19. In this context, a simple method of pretreatment with ABA followed by desiccation appears to be interesting in view of the storage potential at low temperatures. Extensive studies are required for demonstrating such storage potential in a wide variety of plant species. In addition to the well-advocated cryopreservation techniques, the simple method as presented in this study, may offer a new and alternative choice for the low temperature preservation of embryogenic cultures. References 1 Bapat V A & Rao P S, Maturation and desiccation of somatic embryos, in Trends in Plant Tissue Culture & Biotechnology, edited by L K Pareek and P L Swarankar (Agro Botanical Publishers, Bikaner, India) 1996, Redenbaugh K, Fuji J, Slade D, Viss P & Kossler M, Artificial seeds Encapsulation of somatic embryos, in Biotechnology in agriculture & forestry, vol 17, High tech micropropagation 1, edited by Y P S Bajaj (Springer-Verlag, Belrin) 1991, Tetteroo F A A, Hoekstra F A & Karssen C M, Induction of complete desiccation tolerance in carrot (Daucus carota L.) embryoids, J Plant Physiol, 145 (1995) Anandarajah K & McKersie B D, Manipulating the desiccation tolerance and vigour of dry somatic embryos of Medicago sativa L. with sucrose, heat shock and abscisic acid, Plant Cell Rep, 9 (1990) Bapat V A & Rao P S, Plantlet regeneration from encapsulated and non-encapsulated desiccated somatic embryos of a forest tree: Sandalwood (Santalum album L.), J Plant Biochem Biotechnol, 1 (1992) Lecouteux C, Lai F M & McKersie B D, Maturation of alfalfa (Medicago sativa L.) somatic embryos by abscisic acid, sucrose and chilling stress, Plant Sci, 94 (1993) Senaratna T, McKersie B D & Bewley S R, Desiccation tolerance of alfalfa (Medicago sativa L.) somatic embryos: Influence of abscisic acid, stress pretreatments and drying rates, Plant Sci, 65 (1989) Capuana M & Debergh P C, Improvement of the maturation and germination of horse chestnut somatic embryos, Plant Cell Tissue Organ Cult, 48 (1997)

6 526 INDIAN J BIOTECHNOL, OCTOBER Neives N, Martinez M E, Castillo R, Blanco M A & Gonzalez-Olmedo J L, Effect of abscisic acid and jasmonic acid on partial desiccation of encapsulated somatic embryos of sugarcane, Plant Cell Tissue Organ Cult, 65 (2001) Fujii J A, Slade D, Olsen R, Ruzin S E & Redenbaugh K, Alfalfa somatic embryo maturation and conversion to plants, Plant Sci, 72 (1990) Attree S M & Fowke L C, Embryology of gymnosperms: Advances in synthetic seed technology of conifers, Plant Cell Tissue Organ Cult, 3 (1993) Carman J G, Improved somatic embryogenesis in wheat by partial simulation of the in ovulo oxygen, growth regulator and desiccation environments, Planta, 175 (1988) Etienne H, Montoro P, Michaux-Ferriere N & Carron M P, Effects of desiccation, medium osmolarity and abscisic acid on the maturation of Hevea brasiliensis somatic embryos, J Exp Bot, 44 (1993) Desai N S, Suprasanna P & Bapat V A, Partial desiccation of embryogenic calli improves plant regeneration in sugarcane (Saccharum spp.), J Plant Biotechnol, 5 (2004) Attree S M, Pomeroy M K & Fowke L C, Development of white spruce [Picea glauca (Moench.) Voss] somatic embryos during culture with abscisic acid and osmoticum, and their tolerance to drying and frozen storage, J Exp Bot, 46 (1995) Lydia R T, Diemer F, Sourdiox M, Chapelain K & Grenier- De March G, Improvement of somatic embryogenesis in wild cherry (Prunus avium): Effect of maltose and ABA supplements, Plant Cell Tissue Organ Cult, 55 (1999) Quatrano R S, The role of hormones during seed development, in Plant hormones and their role in plant growth and development, edited by P J Davis (Martinus Nijhoff, Dordrecht.) 1987, Senaratna T, MacKersie R D & Bowley S R, Artificial seeds of alfalfa (Medicago sativa L.): Induction of desiccation tolerance in somatic embryos, In Vitro Cell Develop Biol, 26 (1990) Shiota H, Tachibana K, Watabe K & Kamada H, Successful long-term preservation of abscisic acid-treated and desiccated carrot somatic embryos, Plant Cell Rep, 18 (1999) Sagi L, May G D, Remy S & Swennen R, Recent developments in biotechnological research on bananas (Musa spp.), Biotechnol Genet Eng Rev, 15 (1998) Rout G R, Samantaray S & Das P, Biotechnology of the banana: A review of recent progress, Plant Soil, 2 (2000) Ganapathi T R, Suprasanna P & Bapat V A, Biotechnological approaches for banana propagation and improvement, in Proc Natl Sem Biotechnol - Challenges and prospects, edited by L D Souza, M Anuradha, S Hegde, S Nivas & K Rajendra (St. Aloysius College, Mangalore) 2003, Srinivas L, Ganapathi T R, Suprasanna P, Iyer R K & Bapat V A, Ameliorative effect of cyanobacterial extract on the conversion of somatic embryos to plantlets in banana, J New Seeds, 4 (2002) Ganapathi T R, Suprasanna P, Bapat V A & Rao P S, Propagation of banana through encapsulated shoot tips, Plant Cell Rep, 11 (1992) Murashige T & Skoog F, A revised medium for rapid growth and bioassay with tobacco tissue cultures, Physiol Plant, 15 (1962) Ganapathi T R, Higgs N S, Balint-Kurti P J, Arntzen C J, May G D & Van Eck J M, Agrobacterium mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB), Plant Cell Rep, 20 (2001) Guy C, Haskell D, Neven L, Klein P & Smelser C, Hydration state-responsive proteins link cold and drought stress in spinach, Planta, 188 (1992) Kim Y H & Janick J, ABA and Polyox encapsulation of high humidity increases survival of desiccated somatic embryos of celery, Hortic Sci, 24 (1990) Takahata Y, Brown D C W, Keller W A & Kaizuma N, Dry artificial seeds and desiccation tolerance induction in microspore derived embryos of broccoli, Plant Cell Tissue Organ Cult, 35 (1993) Attree S M, Tautorus T E, Dunstan D I & Fowke L C, Somatic embryo maturation, germination and soil establishment of plants of black and white spruce (Picea mariana and P. glauca), Can J Bot, 68 (1990) Bornman C R, Maturation of somatic embryos, in, Synseeds, application of synthetic seeds to crop improvement, edited by K Redenbaugh (CRC Press, Boca Raton, USA) 1993, Morris P C, Kumar A, Bowles D J & Cuming A C, Osmotic stress and abscisic acid induce expression in wheat E M genes, Eur J Biochem, 190 (1990) Roberts D R, Finn B S, Webb D T, Webstar F B & Sutton C S, Abscisic acid and indole-3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce, Physiol Plant, 78 (1990) Gutmann M, von Aderkas P, Label P & Lelu M A, Effects of abscisic acid on somatic embryo maturation of hybrid larch, J Exp Bot, 47 (1996) Guy C L, Cold acclimation and freezing stress tolerance: Role of protein metabolism, Ann Rev Plant Physiol Plant Mol Biol, 41 (1990) Nitzsche W, One year storage of dried carrot callus, Z Pflanzenphysiol, 100 (1980) Pearce R S, Extracellular ice and cell shape in frost stressed cereal leaves: A low temperature scanning electron microscopy study, Planta, 175 (1988) Sakai A, Kobayashi S & Oiyama I, Cryopreservation of nucellar cells of naval orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification, Plant Cell Rep, 9 (1991) Panis B, Schoofs H, Remy S, Sagi L & Swennen R, Cryopreservation of banana embryogenic cell suspensions: An aid for genetic transformation, in Cryopreservation of tropical plant germplasm: Current research progress and application, edited by F Engelmann & H Takagi (IPGRI, Italy) 2000,