Cryopreservation of shoot primordia cultures of melon using a slow prefreezing procedure

Plant Cell, Tissue and Organ Culture 49: 171 177, 1997. 171 cæ 1997 Kluwer Academic Publishers. Printed in the Netherlands. Cryopreservation of shoot primordia cultures of melon using a slow prefreezing procedure Rie Ogawa 1, Masaya Ishikawa, Eiko Niwata 2 & Katsuji Oosawa National Institute of Agrobiological Resources, Kan nondai, Tsukuba, Ibaraki, Japan 305 ( 1 Present address: Aichi Agriculture Research Center, Sagamine, Yazako, Aichi, Japan 480-11; 2 Present address: Aomori Green BioCenter, Yamaguchi, Nogi, Aomori, Japan 030-01) Received 28 April 1996; accepted in revised form 7 April 1997 Key words: Cucumis melo, mass propagation, storage Abstract Tissue-cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) were successfully cryopreserved in liquid nitrogen (LN) using a slow prefreezing method. The highest survival and recovery were obtained with the following procedure. Three week-old shoot primordia clumps were dissected into pieces of 2-3 mm of diameter and precultured in standard medium for 3 days. They were directly soaked in CSP1 cryoprotective solution (10%w/v sucrose, 10%w/v dimethylsulfoxide and 5%w/v glycerol) and incubated at room temperature for 30 min. Samples were ice-inoculated at -8 æ C and cooled at a rate of between 0.3 and 1 æ Cmin,1 with a programmable freezer to -30 æ C for prefreezing. They were then plunged into LN for storage. After rapid thawing in 40 æ C water, the cryoprotective solution was slowly diluted 5 fold in a dropwise manner with 3% sucrose and the shoot primordia were transferred onto regeneration medium. Under optimal conditions, more than 80% of cryopreserved shoot primordia were viable and 50 to 80% regenerated shoots after one month of reculture. Cryopreserved shoot primordia could be used both for reproducing a shoot primordia culture and for regenerating plants. Abbreviations: BA 6-benzyladenine; CSP1 cryoprotective solution for slow prefreezing method #1; DMSO dimethylsulfoxide; FDA fluorescein diacetate; LN liquid nitrogen Introduction Shoot primordia culture was first established with Haplopappus gracilis (Tanaka and Ikeda, 1983). Since then, shoot primordia cultures have been developed for several other plants (Kondo et al., 1991; Takano et al., 1991), including melon (Nagai et al., 1989). They are usually maintained in liquid medium containing cytokinin and gently agitated on a slanted drum revolving at a very slow rate. A shoot primordia culture consists of small pieces of cultured tissues (hereafter referred to as a shoot primordia clump), each of which has numerous apical meristems scattered over a calluslike tissue. Within a clump, daughter apical meristems develop at the base of the mother apical meristems. One shoot primordia clump can regenerate into a large number of shoots with marginal rate of variant emergence (Tanaka and Ikeda, 1983; Ezura et al., 1992). Thus, shoot primordia cultures have drawn attention as an effective method for mass propagation. For the same reasons, they can be used to provide an efficient system for maintaining genetic resources in vitro and is a good system for transformation (Yoshioka et al., 1992). To date, most shoot primordia cultures are maintained by repeated subculture. Short-term in vitro conservation of shoot primordia has been attempted by retarding growth with abscisic acid and low temperature (Suzuki et al., 1991). Cryopreservation, if successful, would provide an efficient method with minimal inputs for long term storage. Cryopreservation of shoot primordia cultures would provide a stand-by supply of inocula for mass propagation and preserve the genetic integrity of the germplasm stored. However, there are only a few reports on cryopreservation of cultured

172 Figure 1. Effect of various cryoprotective solutions on the viability and shoot regeneration of cryopreserved melon shoot primordia. Dissected shoot primordia clumps (2-3 mm in size) were precultured on standard medium at 25 æ C for 3 days. Following incubation in cryoprotective solutions at room temperature for 30 min, they were ice-inoculated at -8 æ C and prefrozen to -30 æ Cat0.3 æ Cmin,1 prior to immersion in LN (LN). TC (treated control): specimen treated with cryoprotective solutions only and not frozen. Regeneration medium: MS + 3% sucrose + 0.2 mg l,1 BA + 0.5 mg l,1 GA 3. The data are the mean of two determinations with their ranges (only positive ranges are shown). Figure 2. Effect of cooling rate on the viability and shoot regeneration of cryopreserved melon shoot primordia. Experimental details were identical to Figure 1, except that CSP1 was used for cryoprotection and the cooling rate during prefreezing to -30 æ C was varied. Treated control: specimen treated with CSP1 only and not frozen. The data are mean æ ranges (n=2). shoot primordia (Taniguchi et al., 1988; Kohmura et al., 1992). Apical meristems of many plant species have been successfully cryopreserved using slow prefreezing (two-step) procedures (Bajaj, 1995; Grout, 1995; Kartha, 1985). Since cultured shoot primordia clumps contain numerous apical meristems, slow prefreezing protocols could be applied to cryopreservation of shoot primordia culture. We reported previously the successful cryopreservation of melon somatic embryos by slow prefreezing (Niwata et al., 1991) and by desiccation (Shimonishi et al.,1991). We developed an efficient cryoprotective solution (CSP1) for cryopreservation of bromegrass cell suspensions using a slow prefreezing procedure (Ishikawa et al., 1991; 1996). In the present study, we attempted to cryopreserve cultured shoot primordia of melon using CSP1 for cryoprotection and a slow-step freezing protocol.

173 Figure 3. Effect of prefreezing temperature on the viability and shoot regeneration of cryopreserved melon shoot primordia. Experimental conditions were the same as in Figure 2 except that specimens were cooled at 0.3 æ Cmin,1 to the designated prefreezing temperature and they were incubated in CSP1 for 1 h prior to cryopreservation. -30 æ C without PC: dissected shoot primordia clumps were used directly for cryopreservation (prefreezing to -30 æ C) without preculturing on standard medium. The data are mean æ range (n=2). Materials and methods Induction of shoot primordia Shoot primordia cultures were initiated by inoculating shoot apices of melon (Cucumis melo L. cv. prince melon) into Murashige and Skoog (1962) liquid medium containing sucrose (3%), BA (1 mg l,1 ) and NAA (0.01 mg l,1 ). They were subcultured by transferring dissected pieces (5 mm) of shoot primordia clumps into MS medium containing 3% sucrose and 1 mg l,1 BA at 3-week intervals (Nagai et al., 1989; Shimonishi et al., 1989). Cultures were incubated at 25 æ C under continuous light with cool-white fluorescent illumination (37.5 çmol m,2 s,1 ) on a slanted rotary incubator revolving at 2 rpm. They were maintained under these conditions for one and one-half years before use. A clump of melon shoot primordia cultured for 3 weeks was about 15 mm in diameter and consisted of numerous apical meristems on the surface (green color) and underlying callus (light green). Each apical meristem usually had a dome with 0-1 primordial leaf. Preculture and cryopreservation procedures Three week-old cultured shoot primordia clumps were dissected into pieces of 1-2 mm or 2-3 mm of diameter and precultured for 3 days in standard medium (MS liquid medium with 3% sucrose and 1 mg l,1 BA) unless otherwise noted. The precultured shoot primordia were placed in 10 ml glass centrifuge tubes. One ml of cryoprotective solution, comprising sucrose, dimethylsulfoxide (DMSO) and glycerol at various concentrations indicated in Figure 1, was added directly to specimens in one time at room temperature. Samples were incubated at room temperature for 0.5 to 3 h. The volume of the cryoprotective solution in each tube was reduced to 0.5 ml before transfer into a programmable freezer (KRYO 10 model 10-21 of Planer Biomed Co. Ltd., UK or model FPR-120S of Fuji Ika Sangyo Co. Ltd., Japan). Samples were then cooled at 0.5 æ Cmin,1 to -8 æ C, at which temperature crystallization of the cryoprotective medium was induced by touching the tubes with a LN-cooled steel rod. The samples were then held for 30 min at -8 æ C. Subsequently, samples were cooled at 0.3-1.0 æ Cmin,1 to prefreezing temperatures ranging from -25 to -40 æ C, held for 20 min at the prefreezing temperature, then immersed in LN. Thawing and reculture After 1 h storage in LN, samples were thawed rapidly in 40 æ C water. The cryoprotective medium was then diluted 5-fold at room temperature by adding 3% (w/v) or 10% (w/v) sucrose, either slowly (drop by drop addition over 20 to 30 min) or rapidly (direct addition in one time). Cryopreserved shoot primordia were transferred onto the regeneration medium (MS gellan gum medium with 3% sucrose and 0.2 mg l,1 BA and 0.2-1.0 mg l,1 GA 3 ). Viability was estimated by calculating the percentage of individual shoot primordia clumps

174 Figure 4. Effect of size of shoot primordia clumps and incubation period in CSP1 on the viability and shoot regeneration of cryopreserved melon shoot primordia. Experimental conditions were identical to Figure 3 except for the varying periods of incubation and the varying sizes of shoot primordia clumps and the fixed prefreezing temperature (-30 æ C). The data are mean æ range (n=2). which were green after one month of reculture. Viability indicated the overall performance of both apices and underlying calli of shoot primordia clumps. Shoot regeneration was also scored after one month of reculture and represented the regrowth capacity of surviving apices. Callus tissues within a clump proliferated only marginally on the regeneration medium when apices in the clump were viable. Viability was also checked by staining 50-100 çm sections of shoot primordia clumps with FDA after 2 weeks of reculture. Experiments were performed twice and each data point represents the mean (æ range) of two consecutive determinations. For each determination, 10 to 12 shoot primordia were used. Results and discussion The effect of different cryoprotective solutions on survival of cultured shoot primordia following cryopreservation is shown on Figure 1. The highest viability (80%) and shoot regeneration rates (30%) were obtained with a combination of 10% (w/v) sucrose, 10% (w/v) DMSO and 5%(w/v) glycerol termed CSP1. Absence (0%) or high (10%) concentrations of glycerol in the cryoprotective medium resulted in decreased viability (36%). In subsequent experiments, CSP1 was used for cryoprotection; originally, it had been developed for cryopreservation of a bromegrass cell culture (Ishikawa et al., 1991; 1996). To determine the effects of cooling rate, shoot primordia were cooled from -8 æ C (ice inoculation temperature) to -30 æ C (prefreezing temperature) at 0.3,0.5 and 1.0 æ Cmin,1 (Figure 2). These cooling rates gave comparable high survival rates. For cryopreservation of melon somatic embryos, an optimum in survival was noted with a cooling rate of 0.3 æ Cmin,1 (Niwata et al., 1991). This difference may be due to the different cryoprotective solution employed with somatic embryos (10% sucrose and 10% DMSO) and/or differences in size and structure between somatic embryos and shoot primordia. To make the protocol less time consuming, a cooling rate of 1.0 æ Cmin,1 can be used for cryopreservation of melon shoot primordia. The effect of prefreezing temperature on the survival of shoot primordia is shown on Figure 3. All the prefreezing temperatures tested (-25 to -40 æ C) gave high survival rates, but the optimum was at -30 æ C, which gave 80 to 90% viability and 70 to 80% shoot regeneration rates. Therefore, prefreezing to -30 æ C was chosen in subsequent experiments. Preculture of shoot primordia clumps in standard medium for 3 days following dissection into 2-3 mm pieces increased survival after cryopreservation (Figure 3). This may have resulted from wound healing of the dissected surface during preculture. Since our

176 primordia and reduced the survival in both size categories. Large clumps (2-3 mm) incubated in CSP1 for 30 min gave the best survival. In a preliminary experiment, we found that addition of GA 3 in the regeneration medium stimulated regrowth of shoot primordia following cryopreservation. However, the viability and regeneration were found to be similar with GA 3 concentrations between 0.2 and 1.0 mg l,1 (data not shown). The method employed to dilute the cryoprotective solution strongly affected viability of cryopreserved shoot primordia (Figure 5). The highest viability and regeneration (74 and 47%, respectively) of cryopreserved shoot primordia were obtained when CSP1 was diluted slowly with 3% sucrose at room temperature over about 20 to 30 min. When dilution was performed rapidly with 3% or 10% sucrose at room temperature, viability was nil and 22%, respectively. As a diluent for the drop-wise slow dilution, 3% sucrose gave higher viability and shoot regeneration than 10% sucrose (Figure 5). Post-thaw dilution of cryoprotectants has often been considered harmful presumably due to rapid deplasmolysis and/or removal of vital solutes leaked from specimens (Withers, 1985). We obtained higher survival of cryopreserved shoot primordia at the slower dilution rate and also with 10% sucrose when rapid dilution was employed. Our results may be consistent with those of Withers (1985). However, avoidance of post-thaw dilution of cryoprotectant by transferring cryopreserved specimens onto filter paper disks over semi-solid medium improved survival only marginally compared to slow dilution of CSP1 with 3% sucrose (data not shown). In separate experiments with bromegrass cell suspensions (Ishikawa et al., 1991; 1996) and somatic embryos of melon (Niwata et al., 1991), we examined the effect on survival of the method employed for adding CSP1. We found that direct (rapid) addition of CSP1 to the specimens gave better survival than slow (gradual) drop-wise addition of CSP1. To improve efficiency, the cryoprotective solution was added directly to the specimens in the experiments reported here. In most reports of cryopreservation using slow prefreezing, the addition of cryoprotectants was conducted slowly in a gradual manner on ice (Chen et al., 1985; Reed, 1990; Bagniol and Engelmann, 1991; Meijer et al., 1991; Grout, 1995). Direct addition of the cryoprotective solutions at one time at room temperature became possible by using CSP1. This increased the efficiency of the method and the applicability to chilling sensitive plants such as melon. In previous cryopreservation experiments with bromegrass cells, plastic cryotubes were tested as containers for this method (Ishikawa et al., 1991; 1996). However, even under optimal conditions, much lower survival rates were noted, probably due to the lower heat conductivity of plastic tubes compared to glass ones. Therefore, in all experiments with melon shoot primordia reported here, glass tubes were used as containers. There were differences in the survival rates following cryopreservation under similar conditions (Figure 1, Figure 3 and Figure 5), especially in the shoot regeneration rates. However, the data shown are not directly comparable between these figures as they were not obtained at the same time. Data in Figures 1 and 2 were obtained in March, 1991 whereas those in Figures 3, 4 and 5 were obtained in November and December, 1990. The differences in the time of experiments seem to reveal slight changes in the performance of shoot primordia during subculturing, especially in the number of apices per clump. This seemingly affected the shoot regeneration before and after cryopreservation conducted at different time periods. When cryopreserved shoot primordia clumps were recultured on semi-solid medium, they turned whitish in one day, presumably due to chlorosis. However, viable primordia clumps displayed green coloured patches from surviving apices and callus tissues after 2 to 3 days. Within a month, they regenerated shoots as vigorously as unfrozen control (Figure 6). When a shoot primordia clump had viable apices, viable callus within the clump proliferated only marginally on the regeneration medium, irrespective of freeze-thaw treatment (Figure 6). FDA staining of sections of cryopreserved shoot primordia after 2 weeks of reculture revealed that a clump of shoot primordia had both stained apices (radiating fluorescence) and totally unstained apices (data not shown). This suggests that some apical meristems only survived LN exposure and regrew into shoots. Only marginal callus formation from apices was noted during regrowth and regenerated shoots originated directly from the surviving entire apical meristems. Whether regenerants from cryopreserved shoot primordia might display any variation remains to be investigated. When cryopreserved shoot primordia clumps were placed in standard medium (liquid MS medium containing 3% sucrose and 1 mg l,1 BA) and cultured at 25 æ C under light for one month, they regrew and regained the original characters of melon shoot primordia cultures (data not shown). Cryopreserved shoot primordia

177 of melon can be used both as inoculum to initiate a new culture and for direct regeneration of plants. In conclusion, we developed a cryopreservation protocol for cultured shoot primordia of melon (Cucumis melo L. cv. prince melon) using a slow prefreezing (two-step) method. Under the optimal conditions, more than 80% of the primordia clumps remained viable and shoot regeneration rates reached 50 80%. The choice of an appropriate cryoprotective solution (CSP1), the dilution method (slow dilution with 3% sucrose at room temperature) and the use of glass containers were critical to obtain high survival rates. Preculture of dissected shoot primordia pieces for 3 days prior to cryopreservation improved survival. A relatively wide range of incubation duration in CSP1 (30 min to 2 h), of cooling rates (0.3 to 1.0 æ Cmin,1 ) and prefreezing temperatures (,30 to,40 æ C) allowed a high survival rate. Acknowledgements The authors would like to thank Dr. Duncan A. Vaughan of NIAR for critical reading the manuscript. References Bajaj YPS (1995) Cryopreservation of Plant Germplasm I. Biotechnology in Agriculture and Forestry 32. Springer-Verlag, Berlin, Heidelberg, New York Bagniol S & Engelmann F (1991) Effects of pregrowth and freezing conditions on the resistance of meristems of date palm (Phoenix dactylifera L. var. Bou Sthammi Noir) to freezing in liquid nitrogen. Cryo-Lett. 12: 279 286 Chen THH, Kartha KK & Gusta LV (1985) Cryopreservation of wheat suspension culture and regenerable callus. Plant Cell Tissue Organ Cult. 4: 101 109 Ezura H, Amagai H, Yoshioka K & Oosawa K (1992) Highly frequent appearance of tetraploidy in regenerated plants, a universal phenomenon, in tissue cultures of melon (Cucumis melo L.). Plant Sci. 85: 209 213 Grout B (1995) Genetic Preservation of Plant Cells in Vitro. Springer-Verlag, Berlin Heidelberg, New York Ishikawa M, Tandon P, Yamaguishi A & Miyazaki S (1991) Cryopreservation of bromegrass cells using a rapid prefreezing and a slow prefreezing method. 1991 Ann. Meeting of Jpn Plant Physiol. 96 Ishikawa M, Tandon P, Suzuki M & Yamaguishi-Ciampi A (1996) Cryopreservation of bromegrass (Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures. Plant Sci. 120: 81 88 Kartha KK (1985) Cryopreservation of Plant Cells and Organs. CRC Press, Florida Kohmura H, Sakai A, Chokyu S & Yakuwa K (1992) Cryopreservation of in vitro-cultured multiple bud clusters of asparagus (Asparagus officinalis L. cv Hirosimagreen (2n=30) by the techniques of vitrification. Plant Cell Rep. 11: 433 437 Kondo K, Nadamitsu S, Tanaka R & Taniguchi K (1991) Micropropagation of Spinacia oleracea L. through culture of shoot primordia. Plant Tissue Cult. Lett. 8: 1 4 Meijer EGM, van Iren F, Schrijnemakers E, Hensgens LAM, van Zijderveld M & Schilperoort RA (1991) Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.). Plant Cell Rep. 10: 171 174 Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473 497 Nagai T, Nomura Y & Oosawa K (1989) Induction of shoot primordia and plantlet formation in melon. J. Jpn. Soc. Hort. Sci. 58 (suppl. 1): 208 209 Niwata E, Ogawa R, Ishikawa M & Oosawa K (1991) Cryopreservation by prefreezing method for somatic embryos of melon. Jpn. J. Breed. 41 (suppl. 1): 188 189 Reed BM (1990) Survival of in vitro-grown apical meristems of Pyrus following cryopreservation. HortSci. 25: 111 113 Shimonishi K, Nomura Y, Nagai T, Yoshioka K & Oosawa K (1989) Plant regeneration ability of subcultured shoot primordia in melon. Jpn. J. Breed. 39 (suppl. 2): 110 111 Shimonishi K, Ishikawa M, Suzuki S & Oosawa K (1991) Cryopreservation of melon somatic embryos by desiccation method. Jpn. J. Breed. 41: 347 351 Suzuki S, Shimonishi K, Ishikawa M & Oosawa K (1991) In vitro preservation of cultured shoot primordia and somatic embryos in melon by growth retarding method. Plant Tissue Cult. Let. 8: 193 197 Takano H, Hirano M, Taniguchi K, Tanaka R & Kondo K (1991) Rapid clonal-propagation of Matricaria chamomilla by tissuecultured shoot primordia. Jpn. J. Breed. 41: 421 426 Tanaka R & Ikeda H (1983) Perennial maintenance of annual Haplopappus gracilis (2n=4) by shoot tip cloning. Jpn. J. Genet. 58: 65 70 Taniguchi K, Tanaka R, Ashitani N & Miyagawa H (1988) Freeze preservation of tissue-cultured shoot primordia of the annual Haplopappus gracilis (2n=4). Jpn. J. Genet. 63: 267 272 Withers LA (1985) Cryopreservation of cultured plant cells and protoplasts. In: Kartha KK (ed) Cryopreservation of Plant Cells and Organs (pp 243 267). CRC Press, Florida Yoshioka K, Hanada K, Nakazaki Y, Minobe Y, Yakuwa T & Oosawa K (1992) Successful transfer of the cucumber mosaic virus coat protein gene to Cucumis melo L. Jpn. J. Breed. 42: 277 285