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  1. 1. Indian Journal of BiotechnologyVol 5, October 2006, pp 521-526Desiccation and ABA treatment improves conversion of somatic embryos to plant- lets 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 400 085, 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 im- proving 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 em- bryos with ABA and desiccation can improve conversion frequency. Keywords: banana, somatic embryos, abscisic acid, desiccation IPC Code: Int. Cl.8 A01H4/00Introduction These have been found to be effective for the matura- The regeneration of plants through somatic em- tion and stimulation of embryo-plant conversion inbryogenesis represents a remarkable feature of plant wheat12, Hevea13, sugarcane14, white spruce15 and wildcells and this has led to the development of novel ap- cherry16. Often embryos display signs of precociousproaches for plant propagation and genetic manipula- germination, which is due to incomplete maturationtion. Such application often necessitates the produc- phase between the stages of embryo development andtion of somatic embryos in large numbers and high or embryo germination17. Treatment of somatic em-conversion of somatic embryos into plants. In many bryos with ABA to reduce water content and to allowplant species, low conversion of embryos is related to preservation of synthetic seeds was demonstrated bypoor quality of embryos and lack of maturation and Senaratna et al18. Shiota et al19 used a combineddesiccation tolerance1,2. Low vigour and poor quality treatment of desiccation and ABA to examine theirof somatic embryos results in poor development into effect on somatic embryo viability during preserva-plants, which is also attributed to reduced levels of tion of carrot somatic embryos at low or sub-zerostorage reserves3. Provision of an artificial endosperm temperatures.to the encapsulated somatic embryo has been shown In banana, somatic embryogenesis leading to plantto increase vigour4 but not with much success. Alter- regeneration has been demonstrated from bases of leafnatively, enhancing the storage reserves in somatic sheaths and corm tissue, thin sections from in vitroembryos through the acquisition of desiccation toler- proliferating meristems, immature zygotic embryos,ance during maturation process as a means of stabili- male and female flowers20-22. Although somatic em-zation and hardening of somatic embryos offers a po- bryos can be produced with higher frequency, matura-tential choice5. tion and conversion into plants are still critical, requir- Desiccation tolerance in developing somatic em- ing optimization of nutritive and environmental con-bryos may be induced by exogenous signals in the ditions. Considering the importance of somatic em-tissue culture medium such as addition of osmotically bryogenic systems in banana for large-scale propaga-active substances like abscisic acid (ABA), poly eth- tion, genetic manipulation and conservation, experi-ylene glycol (PEG), sucrose and chilling stress6-11. ments on the improvement of conversion of somatic embryos into plants are underway in our laboratory.—————— In an earlier paper, we reported stimulatory effects of*Author for correspondence:Tel: 91-22-25595049; Fax: 91-22-25505151 cyanobacterial extracts on the conversion of somaticE-mail: vabapat@magnum.barc.ernet.in embryos into plants23. In this report, our
  2. 2. 522 INDIAN J BIOTECHNOL, OCTOBER 2006results on the effects of ABA and desiccation on the silica gel in a desiccator. Initial weight of embryosconversion and storage of somatic embryos of banana before desiccation was noted and after every hourcv. Rasthali (AAB) are described. weights were taken and relative water content (RWC) was calculated using the following formula:Materials and Methods Initial weight − Final weight RWC= × 100Establishment of Cultures Initial weight Shoot tip cultures were established in vitro fromfield grown plants of cv. Rasthali (AAB genomic Desiccation of Embryosgroup) as per the method described earlier24. The in 5 g of ABA treated and non-treated embryos werevitro multiple shoot cultures were grown and main- used for desiccation using 20 g autoclaved silica geltained on medium containing MS25 salts supple- in a sterile desiccator. Embryos were desiccated for 0,mented with benzylaminopurine (BAP, 4.5 μM), in- 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 9 h and after each desic-dole 3-acetic acid (IAA, 1.14 μm) and sucrose (87.7 cation period, 500 embryos were taken from the des-mM). Thin (400-500 μM) longitudinal sections were iccator and placed on dried filter paper in Petriplatescut from 1 cm long shoot tips (4-6 whorls of leaves) and sealed with parafilm.under aseptic conditions. These were then transferred Storage of Somatic Embryosto MS medium supplemented with 9.05 μM 2,4- ABA treated and non-treated, desiccated and non-dichlorophenoxyacetic acid (2,4-D), 1 μM zeatin and desiccated embryos were subjected to storage at three1 mg L-1 d-biotin for embryogenic callus develop- different temperatures, 25, 10 and −20°C.ment.Suspension Cultures and Somatic Embryo Development Conversion of Somatic Embryos Suspension cultures were established from 6- After incubation at a given temperature, desiccatedmonth-old, whitish, friable embryogenic callus tissue and non-desiccated, with or without ABA treatment,as described by Ganapathi et al26 in liquid M2 me- embryos were placed every week on conversion me-dium [(MS supplemented with 2,4-D (4.5 μM), d- dium containing MS salts supplemented with 1.62 μMbiotin (1 mg L-1), L-glutamine (100 mg L-1), malt ex- BAP, 87.7 mM sucrose and gelled with 0.2% phytageltract (100 mg L-1), sucrose (87.7 mM)]. These cell (Sigma, USA). In each experiment, about 50 embryossuspensions were plated onto half strength MS me- were checked for conversion. Conversion frequencydium (RR) supplemented with zeatin (9.12 μM) and was calculated as the somatic embryos that developedsucrose (87.7 mM) for embryo development. Globular with good shoot and root system, and for each em-embryos were formed in 2-3 weeks after plating the bryo, shoot length and root lengths were recorded. Allcells and fully developed embryos in 4-6 weeks. the experiments were repeated twice with three repli-These were used for desiccation and/or ABA experi- cations and standard error was calculated for the re-ments. sults.Treatment with ABA Results ABA (10 μM) was added aseptically to MS basal Embryogenic cell suspension (ECS) cultures con-medium containing 87.7 mM sucrose. About 5 g so- sisted of embryogenic cells and clumps (Fig. 1A).matic embryos were transferred to 200 mL MS me- Translucent spheres characterized the highly embryo-dium with ABA in 500 mL Erlenmeyer flask and cul- genic cultures and torpedo shaped embryos were ob-tured on gyratory shaker (100 rpm) at 25°C in dark served after 4-6 weeks of transfer to RR medium (Fig.for 48 h. Following incubation, medium was decanted 1B). The embryos converted into tiny plantlets withand embryos were washed with hormone-free MS the emergence of coleoptiles at a frequency of 56%medium and used for desiccation and calculation of (Fig. 1C). Well-developed embryos derived fromwater content. these ECS were employed for ABA and/or desicca-Determination of Water Content (WC) tion treatments. About 5 g of ABA treated and non-treated embryos Somatic embryos treated with or without ABA ex-were tested for measuring the WC. The embryos were hibited relative water contents in the range of 62.0-kept on dried autoclaved filter paper and laid on 20 g 63.5% in the control (Fig. 2). The pattern of decrease
  3. 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. 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 des- iccation 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 nei- ther ABA treated nor desiccated survived up to 6 weeks at 25OC with a gradual decrease in the conver- sion 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 2ndand 3rd weeks during storage and maximum shoot length (3.06 cm) was observed (Fig. ID, Table 1-1. Embryos that were desiccated forFig, 1--Conversion of somatic embryos of banana cv. Rasthali 2 h showed a reduction in conversion from 48 tofollowing ABA and desiccation treatment: A. Embryogenic cells 16% when stored for 5 weeks. An increase in shootas visible in the cell suspension culture (note the cells with densecytoplasm studded with starch grains); B. 6 week-old somatic length was observed only up to 2 weeks (Table 1).embryos developed on MS medium supplemented with zeatin; C. Immediately after desiccation, conversion was 48%Conversion of somatic embryos (Control) on MS medium while up to 3 weeks the decrease was in the range ofsupplemented with BAP; D. Conversion of ABA treated somatic 36-44%. However, embryo conversion fell rapidlyembryos on MS medium supplemented with BAP; E. Conversionof ABA treated and 2 h desiccated somatic embryos on MS thereafter. In the case of treatment with ABA fol-medium supplemented with BAP. lowed by desiccation for 2 h, somatic embryo conver- sion was observed till 6 weeks of storage; 66% so-was similar for ABA treated and non-treated embryos matic embryos converted in the l" week of storageduring desiccation. The relative ~VC was 44.6% (Fig. 1E) followed by 60% in the 2"d week, 42% in(without ABA) and 42.5% (with ABA) during desic- the 3rd week and 30% in the 4" week. Maximumcation for 2 h. The WC dropped slowly to 4.1-5.8% shoot length of 2.2 cm was observed in embryos
  4. 4. 524 INDIAN J BIOTECHNOL, OCTOBER 2006Fig. 3—Effect of desiccation on conversion of control somaticembryos (without ABA treatment and storage). Conversion wasnoted 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 Control ABA Desiccation ABA+ storage (2 h) desiccation (week) (2 h) 0 2.03± 0.14 0.94± 0.18 1.68± 0.15 1.07± 0.07 1 1.57± 0.13 2.50± 0.15 1.83± 0.16 2.20± 0.14 2 1.25± 0.13 3.06± 0.13 2.41± 0.13 0.93± 0.09 3 1.17± 0.11 2.89± 0.12 1.67± 0.12 0.90± 0.11 4 1.06± 0.10 2.45± 0.11 1.38± 0.13 0.79± 0.10 5 0.97± 0.12 1.46± 0.11 1.05± 0.17 0.58± 0.07 6 0.82± 0.15 — 1.25± 1.08 —Fig. 4—Conversion of somatic embryos treated with ABA and/or 7 — — — —2 h desiccation during storage at 25°C. Discussiontransferred to conversion medium after one week of In this study, an attempt was made to examine thestorage. role of ABA and desiccation on the conversion and In contrast, none of the somatic embryos that had storage of somatic embryos of banana. Resultsneither been treated with ABA nor desiccated sur- showed the significance of ABA treatment in confer-vived when stored at low temperatures. Embryos ring desiccation tolerance to somatic embryos. With-treated with ABA and desiccated for 2 h exhibited out prior ABA treatment and desiccation, embryobetter conversion during storage at 10°C after 5th and conversion dropped from 56% in the first week to 4%6th weeks as compared to those desiccated but without in 6th week when stored at 25°C. Somatic embryosABA treatment (Fig. 5). Compared to low conversion either with ABA exposure or desiccation showed in-rate (14%) during the first week, ABA treated and creased conversion during storage at 25°C in the ini-desiccated embryos showed conversion of 56% after tial 1-3 weeks and thereafter there was a gradual de-storage for 5 weeks. It gradually dropped to 24% in cline, however, the combined treatmentthe 6th week, 12% in the 7th week, and beyond this, all (ABA+desiccation) resulted in higher conversionthe embryos died with no conversion. Treatment with (66%) only during initial 1-2 weeks. Such a decreaseABA followed by desiccation up to 4 h did not show in conversion might be due to the high metabolic ac-any conversion while 6 h desiccation resulted in 2-4% tivity of cells and lack of nutrition. Embryos that wereconversion during storage at −20°C up to 5 weeks stored at 10°C showed comparatively longer storabil-(data not shown). ity (up to 6 weeks) with 56% conversion (Fig. 5),
  5. 5. SRINIVAS et al: CONVERSION OF SOMATIC EMBRYOS TO PLANTLETS IN BANANA BY DESICCATION 525suggesting that in such somatic embryos, metabolic desiccation could be preserved at low temperaturesactivity might be very low and hence high germinabil- for 7-8 weeks.ity was noticed. Somatic embryos that were desic- Preservation of plant tissue through cryopreserva-cated showed decline in conversion (< 10%) upon tion is done by the addition of cryoprotectants andstorage at −20°C. This could be due to freezing injury gradual freezing to ensure the growth of ice crystals incaused by the formation of ice crystals between intra- the extracellular spaces37. Additionally, vitrificationcellular spaces27. has also been used successfully for the preservation of The positive effects of desiccation on the conver- plant cells. In this method, cells are exposed to a vitri-sion of somatic embryos to plantlets have been dem- fication solution, cooled rapidly in liquid nitrogen andonstrated in many plants. In horse chestnut, somatic rewarmed rapidly in order to avoid the formation ofembryos were subjected to different desiccation peri- ice crystals38,39. Although these methods have suc-ods after a pretreatment with 80 μM ABA and/or 50 g cessfully been employed for the preservation of plantL-1 PEG. Although heat and chilling stress can induce cells and tissues, rapid freezing conditions or ultradesiccation tolerance in somatic embryos, ABA plays temperatures or thawing steps generally used restricta major role in conferring tolerance as reported for their large-scale and routine application19. In this con-various species including Apium graveolens28, Bras- text, a simple method of pretreatment with ABA fol-sica oleracea29 and Picea spp.30. Although unclear, lowed by desiccation appears to be interesting in viewABA by suppressing abnormal development, inhibit- of the storage potential at low temperatures. Extensiveing precocious germination, conferring desiccation studies are required for demonstrating such storagetolerance and promoting accumulation of storage lip- potential in a wide variety of plant species. In additionids and proteins, induces a synchronized maturation to the well-advocated cryopreservation techniques,that results, upon its withdrawal, in uniform high fre- the simple method as presented in this study, may of-quency germination of somatic embryos31. ABA has fer a new and alternative choice for the low tempera-been known to induce the expression of maturation ture preservation of embryogenic cultures.genes in wheat embryos32 and to inhibit precociousgermination resulting in accumulation of storage pro- References 1 Bapat V A & Rao P S, Maturation and desiccation of somaticteins in interior spruce somatic embryos33. ABA embryos, in Trends in Plant Tissue Culture & Biotechnology,seems to play, at the structural level, a role in cell ex- edited by L K Pareek and P L Swarankar (Agro Botanicalpansion; ABA treated embryos have small and Publishers, Bikaner, India) 1996, 53-64.densely cytoplasmic cells, whereas ABA untreated 2 Redenbaugh K, Fuji J, Slade D, Viss P & Kossler M, Artifi- cial seeds–Encapsulation of somatic embryos, in Biotechnol-embryos have vacuolated and expanded cells34. ogy in agriculture & forestry, vol 17, High tech micropropa- In addition to the known effects on embryo conver- gation 1, edited by Y P S Bajaj (Springer-Verlag, Belrin)sion, studies have also indicated that ABA treated 1991, 395-416.and/or desiccated somatic embryos can avoid damage 3 Tetteroo F A A, Hoekstra F A & Karssen C M, Induction of complete desiccation tolerance in carrot (Daucus carota L.)caused by the formation of ice crystals in intracellular embryoids, J Plant Physiol, 145 (1995) 349-356.spaces, when stored at sub-zero temperatures19. The 4 Anandarajah K & McKersie B D, Manipulating the desicca-damage in the frozen state is generally due to the for- tion tolerance and vigour of dry somatic embryos of Medi-mation of ice crystals in the intracellular spaces in cago sativa L. with sucrose, heat shock and abscisic acid,plant cells35. Carrot callus grown on medium contain- Plant Cell Rep, 9 (1990) 451-455. 5 Bapat V A & Rao P S, Plantlet regeneration from encapsu-ing high sucrose or ABA reduced water content and lated and non-encapsulated desiccated somatic embryos of acould be preserved at −80°C for a year36. The results forest tree: Sandalwood (Santalum album L.), J Plant Bio-in this study also indicate that desiccation preceded by chem Biotechnol, 1 (1992) 109-113.exposure to ABA can avoid such freezing due to low 6 Lecouteux C, Lai F M & McKersie B D, Maturation of al- falfa (Medicago sativa L.) somatic embryos by abscisic acid,water content in the somatic embryos. The 6-8 h des- sucrose and chilling stress, Plant Sci, 94 (1993) 207-213.iccated embryos had less than 10% conversion and 7 Senaratna T, McKersie B D & Bewley S R, Desiccation tol-could be stored at −20°C. Shiota et al19, demonstrated erance of alfalfa (Medicago sativa L.) somatic embryos: In-long-term preservation of ABA treated and desiccated fluence of abscisic acid, stress pretreatments and drying rates, Plant Sci, 65 (1989) 253-259.embryos with viability for more than three years at – 8 Capuana M & Debergh P C, Improvement of the maturation25°C. Our results showed that banana somatic em- and germination of horse chestnut somatic embryos, Plantbryos exposed to a combined treatment with ABA and Cell Tissue Organ Cult, 48 (1997) 23-29.
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