Cryopreservation of Papaya (Carica papaya L.) Seed: Overcoming

Cryopreservation of Papaya (Carica papaya L.) Seed: Overcoming Dormancy and Optimizing Seed Desiccation and Storage Conditions S.E. Ashmore, R.A. Drew, C. O'Brien and A. Parisi Centre for Forestry and Horticulture Research Griffith University, Nathan, Queensland 4111 Australia Keywords: seed dormancy, seed desiccation, seed storage, gibberellic acid, potassium nitrate Abstract Protocols for efficient storage and germination of fresh and desiccated seeds of Carica papaya are needed for improved management of seed stocks by the industry. Two genotypes of papaya of importance to Australia were investigated: the tropical cultivar ‘Solo’ and a Queensland line ‘007’. Germination of fresh seed was close to zero, but high proportions of the seeds were found to be viable by tetrazolium staining: 93% for ‘Solo’ and 94% for ‘007’. We aimed to overcome the dormancy blocking by pre-soaking seeds in gibberellic acid (for up to 60 min) or potassium nitrate (for up to 24h). The most effective treatments were: 2 mM GA3 for 30 min; and 2 or 3h in 0.25 M KNO3. All treatments resulted in 61-65% germination. Seeds of both cultivars tolerated drying to low moisture contents, but required GA3 pre-treatment to stimulate 30-60% germination; KNO3 pre-treatment was much less effective in stimulating germination in desiccated seeds. Desiccated seeds (5, 15 and 30% RH) germinated (>70%) after storage in liquid nitrogen(-196°C) for 3 months if GA3 pre-treatment was applied. Recovery from storage at the standard genebank storage temperature of -20°C was very low (1-36%). These results provide valuable information for improved seed handling practices in the papaya industry. INTRODUCTION The fruit papaya (Carica papaya L.) is one of the most popular crops in many countries of subtropical and tropical countries (Litz, 1989). It is exclusively raised from seed for commercial propagation and the supply of viable, readily germinated seeds (fresh and stored) is thus critical to the industry. However, stored seed can be difficult to germinate and seed germination is commonly slow and sporadic (Perez et al., 1980). Fresh seed may also be difficult to germinate as a result of dormancy, although this is reputed to occur less in cultivated papaya than in wild populations (Paz and VázquezYanes, 1998). Wood et al. (2000) have observed the induction of dormancy upon desiccation of papaya seeds. Papaya seeds have generally been classified as having intermediate seed storage capacities and storage for periods greater than 5 years is difficult (Ellis et al., 1991). Loss of viability has been reported at moisture contents below 8 to 10% (Ellis et al., 1991), although viability has been reported by Magill et al. (1994) when seed was desiccated to 5% moisture content. Previous reports have described a range of treatments to improve germination in papaya. These have included removing the sarcotesta (Gherardi and Valio, 1976; Perez et al., 1980; Reyes et al., 1980); leaching chemical inhibitors from the endosperm (Bertocci et al., 1994), the sarcotesta and seed coat (Chow and Lin, 1991); soaking seeds in a GA3 solution (Furutani and Nagao, 1987; Perez et al., 1980; Yahiro and Oryoji, 1980; Nagao and Furutani, 1986), the application of KNO3 solution (Furutani and Nagao, 1987) and the use of heat shock treatment in desiccated seed (Wood et al., 2000). The objective of this research was to investigate and enhance seed germination in two commercially grown genotypes of importance in Queensland and the effects of GA3 and KNO3 on breaking dormancy and improving germination of fresh and desiccated seed both pre- and post-storage at a range of temperatures. Outcomes of this work were aimed at improving germinability and storability of seed lots to assist the papaya industry. Proc. 1st IS on Biotechnol. of Fruit Species Eds.: M.-V. Hanke et al. Acta Hort. 839, ISHS 2009

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MATERIALS AND METHODS Plant Material For all experiments, mature seeds from two genotypes of Carica papaya were selected; ‘Solo’ and ‘007’. The ‘Solo’ cultivar, originally from Hawaii, is a red-fleshed hermaphrodite papaya, which grows well in North Queensland and ‘007’ is a yellowfleshed dioecious papaya, which grows well in Central Queensland. The seed was extracted from the fruit and the sarcotesta was removed by gently rubbing the seed in a sieve under running water. After the sarcotesta was removed the seeds were washed with distilled water and blotted dry with paper towel prior to treatment. All seeds used for this experiment were harvested in June and July (Australian winter period). Seed Viability Testing A random sample of 25 seeds each of ‘Solo’ and ‘007’ varieties were viability tested using 2,3,5-Triphenyltetrazolium chloride (TTC) (International Seed Testing Association, 1985). The test solution was a 0.5% solution of TTC at pH 6.5-7.0. Seed viability was assessed after incubation of cut seeds at 35°C for 1h. Germination of Fresh Seed Germination testing was undertaken by placing seeds on wet filter paper in Petri dishes in an environmental chamber at 35±2ºC in the dark. Seeds were pre-soaked in aqueous solutions of GA3 at a range of concentrations (0, 0.5, 1, 2 mM) for 0, 15, 30 or 60 min prior to germination testing or in aqueous solutions of KNO3 at a range of concentrations (0, 0.25, 0.5, 1, 1.5 M) for 0, 15, 30, 60 min, 2, 3, 6, 14 or 24h prior to germination testing. Ten replicates of 25 seeds were used for each germination treatment and germination was recorded (days 7-14 post incubation) as the percentage of seeds showing radicle emergence. Germination of Desiccated Seed Washed seeds were desiccated to eRH (equilibrium Relative Humidity) levels between 100 and 10% prior to germination testing. Seeds were germination tested as described above, including pre-soaking in 2 mM GA3 for 15 or 30 min, or 0.25 M KNO3 for 2 or 3h. Control samples were not pre-soaked. Seed Storage Following desiccation, seeds were stored in sealed plastic coated aluminium bags at a range of temperatures (-196, -80, -20, +4, +15°C) for a period of 3 months. Seeds were germination tested as described above after pre-soaking in 2 mM GA3 for 15min. RESULTS AND DISCUSSION Seed Viability High proportions of the embryos from both seed lines stained uniformly using the TTC test; 92.8% for ‘Solo’ and 94% for ‘007’. Germination of Fresh Seed Mean germination percentages of fresh seeds were very low (0 and 2.4% for ‘Solo’ and ‘007’, respectively) despite the high levels of seed viability determined by TTC testing. This suggested that fresh seeds were dormant and is consistent with at least one previous report in cultivated papaya (Paz and Vánquez-Yanes, 1998). The mean germination percentage increased above control levels with GA3 pretreatment at all concentrations and all durations for both varieties, although the response was greatest in ‘Solo’ seeds (Fig. 1). The highest mean percentage of germination was 61.2 for ‘Solo’ after pre-treatment at 2 mM GA3 for 30 min and 38 for ‘007’ after pretreatment at 2 mM GA3 for 15 min (Fig. 1). The mean percentage of germination 230

increased above control levels for both varieties after pre-treatment in either 0.25 M or 0.5 M KNO3 (Fig. 2). The highest mean percentage of germination was seen after pretreatment at 0.25 M KNO3 for 2 or 3h (64 and 65.2% for ‘Solo’, 58 and 64% for ‘007’). Germination of Desiccated Seed Desiccation of seeds of both varieties did not improve germination percentages in control samples and KNO3 pre-treatments were ineffective in enhancing germination percentages above control levels (Fig. 3). Germination was achieved after pre-treatment in 2 mM GA3 for 15 or 30 min for seed of both varieties desiccated to all levels between 70 and 10% RH. Mean germination percentages dropped to between 30 and 60 at RH below 25% for ‘Solo’ seeds and were lower than this (15-30%) for ‘007’ seeds desiccated below 70% RH (Fig. 3). Germination Post Storage Table 1 shows the germination of seeds at a range of RH levels after storage for 3 months. It can be seen that the mean percentage of germination after cryo-storage for 3 months is 70% or greater, at least for seeds at RH of 5 or 30%. The lowest mean percentage of germination (1-36%) is seen in seeds stored at the standard seed banking temperature of -20°C. Whilst germination percentages are relatively high after storage at either -80°C or at +15°C, it is suggested that long-term storage of papaya seed would be best in LN. CONCLUSION This study has demonstrated that fresh seeds of papaya cultivars can be dormant when freshly harvested, at least in seed harvested in the Australian winter. This dormancy is likely to be related to the light requirement demonstrated in wild papaya (Paz and Vánzquez-Yanes, 1998) as it could be broken to give acceptable levels of germination when either GA3 or KNO3 were used; KNO3 gave the highest level of germination for ‘007’ seeds and may be the preferred treatment for application in the industry. However, once seeds were desiccated, KNO3 was no longer effective in promoting germination in either ‘Solo’ or ‘007’ seeds, but GA3 still promoted germination at all desiccation levels and was particularly effective in ‘Solo’ seeds. Some reduction in germination percentage was seen as seeds were desiccated below 25% RH for ‘Solo’ and 70% RH for ‘007’, but the results support the findings of Wood et al. (2000), that papaya seeds are tolerant to the desiccation required for seed storage. Desiccated seeds were germinated to high levels (>70%) following cryo-storage for 3 months, compared to storage for the same period at the standard temperature for seed banking, -20°C. This data furthers our knowledge of papaya seed germination and storage and provides valuable information for application in long-term conservation and for management of seed stocks for the industry. Literature Cited Bertocci, F., Vogler, J.N. and Drew, R.A. 1994. Influence of Culture Media on the Germination of Papaya (Carica papaya) Whole Seeds, Mature and Immature Embryos. VIIIth Int. Cong. Plant Tissue and Cell Culture S1-161, p.46. Chow, Y.J. and Lin, C.H. 1991. p-Hydroxy benzoic Acid as the Major Phenolic Germination Inhibitor of Papaya Seed. Seed Sci. Technol. 19:167-174. Ellis, R.H., Hong, T.D. and Roberts, E.H. 1991. Effect of storage temperature and moisture on the germination of papaya seeds. Seed Science Research 1: 69-72. Furutani, S.C. and Nagao, M.A. 1987. Influence of Temperature, KNO3, GA3 and Seed Drying on Emergence of Papaya Seedlings. Sci. Hort. 32:67-72. Gherardi, E. and Valio, I.F.M. 1976. Occurrence of Promoting and Inhibitory Substances in the Seed Arils of Carica papaya L. J. Hort Sci. 51:1-4. International Seed Testing Association. 1985. Handbook on Tetrazolium Test, International Seed Testing Association. Zurich, Switzerland. Litz, R.E. 1989. Papaya. In: Handbook of Plant Cell Culture V.2, 1989. Macmillan 231

Publishing Company. New York. USA. Magill, W., Deighton, N., Pritchard, H.W., Benson, E.E. and Goodman, B.A. 1994. Physiological and biochemical studies of seed storage parameters in Carica papaya. Proc. Royal Soc. Edinburgh 102B:239-442. Nagao, M.A. and Furutani, S.C. 1986. Improving Germination of Papaya Seed by Density Separation, Potassium Nitrate and Gibberellic Acid. Hort Science 21:1438-1449. Paz, L. and Vazquez-Yanes, C. 1998. Comparative Seed Ecophysiology of Wild and Cultivated Carica papaya Trees from a Tropical Rain Forest Region in Mexico. Tree Physiology 18:227-280. Perez, A., Reyes, M.N. and Cuevas, J. 1980. Germination of Two Papaya Varieties: Effect of Seed Aeration, K-Treatment, Removal and Sarcotesta, High Temperature, Soaking in Distilled Water, and Age of Seed. J. Agric. Univ. Puerto Rico 64:173-180. Reyes, M.N., Perez, A. and Cuevas, J. 1980. Detecting Endogenous Growth Regulators on the Sarcotesta, Endosperm and Embryo by Paper Chromatography on Fresh and Old Seeds of Two Papaya Varieties. J. Agr. Univ. Puerto Rico 64:164-172. Wood, C.B., Pritchard, H.W. and Amritphale, D. 2000. Desiccation-induced Dormancy in Papaya (Carica papaya L.) Seeds is alleviated by Heat Shock. Seed Science Research 10:135-145. Yahiro, M. and Oryoji, Y. 1980. Effects of Gibberellin and Cytokine Treatments on the Promotion of Germination in Papaya, Carica papaya L., Seeds. Mem. Fac. Agric. Kagoshima Univ. 16:45-51.

Tables

Table 1. Mean percentage germination (± SEM) of C. papaya ‘Solo’ seeds following 3 months storage at a range of relative humidities (%RH) and a range of temperatures. Results are based on 10 replicates of 25 seeds in each case. % RH 5 15 30

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-196°C (LN) 80±2.8 10±2.3 71±4.6

Temperature of Storage (°C) -80°C -20°C +4°C 74±3 36±3.2 92±1.2 39±5.7 1±0.5 47±3.9 54±2.2 2±1.6 10±1.4

+15°C 57±3.3 62±3.4 61±3.6

Figures a 70

% Germination

60 50 40 30 20 10 0 Control

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Time 0.5mM 1mM 2mM

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Fig. 1. Mean percentages of germination of fresh seed of (a) solo and (b) 007 varieties of Carica papaya after pre-treatment for various times in a range of concentrations of GA3. Each data point is the mean of 10 replicates of 25 seeds. Error bars are standard errors of the means (SEM).

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a 80 70

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Fig. 2. Mean percentages of germination of fresh seed of (a) solo and (b) 007 varieties of Carica papaya after pre-treatment for various times in a range of concentrations of KNO3. Each data point is the mean of 10 replicates of 25 seeds. Error bars are standard errors of the means (SEM).

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a 90 80 % Germination

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Fig. 3. Mean percentages of germination of fresh seed of (a) solo and (b) 007 varieties of Carica papaya dried to a range of eRH levels after pre-treatment with GA3 and KNO3. Each data point is the mean of 10 replicates of 25 seeds. Error bars are standard errors of the means (SEM).

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