PlantceU Reports

Plant Cell Reports (1992) 11:175-178

9 Springer-Verlag 1992

Cryopreservation of sweet potato (Ipomoea batatas ILl Lam.) shoot tips by vitrification Leigh E. Towill ~ and Robert L. Jarret 2 1 U S D A / A R S , N a t i o n a l Seed Storage Laboratory, Colorado State University, F o r t Collins, CO 80523, U S A

2 USDA/ARS, Southern Regional Plant Introduction Station, 1109 Experiment Street, Griffin, GA 30223, USA Received October 1, 1991/Revisedversion received February 19, 1992 - Communicated by G. C. Phillips SUMMARY. Vitrification is a technically simple method for cryopreserving plant germplasm, requiring only the application of suitable cryoprotectants and rapid cooling rates. Sweetpotato (Ipomoea batatas [L.] Lam.) shoot tips obtained from in vitro plants survived liquid nitrogen (-196~ exposure following a vitrification-inducing pretreatment. Shoot tips were treated in a stepwise manner with a vitrification solution containing 30% glycerol, 15 % ethylene glycol and 15 % dimethylsulfoxide in growth medium. Incubation of shoot tips for 1 to 2 h in low concentrations of the vitrification solution enhanced survival. Most surviving shoot tips developed callus, and a variable percentage subsequently formed shoots. Survival was not achieved using two-step cooling procedures. The percentage of shoot tips surviving vitrification and those subsequently forming a shoot varied widely among replications.

Key words: clones - cryoprotectants - germplasm preservation - shoot tip culture

Abbreviations: BA, N6-benzyladenine; IBA, indole-3butyric acid; EG, ethylene glycol; DMSO, dimethylsulfoxide; MS, Murashige and Skoog (1962) minerals and vitamins; LN, liquid nitrogen; PI, plant introduction

ceils and shoot tips of selected species (Withers 1987; Towill 1990b). Vitrification (glass formation) is an alternative method for attaining cryopreservation (Fahy et al. 1987; Fahy 1988) and has been successfully applied to plant protoplasts, cells and shoot tips of various genera (Langis et al. 1989; Langis and Steponkus 1990, 1991; Uragami et al. 1989; Sakai et al. 1990; Towill 1990a). Vitrification is achieved by exposure of the propagules to high concentrations of cryoprotectants. Glass formation occurs during subsequent cooling (usually rapid). Ostensibly, survival is enhanced by avoiding the damaging effects of ice formation, but exposure of propagules to high concentrations of cryoprotectants may, in itself, be damaging due to osmotic or biochemical effects. In a practical sense, vitrification simplifies explant handling (cooling rate devices are not needed), and has allowed survival of shoot tips of species where other cryopreservation techniques have failed (Towill, unpublished). To date, both two-step freezing and vitrification protocols have proven inadequate for preservation of sweetpotato shoot tips or cell suspensions. This report describes the survival of sweetpotato shoot tips after exposure to LN using modifications of a vitrification procedure described by Sakai and co-workers (Uragami et al. 1989; Sakai et al. 1990).

MATERIALS AND METHODS

INTRODUCTION Cryopreservation of shoot tips may be useful for the longterm storage of vegetatively propagated plant germplasm (Towill 1988, 1989). Slow, or two-step, cooling protocols have been used to cryopreserve protoplasts, Correspondence to: L. E. Towill

Micropropagated sweetpotato 0pomoea batatas [L.] Lain.) plants (PI 508515 and PI 290657) were obtained from the Southern Regional Plant Introduction Station, Griffin, Georgia and were propagated on a MS basal medium, 2% w/v sucrose and 0.7% (w/v) agar. Plants were maintained in 'GA 7' vessels (Magenta Corp.y and nodal segments containing a single axillary bud were subcultured at 8 to 12 week intervals. Cultures were incubated at 25~ under cool-white fluorescent lights (ca. 40-60 uE m "z s"l) using a 16 h light/8 h dark photoperiod. Immediately prior to use, shoot tips, 0.5-0.7 mm long containing 3 to 4 leaf primordia, were excised from axillary buds of in vitro plants.

176 Three shoot tips were placed in 0.2 ml within a 75 x 10 mm culture tube. Four to 8 test tubes were used per treatment. The vitrification-inducingprotocol was a modificationof that described by Sakai et al. (1990). A stock solution of 30% (w/v) glycerol, 15% (w/v) EG, and 15% (w/v) DMSO was prepared in MS and 0.4M sucrose (termed '100%' PVS2). This stock solution was diluted with MS containing 0.4M sucrose (no growth regulators) to obtain 10, 20, 40, 60, and 80% of PVS2 (v/v). Examinationof 100% PVS2 using differential scanning calorimetry confirmed the data of Uragami et al. (1989) showing that the solution does vitrify, ie. it forms a glass upon cooling. Shoot tips were exposed gradually to PVS2 by draining off the liquid mediumand adding about 0.25 ml of 10% PVS2. Aftertreatmentof the shoot tips for the predeterminedtime period, the solutionwas removed with a Pasteur pipette and about 0.25 ml of the next higher percentage PVS2 solutionwas added. Shoot tips were exposed to 10, 20, and 40% PVS2 at about 22~ and to 60, 80, and 100% PVS2 at 0"C. The lower temperature was used with the higher concentrations to minimize toxicity. Following exposure to PVS2, shoot tips were removed from the test tube with a Pasteur pipette and placed on a small strip (ca. 5 mm x l0 mm) of sterile tissue paper moistenedwith PVS2. The paper strip was folded to enclose the shoot tips, immersed in LN (cooling rate about 4000`C/rain), and held for about 1 h. Shoot tips were thawed by rapidly transferring the paper strip from LN into 3 ml of 1.2M sucrose in MS (MS2) held at 30"C (thaw rate of approximately 9000`C/rain). Test tubes containing shoot tips were immediatelytransferred from 30'C to about 22~ and held for 30 to 45 rain prior to reculture of the shoot tips to the recovery media (RM) described below. Vitrification solutionexposed control shoot tips were treated as describedabovebut were then transferred directly into MS2 at 30"C without immersion in LN. Unexposed controls were not treated with the cryoprotectantsbut were exposed to MS2. Preculture, when used, consistedof incubatingisolated shoot tips at 25~ for 2 d in MS containing3 % EG prior to exposureto the vitrificationtreatment. Treated and control shoot tips were recultured into 35 x 10 mm petri dishes containing2 ml of RM consistingof MS supplementedwith 0.5 mg/1 BA, 0.1 mg/l IBA, 3% sucrose, and 0.7% agar at pH 5.8. Cultureswere held at 25~ under a 16 h light/8 h dark photoperiod (cool white fluorescent lights, ca. 40-60 uE m2 sl). Growth observations were made over a 2 month period.

R E S U L T S AND D I S C U S S I O N Several modifications o f the two-step cooling procedures, as described b y TowiU (1990b) for shoot tip cryopreservation, were tested o n sweetpotato shoot tips. Although eryoprotectant-exposed shoot tips survived in high percentages, subsequently LN-treated samples did not survive (data not presented). Vitrification methods, which have been successfully applied to other animal and plant systems (Fahy et al. 1987; Fahy 1988; Sakai et al. 1990), were evaluated. Success in attaining vitrification depends on the application o f an effective vitrification-inducingprotocol. The kinetics o f the exposure of explants to the vitrification solutions are critical for explant survival. Thus, exposure o f shoot tips to the vitrification solution is t Mention of trade products, equipment,or commercialcompaniesin this publication does not imply endorsement by the U.S. Dept. of Agriculture over similar products or companiesnot named.

usually performed in two steps, first placing them in low concentrations of cryoprotectants to facilitate permeation o f individual solutes, and subsequently transferring them to higher concentrations to provide the desired degree o f desiccation. Exposure o f shoot tips to vitrification solutions is potentially injurious due to the phytotoxie effects of individual solution components or their combined osmotic effects o n cell viability. Survival of shoot tips that were not exposed to PVS2, but were exposed to MS2, approached 100 %. PVS2 concentrations ranging from 20 to 80% were not toxic within the exposure times examined (Table 1). Exposure of shoot tips for 5 rain to 100% PVS2 greatly decreased their survival whereas a 2 minute exposure did not. In contrast, increased exposure times in the presence of 10, 20, 40, and 80% PVS2 did not decrease explant survival. These observations suggest that shoot tips from sweetpotato are sensitive to severe osmotic stress, much moreso than shoot tips from Mentha (Towill 1990a).

Table 1. Effectof exposure times to different percentages of PVS2 on survival of shoot tips from I. batatas PI 508515. Percentage of PVS2 20 40 60 80 100

Survival callus~ shootsb

Experiment I . 5c 5 5 5 5

. . 5 5 5 5

20" 60 60 I0 60

10 10 10 5

. .

5

100 I00 100 100 100 33

42 25 25 25 25 0

5 5 5 5 5 10 5 5 2 5 5 2

100 100 100 100 100

64 67 83 50 54

5 5 5

5 5

Experiment2

9 percentage of treated shoot tips forming a callus. b percentage of treated shoot tips producing an elongatingshoot. minutes of exposure to the stated percentage of PVS2.

Shoot tips o f PI 508515 were exposed to stepwise additions o f PVS2 and subsequently immersed in LN. Increased survival o f shoot tips following immersion and storage for 1 h in L N was achieved when explants were exposed for longer time periods at the lower concentrations of PVS2 prior to L N immersion (Table 2). EG and D M S O are the permeating components o f the vitrification solution. Preculture of shoot tips for 2 d in 3 % EG was examined to determine whether gradual permeation prior to PVS2 exposure enhanced survival. This exposure to 3% EG did slightly increase the

177 percentage of shoot tips surviving LN exposure (Table 3); statistically, however, the difference is not significant. Other experiments gave the same trend. Table 2. Survival ofI. batatasP1508515 shoot tips aRer vitrification. Percentage of PVS2 10 20 40 60 80 100

Experiment 1 10~ 5 5 10 5 5

5 5

20 5 5 20 10 5

Survival Control Liquid nitrogen callus~ shootb callus shoots

5 5

100 100 100 100

54 (8) 42(43) 50 (9) 68(13)

3(10) 0 3(8) 0 57(4)38(38)

Experiment 2 20 10 5 60 10 5 60 10 5

5 5 10

100 100 100

67 (0) 67 (0) 89(16)

40(12) 2104) 72(10) 12(18) 91 (9)64(37)

Experiment 3 60 10 5 60 10 5 60 60 10 5 60 60 10 5

5 5 5 5

2

2 2

100 100 100 100

_d

5(7)

0

12(21) 13(21) 27(33) 21(22)

" percentage of shoot tips forming a callus; standard error in parentheses. Twelve and 24 shoot tips (3 per replicate) were used for each control and LN-treated group respectively. b percentage of growing shoot tips that form an elongating shoot; standard deviation in parentheses. ~ minutes of exposure to the stated percentage of PVS2. d not determined; pieces used to test different media for regeneration.

Table 3. Effect of preculture in ethylene glycol upon subsequent survival using vitrification of shoot tips from I. batatas PI 508515.

Days of preculture 0 2

Survival" Control Liquid nitrogen 83(8) 75(14) 100(0) 95(11)

" Percentage of shoot tips developing a callus. The exposure protocol was 60, 60, 10, 5, 5, and 1 minutes of exposure to 10, 20, 40, 60, 80 and 100% PVS2 respectively.

Folded tissue paper strips were used for exposing shoot tips to LN. This facilitated handling of the many shoot tips used in an experiment. Semen straws (0.25 ml) have been used for subsequent studies. The survival percentages and manner of growth observed are similar to those obtained using paper strips. We observed considerable variation in survival among experiments, whether using paper strips or semen straws. This also was observed with other species using two-step cooling procedures (Towill 1990b). No explanation is apparent. Increased variation for plantlet growth within a

treatment is common when sweetpotato plants are exposed to growth-inhibiting culture conditions (Jarret and Gawel 1991). The in vitro propagated plants utilized in this study were virus-free and uniform in age and size. In addition, the axillary buds used for dissection experiments were uniform in size. However, extended durations of in vitro culture decreased survival of cryopreserved Solanum tuberosum shoot tips (Harding et al. 1991). Additional experiments are needed to optimize time exposures to cryoprotectants in order to attain reproducible high levels of survival. Shoot tips of sweetpotato PI 290657 also survived LNexposure using vitrification. Survival of vitrificationsolution exposed and LN-treated samples was 91% and 26 %, respectively. Growth characteristics of treated shoot tips were similar to those described below for PI 508515. Vitrification solution-treated shoot tips, and shoot tips subsequently exposed to LN, grew considerably slower than untreated controls. The green coloration of the shoot tips was evident immediately following treatment but was lost within 24 h. Those shoot tip that were subsequently to grow regained a green color during the next 5 to 10 d. Growth of shoot tips on RM was characterized by the development of a slowly proliferating callus that encircled the explant. Untreated control-, vitrification solutiontreated control-, and LN exposed-shoot tips all exhibited this developmental pattern. Shoots, when present, arose from the central portion of the disc of callus (Figure 1). The callus usually originated from the bottom portion of the shoot tip in contact with the culture media (Fig. 2). Not all calli formed shoots within the 2 month observation period. However, a dark green region was observed in the top-center of all calli. This response, which varies with genotype, is typical of sweetpotato meristems cultured on MS with 0.5 mg/l BA (R.L. Jarret, unpublished). The regeneration of shoots from untreated controls, vitrification solution-treated controls and vitrified samples, was variable. For example, 100% of the shoot tips exposed to only 1.2M sucrose survived; however, subsequent shoot formation from these ranged from 42 to 100 %. Subculture of only the green central portion of the callus surrounding the original explant increased the frequency of shoot formation in both controls and in the LN-exposed samples. It was not determined whether shoots that did develop were axillary or adventitious in origin. Cryoprotectant-exposed and LN-treated shoot tips did regain their green color prior to callus proliferation. This suggests that shoots may arise from preexisting axillary or terminal meristems within the explants. Culture of fragments taken from the periphery of the callus disc (not containing the central dark green area) did not form shoots on RM. The effect of an MS-based medium (without growth regulators) on shoot tip recovery was examined. In general, a very low percentage of vitrification solution-

178 treated shoot tips survived. As was observed in potato (Towill 1983), the development of a suitable shoot tip recovery media is essential to minimize callus formation prior to shoot initiation in LN-treated shoot tips. As with any clonally propagated crop, germplasm of sweetpotato maintained in the field or greenhouse is susceptible to infection by numerous disease agents. In vitro maintenance permits retention of a disease-free state for short- and medium-term storage (Jarret 1990). However, long-term preservation is best accomplished using cryogenic storage. We have utilized micropropagated plants for these studies because of the axenie nature of the system. The development of cryopreservation using vitrification techniques should

augment existing maintenance strategies and, thus, enhance the availability of sweetpotato germplasm. To our knowledge, this is the first report of the successful cryopreservation of sweetpotato shoot tips. Induction of vitrification is a relatively simple procedure and cryopreservation of vitrified materials does not require a controlled cooling apparatus. Thus, these techniques can be applied at all locations where a suitable cryogen is available. Further studies are needed to identify factors affecting the high levels of within and between treatment variability for explant survival and shoot recovery, to determine the stability of glasses to nucleation and cracking at low temperatures, and to determine the applicability of these protocols to a wider array of genotypes. ACKNOWLEDGEMENTS. The authors acknowledge the expert

technical assistance of Genevieve Valdez. This work was supported in part by a gift from RJR-Nabisco.

REFERENCES

Fig. 1 Vitrification solution-treated (A) and vitrified (B) shoot tips from Ipomoea batatas PI 508515 after 8 weeks of culture. The callus is approximately 1 cm in diameter.

Fig. 2. Vitrified shoot tip from Ipomoea batatas PI 508515 after 2 weeks of culture. The callused shoot tip is approximately 2 mm in diameter.

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