Ultralow-temperature preservation method for grapefruit germplasm pollen

[0049] Example 1 The effect of thawing method and germination medium on pollen germination rate after ultra-low temperature storage

[0050] 1. Experimental materials

[0051] The materials used in the experiment are the three pomelo germplasm pollens of "Zuoshiyou", "Kuiyou" and "Beizhuanyou" provided by the National Fruit Tree Germplasm Citrus Nursery (Chongqing).

[0052] 2. Experimental methods and specific steps

[0053] -Pollen drying treatment: The collected anthers are dried under an incandescent lamp (temperature is about 30°C), and dried to a water content of 10-20%.

[0054] —Storage in sealed packaging with liquid nitrogen: After drying the pollen properly, put it into a 1.8ml cryotube (Nunc, USA) and put it in liquid nitrogen (CBS S3000-AB SERIES) for storage.

[0055] —Thawing: remove the cryotube containing pollen from the liquid nitrogen tank and thaw it in two ways: (1) Rinse and thaw with tap water at 25°C, the time is about 15min; (2) quickly thaw in the 37°C water bath, the time is about For 5min.

[0056] -Pollen germination statistics in vitro after cryopreservation: adopt hanging drop germination method or solid medium germination method to carry out in vitro germination culture on pollen in vitro culture medium. The specific pollen in vitro culture medium formula is 100~150g/L sucrose , 0.1g/L boric acid, 0.1g/L potassium nitrate, 0.3~1.0g/L calcium nitrate, 0.0~0.3g/L magnesium sulfate, 0~10g/L agar (in vitro germination medium number is M2, M3, M4, and M5, see Table 1). In vitro pollen culture was cultured overnight at 25°C in the dark. A microscope (OLYMPUS SZ×16) was used for statistics and photography of pollen germination rate in vitro. In statistics, pollen tube length exceeding half the diameter of pollen grains is regarded as viable pollen. 4 fields of view are randomly selected each time, with an average of about 100 pollen per field of view, and 4 replicates for each group to calculate the pollen germination rate.

[0057] Table 1 Formulation of pollen germination medium

[0058]

[0059] 3. Test results

[0060] Experiments have shown that the method of thawing and the composition of the recovery medium have a significant effect on the in vitro germination rate of pomelo germplasm pollen stored in ultra-low temperature. figure 1 As shown, the samples of Zuoshi pomelo germplasm with pollen water content of 20%, after being stored in liquid nitrogen, thawed at 37°C, in 100-150g/L sucrose, 0.1g/L boric acid, 0.1g/L potassium nitrate, 0.3 -1.0g/L calcium nitrate, 0.0-0.3g/L magnesium sulfate, 10g/L agar in vitro germination medium (M3 and M5), the average germination rate of pollen in vitro is 32-40%; Kuiyou germplasm A sample with a pollen water content of 10%, stored in liquid nitrogen and thawed at 25°C, contains 150g/L sucrose, 0.1g/L boric acid, 0.1g/L potassium nitrate, 1.0g/L calcium nitrate, 0.3g/L L magnesium sulfate, 0-10g/L agar in vitro germination medium (M2 and M3 medium), the average germination rate of pollen in vitro is 39-43%; samples with pollen water content of 10% in Beizhuan pomelo germplasm After being stored in liquid nitrogen, it contains 150g/L sucrose, 0.1g/L boric acid, 0.1g/L potassium nitrate, 0.3-1.0g/L calcium nitrate, 0.2-0.3g/L magnesium sulfate, 0-10g/L On agar in vitro germination medium (M3 and M4 medium), after thawing at 37°C, the average germination rate of pollen in vitro is 34-42%.

[0061] Example 2 Effect of rewetting on the germination rate of grapefruit pollen

[0062] 1. Experimental materials

[0063] The varieties used in the experiment were the pollen of two grapefruit germplasms, "Zuoshiyou" and "Kuiyou" provided by the National Fruit Tree Germplasm Citrus Nursery (Chongqing).

[0064] 2. Experimental methods and specific steps

[0065] -Pollen drying treatment: The collected anthers are dried under an incandescent lamp (temperature is about 30°C), and dried to a water content of 10-20%. Three samples of "Left Pomelo-Water Content 10%", "Left Pomelo-Water Content 20%" and "Kui Pomelo-Water Content 10%" were obtained.

[0066] —Storage in sealed package with liquid nitrogen: After the pollen is properly dried, put it into a 1.8ml cryotube (Nunc, USA) and put it into liquid nitrogen (CBS S3000-AB SERIES) for storage.

[0067] —Thawing: remove the cryotube containing pollen from the liquid nitrogen tank and thaw it in two ways: (1) Rinse and thaw with tap water at 25°C, the time is about 15min; (2) quickly thaw in the 37°C water bath, the time is about For 5min.

[0068] —Remoisture: Use a dissecting needle to take 10 mg of pollen from the cryotube and place it in an ampere bottle, place it in a closed container containing supersaturated copper sulfate, and rewet for 3 hours at room temperature.

[0069] -Pollen germination statistics in vitro after cryopreservation: adopt hanging drop germination method or solid medium germination method to carry out in vitro germination culture on pollen in vitro medium (M3 and M5). The specific pollen in vitro medium formula is: 100 ~150g/L sucrose, 0.1g/L boric acid, 0.1g/L potassium nitrate, 0.3~1.0g/L calcium nitrate, 0.0~0.3g/L magnesium sulfate, 10g/L agar. In vitro pollen culture was cultured overnight at 25°C in the dark. A microscope (OLYMPUSSZ×16) was used for statistics and photography of pollen germination rate in vitro. In statistics, pollen tube length exceeding half the diameter of pollen grains is regarded as viable pollen. 4 fields of view are randomly selected each time, with an average of about 100 pollen per field of view, and 4 replicates for each group to calculate the pollen germination rate.

[0070] 3. Test results

[0071] Experiments showed that the 37℃ thaw and rewetting treatment had a significant effect on the ultra-low temperature preservation of pomelo germplasm pollen. The average germination rate of the pollen used in the experiment can reach 70% after ultra-low temperature storage. The result is figure 2 As shown, the samples of Zuoshi pomelo germplasm with pollen water content of 10%, after being stored in liquid nitrogen, thawed at 37°C and subjected to rewetting treatment, the average germination rate of pollen in vitro is 40-60%; Zuoshi pomelo germplasm pollen The samples with a water content of 20%, after being stored in liquid nitrogen, thawed at 37°C and subjected to rewetting treatment, the average germination rate of pollen in vitro is 45-52%; samples with a water content of 10% of Kuiyou germplasm pollen, menstrual fluid After being stored in nitrogen, thawed at 37°C and remoisturized, the average germination rate of pollen in vitro is 70%.

[0072] In addition, the experiment found that Beizhuan pomelo was thawed at 37°C for 5 minutes, without rewetting step, and the resuscitated pollen was placed in 150g/L sucrose, 0.1g/L boric acid, 0.1g/L potassium nitrate, 0.3 -1.0g/L calcium nitrate, 0.2-0.3g/L magnesium sulfate, 0-10g/L agar pollen in vitro germination medium cultured in the dark at 25℃ for 16-20h, which can have the highest germination rate.