Effects of calcium, magnesium, potassium, light and temperature on pollen germination and pollen tube elongation of passion fruit in vitro

Abstract: 【Objective】Pollen germination in vitro is an important means to detect pollen activity. So far, the effect of calcium, magnesium, and potassium concentrations in the medium, and light and tem-perature conditions on Passiflora pollen germination and tube elongation are not yet clear, and there-fore, the research on these influence factors not only leads to further optimizing the germination condi-tions, but also provides theoretical guidance for the Passiflora cultivation.【Methods】The experiment was conducted from September to November, 2020 in the Experimental Station (N 22.85°, E 108.24°), Horticulture Institute, Academy of Agricultural Sciences in Guangxi Zhuang Autonomous Region. The maximum temperature during the period was around 37 ℃, and the minimum temperature was 20 ℃. The fresh pollen of Passiflora caerulea L.‘Tainong No. 1’as a China’s main variety was used as the  experimental material, the flowers were picked up when they were just opened, and two fully-bloomed flowers were collected from each of the 10 plants. The flowers were put into paper bags and brough to the laboratory immediately. The anther was gently removed in the laboratory with air conditioner of 25 ℃, and placed in a petri dish lined with weighing paper, with dried silica gel under the paper. The pe- tri dish was placed in a ventilated and dry place for about 2 h, until the anthers were fully cracked, and then the pollen grain was swept down gently with a soft brush, mixed with all the pollen for later use. The medium consisted of 150 g · L^-1 sugar, 150 g · L^-1 PEG-4000, 25 mg·L^-1 H3BO3, 300 mg·L^-1 Ca(NO3)2·4H2O, 200 mg·L^-1 MgSO4·7 H2O, and 100 mg·L^-1 KNO3was used as a basic medium. 0, 100, 300, 600, 1200, 2400 mg·L^-1 of Ca(NO3)2·4H2O and 0, 200, 400, 800, 1600, 3200 mg·L^-1 of MgSO4·7 H2O and 0, 100, 200, 400, 800 and 1600 mg·L^-1 of KNO3 were set to study the effect of calcium, magnesium and potassium concentrations on pollen germination in vitro under 25 ℃ and dark condition. 0 lx (dark), 600 lx, 1150 lx and 2210 lx were set to study the effect of illumination under 25 and 5 ℃, 10, 15, 20, 25, 30, 35, 40 and 45 ℃ were set to study the effect of temperature under dark condition, and each process was set to have three replicates. Liquid medium was used in this study, 3.5 mL liquid medi-um was put into a petri dish with 6 cm in diameter, the petri dish was shaken gently and the liquid medi-um would distribute evenly to form a thin layer. The petri dish was placed in an artificial climate cham-ber at preset temperature and under light conditions. Once the temperature equilibrated, a soft brush was used to sprinkle the pollen on the medium and it was incubated for 4 h. Petri dish with germinated pollens were examined under 40× magnification of Olympus microscope BX53, 5 microscopic fields were observed in different positions of the petri dish for each replicate, and the number of pollens in each field was no less than 20, so the number of pollens observed with each treatment was no less than 300. A pollen grain was considered to germinate when the elongation length of the germinated pollen tube was equal to or longer than the diameter of the pollen. The tube elongation length was measured at the end of 4 h incubation with all the treatments using the polyline length measurement tool and expressed in micrometer. The replicated values of pollen germination and tube elongation length from all tests were analyzed using the one-way ANOVA procedure of SPSS 17.0, the Duncan method was used for multiple comparisons, and curve fitting analysis was done using SPSS 17.0 to the data from tempera- ture treatments.【Results】(1) Calcium, magnesium and potassium were not essential components for pollen germination of Passiflora in vitro, because pollen can also germinated in the medium lacking of them, but calcium had significant effects on pollen germination and pollen tube growth of Passiflora in vitro, the optimum concentration was 300-1200 mg·L^-1, excessively high or low concentration was not conducive to pollen germination and significantly reduced pollen tube elongation. Magnesium and po- tassium had no significant effect on pollen germination and pollen tube growth. (2) Light intensity had a significant effect on the pollen germination and tube growth of Passiflora pollen, 0 lx was the best light condition, and the germination rate and pollen tube elongation decreased significantly with the increase of light intensity (3) Temperature had a significant effect on pollen germination and pollen tube elonga- tion of Passiflora pollen. It was found that the quadratic equation can fit the germination rate and pollen tube elongation well, with the P value of 0.000<0.05, reaching a significant level, the R2 was 0.95 and 0.98, respectively. The fitting equation of pollen germination rate was as follows: Y = -0.21x2+10.78x-58.71, the fitting equation for pollen tube growth was: Y= -0.56x2 + 28.38x-124.52, and according to the fitting equation, the Tmin (the minimum temperature ) of pollen germination was 6.19 ℃, the Topt (the optimum temperature) was 25.69 ℃, and the Tmax (the maximum temperature) was 45.13 ℃. The Tmin of pollen tube growth was 4.85 ℃, the Topt was 25.34 ℃, and the Tmax was 45.55 ℃.【Conclusion】Calcium, light and temperature had significant effects on pollen germination in vitro, but magnesium and potassium had no significant effect. The optimal concentration of calcium was 300-1200 mg·L-1, and the opti-mal light was 0 lx. The quadratic equation can well fit the effect of temperature on pollen germination rate and pollen tube elongation length in vitro of Passiflora pollen.