Effective long-term storage methods for soft X-ray irradiated pollen, and the use of storage pollen 1

Transcription

1 Effective long-term storage methods for soft X-ray irradiated pollen, and the use of storage pollen 1 M. Akutsu *, D. Kami, and K. Sugiyama National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohiraku, Sapporo, Hokkaido, , Japan * Corresponding author masakoa@affrc.go.jp Keywords: Nitrogen, temperature, humidity Abstract An effective long-term storage method for active soft X-ray irradiated pollen for the production of seedless watermelon (Citrullus lanatus) in diploid plants by using gas treatments is described. Soft X-ray irradiated pollen stored under N 2 or CO 2 gas, or under a vacuum for 1 year had high pollen germination abilities. Also, pollen stored at -25ºC under N 2 gas for 1 year and mixed with an equal amount of Marriage powder had similar results in the fruit produced. This result indicated that Marriage powder was useful as a pollen extender medium for watermelon. Soft X-ray irradiated pollen was stored at -25ºC for about 1 week, after which the pollen was set in an incubator at 2, 25, 3, 35, or 4ºC under 5,, or 8 % relative humidity for 1 to 6 hours. It is suitable to use pollen that is stored at a low temperature, less than 3ºC, for less than 3 hours under less than 8 % humidity during pollination. INTRODUCTION Sugiyama and Morishita (2) produced seedless watermelons by pollination with soft X-ray irradiated pollen. Using irradiated pollen is advantageous because seedless watermelons can be produced by ordinary cultivation methods due to the use of common diploid plants. The mechanism of producing seedless watermelons using soft X-ray irradiated pollen was published in a previous paper (Sugiyama et al. 22). Mass production of seedless watermelon seed by this new method will require a large number of irradiated staminate flowers (pollen) prior to pollination. Therefore, it is necessary to use stored irradiated pollen and we need to determine an efficient method of storing irradiated pollen. Miyaji and Shirazaki (1977) reported that the best storage conditions for watermelon pollen were 5ºC at 2 to 4 % humidity, with the duration of viable pollen extending to days. Preservation methods by temperature and humidity control had tendency to decrease the germination rate rapidly. Therefore, it was supposed that it was improper to the maintenance of long-term pollen germination vigor in watermelon. Using organic solvents allowed to store pollen for 79 days (Sugiyama et al. 22). Until now, pollen storage by using organic solvent is the most suitable for long-term storage of watermelon pollen. But, we are interested in developing a simple and eco-friendly protocol for pollen storage which produces a stable germination ability after long-term storage because we want to transport and/or import pollen directly to producers. 1 Cucurbitaceae 28, Proceedings of the IX th EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May th,

2 In this study, we determined the most effective storage parameters with regard to duration of pollen viability, and we evaluated fruit quality after pollination with stored, and with soft X-ray irradiated pollen. We also checked the relationship between pollen germination ability and the time and temperature between terminating storage and use, on the assumption that farmers are using it in the field. MATERIALS AND METHODS Plant Materials and Cultivation methods The Fujihikari TR watermelon cultivar was used. Seeds were sown in pots in April, 27 in a greenhouse. Seedlings with 5 to 6 leaves were transplanted 5 cm apart in a bed (2.3m x 35m) in a greenhouse in May, 27. Flowers were pollinated starting at about the 15 th node of the lateral branches. Methods of soft X-ray treatments, packaging, storage of pollen, and measurements Watermelon cultivar Green Seeded seeds were sown as a source of pollen for the storage experiments before Fujihikari TR was sown. Staminate flowers of Green Seeded were harvested in the morning to obtain pollen that would be stored for 14, 28, 9, 18, and 365 days. The collected pollen was packed in paraffin paper and irradiated with Gy soft X-ray (Soft X-ray Unit OM-R, OHMIC Ltd.) at 14.5 Gy min -1. Irradiated pollen was packed with a vacuum packaging machine ( TOSPACK V-38G, TOSEI, Shizuoka, Japan), either with N 2, CO 2, or O 2 gas, or with air, or under vacuum, and stored at either 4 or -25ºC. The pollen stored under N 2 was mixed with an equal amount of pollen extender medium, Marriage Powder. At anthesis, they were artificially pollinated with stored pollen and re-covered to prevent contamination by normal pollen. The mature fruits were harvested and fruit weight, fruit shape, rind thickness and sugar content were investigated. Pollen was assayed for germination ability on germination medium before pollination. Comparison of storage methods for the time, temperature, and humidity Collected watermelon pollen was irradiated with soft X-rays, packed under vacuum, and then stored at -25ºC for about one week. After that, stored pollen was incubated at 2, 25, 3, 35, or 4ºC under 5,, or 8 % relative humidity for 1 to 6 hours. Pollen from each treatment was assayed for germination ability on germination medium. RESULTS AND DISCUSSION Long- term storage for soft X-ray irradiated pollen The percentage of the pollen which germinated was assayed on germination medium. Figure 1 illustrates the relationship between soft X-ray irradiated pollen storage conditions: -25ºC with N 2, CO 2, or O 2 gas; air; or under vacuum, and the storage period length. After a year of storage, the soft X-ray irradiated pollen placed under N 2 or CO 2 gas, or under a vacuum, had a high germination percentage. The aniline blue staining method was used to observe pollen tube elongation. These results for pollen elongation in the ovaries showed that pollen stored with N 2 elongated more than pollen stored under vacuum (data not shown). Thus, N 2 gas provides the best conditions for storing watermelon pollen. 366

3 Cultivation using long-term stored pollen mixed with Marriage powder as a pollen extender medium The pollen stored at -25ºC with N 2 gas for 1 year, and the pollen stored at - 25ºC with N 2 gas for 1 year and mixed with an equal amount of Marriage powder, were not significantly different in the weight, shape, rind thickness, flesh color, or sugar content (Brix) of the fruits they produced (Tab. 1). These results indicate that pollen stored at -25ºC with N 2 gas for 1 year had a sufficient ability to produce fruit set, and using an equal amount of Marriage powder as a pollen extending medium was just as useful as when the non-stored soft X-ray irradiated pollen was used (Tab. 1). Table 1. Fruit quality of seedless watermelon pollination with irradiated pollen in several conditions. Storage Fruits Fruit Rind Flesh Brix Treatment weight shape z thickness color y Temp Time (days) (kg) (mm) (%) Control ±2.6 x 1.2±. 17.9± ± ±1. Nitrogen -25 C ± ±. 17.± ± ±1.1 Nitrogen C ± ± ± ± ±1. M w z Ratio length/diameter y a* : Hue relates to red (+) green (-) color axes x Mean ± SE w When pollinating, pollen stored with nitrogen mixed with once amount of Marriage powder The influence of temperature and humidity conditions on the ability of stored pollen to germinate after storage Soft X-ray irradiated pollen was stored at -25ºC for about 1 week, after which the pollen was placed in an incubator at 2, 25, 3, 35, or 4ºC under 5,, or 8 % relative humidity for 1 to 6 hours (Fig. 2-4). When the pollen was incubated at less than 3ºC, up to a maximum of 3 hours, it had a similar pollen germination ability, but this depends on the humidity. Furthermore when pollen was incubated under 8 % humidity for 6 hours at 2ºC, the pollen germination percentage was more than half of that before treatment. These results indicate that for successful pollination it is best to use pollen which has been stored at a low temperature for less than 3 hours, under less than 8 % humidity. These results indicate that good seedless watermelon fruit can be produced in any season, and this storage method eliminates the need for organic solvents and chemicals, thus providing an eco-friendly storage system. 367

4 8 Percentage of germinating pollen (%) N2 O2 CO2 Vacuum air days 14 days 28 days 3 months 6 months 1year Periods for the storage Figure 1. Difference of pollen germination ability between term and condition for storage. Percentage of germinated pollen (%) Storage time (hours) Figure 2. Influence of storage time and temperature on the percentage of germination of soft X-ray irradiated pollen. 368

5 Percentage of germinated pollen (%) Storage time (hours) Relative humidity % Figure 3. Influence of storage time, temperature, and relative humidity ( %) on the percentage of germination of soft X-ray irradiated pollen. Pecentage of pollen germination (%) Storage time (hours) Relative humidity 8% Figure 4. Influence of storage time, temperature, and relative humidity (8 %) on the percentage of germination of soft X-ray irradiated pollen. 369

6 ACKNOWLEDGEMENT The authors are thankful to Mr. Y. Horiuchi, Mrs. S. Ando, Mrs. R. Hirama and Mrs. A. Tanii of National Agricultural Research Center of Hokkaido Region of experimental helps. And we did this research in Research and Development Program for New Bio-Industry Initiatives. Literature cited Miyaji R, Shirazawa M (1977) Influence of temperature and the humidity for melon s and watermelon s pollens on their storage. Bulletin of the Kagoshima Agric Exp Stn (in Japanese). Sugiyama K, Morishita M (2) Production of seedless watermelon using soft-x-irradiated pollen. Scientia Horticult 84: Sugiyama K, Morishita M, Nishino E (22) Seedless watermelons produced via soft-xirradiated pollen. HortScience 37: Sugiyama M, Sajata Y, Kitadani E, Morishita M, Sugiyama K (22) Pollen storage for production of seedless watermelon (Citrullus lanatus) using soft X-ray irradiated pollen. Acta Horticult 588: