A PRELIMINARY STUDY ON CRYOPRESERVATION PROTOCOL APPLICABLE TO ALL TYPES OF DIOSPYROS KAKI THUNB.

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1 Article DOI: /v A&EB A PRELIMINARY STUDY ON CRYOPRESERVATION PROTOCOL APPLICABLE TO ALL TYPES OF DIOSPYROS KAKI THUNB. Y.L. Niu 1,2, Y.F. Zhang 1, Q.L. Zhang 1, Z.R. Luo 1 1 Huazhong Agricultural University, Key Laboratory of Horticultural Plant Biology Affiliated to Ministry of Education, Wuhan, China 2 Chinese Academy of Science, Lushan Botanical Garden, Lushan, Jiangxi, China Correspondence to: Qinglin Zhang zhangqinglin@mail.hzau.edu.cn ABSTRACT Diospyros kaki Thunb. was divided into four types based on astringency in the fruit at harvest, presence of seed, and flesh colour. The factors that affected cryopreservation of D. kaki Thunb. were studied by modified droplet vitrification: preculture condition and PVS 2 treatment time. After cryopreservation, different concentration of Pluronic F-68 in the regenerating medium was also investigated. A suitable protocol was established: shoot tips of axillaries were precultured on MS (1/2) medium with 0.5mol/L sucrose and 10g/L Pluronic F-68 for 5 days, which were loaded in loading solution for 20 min at 20 o C, and incubated in PVS 2 for 1.5 h at 0 o C prior to direct plunge into liquid nitrogen. After rapid thawing at 40 o C warm water bath and rinsed, the shoot tips were plated on the regeneration medium supplemented with 1.0mg/L TDZ, 0.6g/L PVP, 0.1% Pluronic F-68, 30g/L sucrose and 7g/L agar for one week in dark prior to exposure to normal conditions. Using this procedure in cryopreservation of D. kaki Thunb., the best regeneration rate was 80%, the average rate was 73% and the difference of four types was not significant at P=0.05. Biotechnol. & Biotechnol. Eq. 2010, 24(3), Keywords: Diospyros kaki Thunb., modified droplet vitrification, cryopreservation Introduction China is one of the primary origins of Diospyros kaki Thunb. and has abundant germplasms of D. kaki Thunb. At present, most of these germplasms have been conserved in situ. A germplasm collection of D. kaki Thunb. has been established in National Persimmon Germplasm Repository which is affiliated with Northwest Sci-tech University of Agriculture and Forestry, Yangling, Shaanxi Province (37). In the Repository there are more than 550 accessions conserved in the field. Their conservation is time-, space- and labour-consuming and bears the risk of biological and abiotic stress or even extinction. So, cryopreservation in liquid nitrogen has been the most promising choice for long-term storage of germplasm. D. kaki Thunb. can be classified into four types depending on the astringency at maturity and the pollination effect, i.e. the presence or absence of seeds in the flesh: pollination constant non-astringent (PCNA), pollination constant astringent (PCA), pollination variant non-astringent (PVNA) and pollination variant astringent (PVA) type. Matsumoto et al. (20) firstly attempted to cryopreserve dormant shoot tips of four types of D. kaki Thunb. by vitrification. The survival rate of dormant shoot tips of 16 accessions native to China, Japan and South Korea reached 89%, while the survival rate of accessions native to subtropical origin was only 37-66% and the difference between the tested genotypes was very significant. Later, Zhang and Luo (38) and Ai and Luo (2), respectively, cryopreserved some accessions of D. kaki Thunb. by encapsulation-vitrification and by vitrification. However, the above materials were not all-inclusive. Ai and Luo (4) cryopreserved the dormant shoot tips of PCNA type and dateplum by vitrification, but the regeneration rate was only 30%. Ai and Luo (5) also conserved three other types except for PVNA type under slow growth conditions and the survival rate was more than 60%, but this method needs more complicated steps. Therefore, if we can establish an economical and practical cryopreservation method, it can be applied to all types of D. kaki Thunb. and if each type is not significantly different for survival and regeneration rate, it will improve the efficiency of germplasm conservation. In this study, all four types were cryopreserved using modified droplet vitrification and the factors that affected the cryopreservation were studied in order to find an efficient method applicable to all four types. Materials and Methods Plant materials Shoot tips from dormant axillary vegetative buds of six varieties of D. kaki Thunb. were served as experimental materials including two PCNA types - Eshi 1 and Huashi 1 and one PCA type- Mopanshi originated in China, as well as one PCNA type- Youhou, one PVNA type- Zenjimaru and one PVA type- Hiratanenashi originated in Japan. In mid-january 2006, dormant twigs with axillary vegetative buds were collected from Persimmon Repository, Huazhong Agricultural University, Wuhan, China. These twigs were wrapped with gauze and placed in 4 o C refrigerator for backup Biotechnol. & Biotechnol. Eq. 24/2010/3

2 Cryopreservation procedures and culture conditions In present study, droplet vitrification (1, 13, 16, 17, 18, 21, 24, 28, 29, 34) combined with encapsulation was carried out. First, shoot tips were precultured in 0.5 mol/l sucrose medium for 5 days. Second, shoot tips were encapsulated in alginate gel beads and suspended in calcium-free MS medium supplemented with 3% (w/v) Na-alginate plus 0.4 M sucrose solution. Next, shoot tips were transferred to a droplet of PVS 2 solution on a strip of aluminum foil around 0 o C during the manipulation. After the PVS 2 treatment, the aluminum strip was plunged into liquid nitrogen. After thawing and unloading, the shoot tips were transferred onto regeneration medium. The first week on the regeneration medium was always in the dark. Both post-thaw survival and post-thaw regeneration were determined after 4-6 weeks. The basic protocol was shown in Fig. 1. Fig. 1. The cryopreservation protocol of modified droplet vitrification Statistical analysis of results Results are presented in mean percentages with their standard error. The rate of survival and regeneration after cryopreservation were subjected to analysis of variance (ANOVA), and the means were compared by the least significant difference (LSD) test (35). Results and Discussion Effect of preculture condition on cryopreserved dormant vegetative shoot tips of D. kaki Thunb. The purpose of preculture treatment is to increase the cytoplasmic concentration of antifreeze and antidehydration strength and to facilitate vitrification formation in the process of cooling. Preliminary test results showed that MS (1/2) medium containing 0.5mol/L sucrose and 10g/L Pluronic F-68 was the best preculture medium. As shown in Fig. 2, the regeneration rate increased within a certain incubation time, but when the time extended more than 1.5 h the regeneration rates declined. The reason may be that the water content of dormant bud meristem cell decreased, soluble sugar and other protective substances content increased when the concentration of Pluronic F-68 in the medium increased and the culture time extended. Thus, the osmotic potential of the cells was increased and freezing point was reduced, so the survival rate was increased. However, if sucrose concentration is too high, it will lead to excessive cell dehydration and osmotic stress. Biotechnol. & Biotechnol. Eq. 24/2010/3 Fig. 2. Effect of preculture time on cryopreserved dormant vegetative shoot tips of D. kaki Thunb. Effect of duration of exposure to PVS 2 on cryopreserved dormant vegetative shoot tips of D. kaki Thunb. The PVS 2 treatment at 0 o C helps to improve the conservation effect. To determine the optimal time of exposure to PVS 2, shoot tips were treated for min with 60% PVS 2 at 0 o C prior to a plunge in LN. Three tested genotypes without PVS 2 treatment did not survive. First, the survival rate was increased when the treatment time was from 0 h to 1.5 h, but when the duration prolonged, survival rate decreased. The trends for regeneration rate were same as these for survival. As shown in Fig. 3, the regeneration rate reached the maximum when the treatment time was 1.5 h. This result indicated that the shoot tips were injured in a greater extent due to an overexposure to the PVS 2 solution during the dehydration. On the contrary, the treatment time was too short for inadequate dehydration and in order to reach vitrification, and thus resulted in reduced regeneration rate. Fig. 3. Effect of duration of exposure to PVS 2 on cryopreserved dormant vegetative shoot tips of D. kaki Thunb. Effect of Pluronic F-68 on cryopreserved dormant shoot tips of D. kaki Thunb. Appropriate media can stimulate cell division and allow shoot tips to recover from freezing injury. Pluronic F-68 added to 1961

3 the culture medium after cryopreservation improved the survival rate. The trend of survival rate was the same as that for the regeneration rate, as shown in Fig. 4. However, when concentration of Pluronic F-68 exceeded a certain range, the regeneration rate was reduced which was also stated by Zhao et al. (39). The reason was that the concentration of Pluronic F-68 was so much higher and the remaining time in the medium was so much longer, that polyphenol oxide was activated and oxidized to quinines that were dispersed in the medium. Thus it affected the recovery of shoot tips and shoot elongation. Fig. 4. Effect of Pluronic F-68 on cryopreserved dormant shoot tips of D. kaki Thunb. The dormant shoot tips cryopreserved of different types of D. kaki Thunb. The modified droplet vitrification had application in four types of D. kaki Thunb. The results were summarized in Table 1: the regeneration rates of non-astringent persimmon types were higher than that of astringent persimmon. But these four types were not significantly different (P=0.05). The shoot tips were kept in the dark for one week prior to exposure to the light (12 h photoperiod cycle). The new leaves can be found after cryopreservation in D. kaki Eshi 1 after two weeks and develop into plantlets after 12 weeks (Fig. 5). TABLE 1 The survival and regeneration rate of cryopreserved dormant vegetative shoot tips of different types of D. kaki Thunb. Genotype Astringent Survival rate Regeneration type (%) rate (%) Mopanshi PCA 81.5±1.83 a 70.4±1.83 a Eshi 1 PCNA 88.9±0 a 79.6±3.70 a Huashi 1 PCNA 79.6±3.70 a 72.2±3.20 a Zenjimaru PVNA 81.5±4.90 a 69.5±2.75 a Hiratanenashi PVA 79.6±1.83 a 68.5±1.83 a Youhou PCNA 87.0±4.88 a 77.8±5.53 a Note: (1) The data is mean ± standard error; (2) Means within columns followed by the same letter are not significantly different by least significant difference (LSD) test (P=0.05); (3) Eighteen shoot tips are tested for each of three replications Fig.5. Plantlet regeneration of cryopreserved dormant shoot tips in D. kaki cv. Eshi 1 D. kaki Thunb. has been conserved in many ways. For example, National Persimmon Germplasm Repository (37), Fukui (9) and Ai and Luo (3) conserved some of D. kaki Thunb. by low temperature storage. These conventional methods have many shortcomings. Field collections are exposed to natural disasters and attacked by pathogens and pests. In vitro cultures also face many risks such as contamination during frequent subculture and soma-clonal variations during conservation. In recent years, cryopreservation in liquid nitrogen (LN) has been a prospective conservation method due to experimental materials in minimal space and few variations (26, 27). Matsumoto et al. (19, 20) and Ai and Luo (2, 4) and Zhang and Luo (38) cryopreserved one or some astringent types, but the above researches did not obtain very high regeneration rates. In the present study, modified droplet vitrification can handle simultaneously a large number of materials, and achieve high regeneration rate. The method would be an ideal cryopreservation technique (22). Moreover, the results for all four types of D. kaki Thunb. were not significantly different. In general, the survival of this organism is very difficult in the absence of cryoprotective substance in the experiment of low-temperature. The use of vitrification solution is the key step of the successful cryopreservation. PVS 2 treatment time is different because of various materials and cryopreservation methods (12, 13, 15, 30, 33). The treatment time of encapsulated explants should be usually longer. This study confirmed that treatment time of 1.5 h with PVS 2 was better than 0.5 h and 1 h, but the effect of cryopreservation was reduced when the processing time was 2 h. This may be related to the concentration of vitrification solution. Xia (32), 1962 Biotechnol. & Biotechnol. Eq. 24/2010/3

4 Wowk et al. (31), Zhao et al. (39), Kami (14) and Esther et al. (8) improved the survival rate respectively by adding some antifreeze protein or vitamin or cryoprotectant substance to their vitrification solution. This is probably done in order to reduce the toxicity of the vitrification solution through a diluting solution and the above additions may also present an obstacle to the formation of intracellular ice crystals during freezing and thawing. Because crystal formation can cause irreversible damage to cell membranes and thus destroy their semi-permeability. Pluronic F-68 is a non-ionic surface active agent and can promote cell differentiation, so it has been widely used in animal and plant tissue culture systems (6, 23). Zhao et al. (39) and Pennycooke et al. (25) significantly improved the survival rate in the cryopreservation of potato using low concentration of Pluronic F-68. This study showed that by adding 0.1% Pluronic F-68 to the regeneration medium best results were achieved. Matsumoto et al. (20) cryopreserved all astringent types of D. kaki Thunb. and the result was different between all tested genotypes. Benelli et al. (7), Zhang and Luo (38) and Ai and Luo (2, 4, 5) studied the cryopreservation of D. kaki Thunb., but the materials referred only to some astringent types. Our results showed that the survival and regeneration rates were not significantly different between all four types by modified droplet vitrification. Papers dealing with the cryopreservation of large numbers of accessions of the same plant species are very scarce. The major merit of our study was not only to obtain the higher respectively survival and regeneration rate fit for all types but also to develop an easy protocol to deal with a large number of accessions. The study is the first report on a cryopreservation protocol applicable to all types of D. kaki Thunb. It could provide a theoretical basis for the establishment for cryopreserved gene bank of D. kaki Thunb. core collection (36). A cryopreserved gene bank of European Elms core collection has been established (11). Conclusions Papers dealing with the cryopreservation of large numbers of accessions of the same plant species are very scarce. The major merit of our study was not only to obtain the higher respectively survival and regeneration rate fit for all types but also to develop an easy protocol to deal with a large number of accessions. The study is the first report on a cryopreservation protocol applicable to all types of D. kaki Thunb. It could provide a theoretical basis for the establishment for cryopreserved gene bank of D. kaki Thunb. core collection (36). A cryopreserved gene bank of European Elms core collection has been established (11). REFERENCES 1. Agrawal A., Swennen R., Panis B. (2004) Cryo-Lett., 25, Ai P.F. and Luo Z.R. (2003) Sci. Agr. Sin., 36, Ai P.F. and Luo Z.R. (2004) Acta Hort. Sin., 31, Biotechnol. & Biotechnol. Eq. 24/2010/3 4. Ai P.F. and Luo Z.R. (2004) Sci. Agr. Sin., 37, Ai P.F. and Luo Z.R. (2005) Acta Hort., 685, Anthony P., Lowe K.C., Power J.B., Davey M.R. (1999) Cryobiol., 35, Benelli C., Carlo A.D., Giordani E., Pecchioli S., Bellini E., Kochanova Z.V. (2009) Acta Hort., 833, Esther E.U., Scott W.L., Maret G.T., Barbara M.R. (2010) Plant Cell Rep., 29, Fukui H. (1994) Combined Proc. Int. Plant Propagators Soc., 44, Halmagyi A. and Pinker I. (2006) Plant Cell Tiss. Org. Cult., 84, Harvengt L., Meier-Dinkel A., Dumas E., Collin E. (2004) Can. J. For. Res., 34, Hirano T., Godo T., Miyoshi K., Ishikawa K., Ishikawa W., Mii M. (2009) Plant Biotechnol. Rep., 3, Hong S.R. and Yin M.H. (2009) Plant Cell Tiss. Org. Cult., 99, Kami D., Kasuga J., Arakawa K., Fujikawa S. (2008) Cryobiol., 57, Kami D., Shi L., Sato T., Suzuki T., Oosawa K. (2009) Sci. Hort., 120, Keller E.R.J. (2005) Cryo-Lett., 26, Kim H.H., Yoom J.W., Park Y.E., Cho E.G. (2006) Cryo- Lett., 27, Leunufna S. and Keller E.R.J. (2003) Plant Cell Rep., 21, Matsumoto T., Kurahashi T., Niino T. (2004) Plant Biotechnol., 21, Matsumoto T, Mochida K, Itamura H, Sakai A. (2001) Plant Cell Rep., 20, Mix-Wagner G., Conner A.J., Cross R.J. (2000) New Zeal. J. Crop Hort., 28, Niu Y.L., Luo Z.Y., Zhang Y.F. (2009) J. Wuhan Bot. Res., 27, Palasz A.T., Thundathil J., de la Fuente J., Mapletoft R.J. (2000) Cryobiol., 41, Panis B., Piette B., Swennen R. (2005) Plant Sci., 168, Pennycooke J.C. and Towill L.E. (2000) Plant Cell Rep., 19, Sakai A. (1995) In: Cryopreservation of Plant Germplasm (Y.P.S. Bajaj, Ed.), Springer-Verlag, Berlin Heidelberg, Sakai A. (1997) In: Conservation of Plant Genetic Resources in Vitro (M.K. Razdan, E.C. Cocking, Eds.), Science Publishers, New Hampshire, Sant R., Panis B., Taylor M., Tyagi A. (2008) Plant Cell Tiss. Org. Cult., 92, Towill L E. and Bonnart R.C. (2003) Cryo-Lett., 24,

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