Pollen tube fluorescence microscopic observation and pollination compatibility analysis of self and cross- pollination in passion fruit (Passiflora edulis)

Abstract: 【Objective】This study summarized the pollen tube growth behavior of different compatibility types by comparing the differences in pollen germination, pollen tube growth and the strength of pollination compatibility after self and cross-pollination among different passion fruit varieties (lines), so as to provide reference for passion fruit pollination and cross breeding.【Methods】In this experiment, the main cultivar of China, Tainong 1 (TN) and passion fruit lines numbered as H2, MB, H6 and ZS were used as test materials. Four self- pollination combinations were set up, namely TN, H2, MB and H6 self-pollination, and six cross-pollination combinations (female parent×male parent): H6×MB, H6×TN, MB×H6, MB×TN, ZS×MB and ZS×H6, respectively. Pollen behaviors 0.5-24 hours after pollination were observed and photographed under a fluorescence microscope. We counted the number of pollen tubes germinating on the stigma surface, entering into the stigma transmitting tissue, reaching the middle of the style and entering the ovary, and calculated the pollination compatibility index. At the same time, the pistil was dissected and observed, and the fruit setting rate of four combinations including H6×MB, H6×TN, MB×H6 and MB×TN was calculated.【Results】(1) The pistil of passion fruit consisted of stigma, style and ovary. The stigma was densely covered with papillae. The pollen tube passed through the gap between the stigma papilla cells and entered the stigma transmitting tissue. The transmitting tissue was funnel-shaped, extending from the stigma to the base of the style. The style had three branches, connecting to the ovary. The ovule in the ovary was pear-shaped, the micropyle was at the base of the funicle, the embryo sac was long oval, and the embryo sac was covered with the integument. (2) We found that in the self and cross-pollination combinations of passion fruit with affinity, the pollen grains attached to the stigma, sprouted pollen tubes into the stigma papilla, then passed through the stigma and style transmitting tissue, and entered the ovule 18-24 hours after pollination. The pollen germination rate and pollen tube growth rate of different combinations were different. The“fast”pollen grains germinated 0.5 hour after pollination and reached ovary 8 hours after pollination, while the “slow”pollen grains germinated 6 hours after pollination and the pollen tube did not reach the ovary 24 hours after pollination. However, in case of incompatible passion fruit, either self or cross- pollination combinations, pollen grains could attach to the stigma, sprout and sent forth pollen tubes, but the bottom of the pollen tube expanded, could not elongate normally, and finally could only stay in the stigma papilla, which showed the characteristics of sporophyte incompatibility type. (3) The average pollination compatibility index of 8 to 24 hours after self-pollination of TN was the highest (1.25), and that of H6×TN was the lowest (0.01). The pollination compatibility trend of the four self-pollination combinations was TN＞H2＞MB＞H6, and H6 was self- incompatibility type. The pollination compatibility trend of the six cross-pollination combinations was H6×MB＞H6×TN, MB×H6＞MB×TN, ZS×MB＞ ZS×H6, and H6×TN was the cross-incompatibility type. Except MB×TN, the fruit setting trend of H6× MB, H6×TN and MB×H6 was consistent with pollen tube observation. In addition, we found that there were differences in compatibility and fruit drop rates of reciprocal crosses combinations. For example, H6 as the female parent and MB as the male parent had higher compatibility and lower fruit drop rate, compared with H6 as the male parent and MB as the female parent. So the selection of parents was related to pollination compatibility and fruit setting of cross-pollination combinations. (4) Pollen tube elongation was accompanied by callose accumulation. Pollen tube growth of compatible combinations was accompanied by regular callose plugs. Callose accumulation in stigma papilla was correlated with pollination compatibility, indicating that callose accumulation in papilla cells increased with a decreasing compatibility. For example, a large amount of callose accumulation was observed in the stigma papilla 24 hours after pollination in H6 self-pollination and H6×TN incompatibility combinations, while no obvious callose accumulation was observed in the stigma papilla in TN self-crossing and H2 self-pollination combinations with the highest self-pollination compatibility.【Conclusion】The pollen tubes of the self and cross-pollination combinations entered the ovules 18-24 hours after pollination, while the pollen tubes of the incompatible combinations could not pass through the stigma papilla, so it belonged to sporophyte incompatibility type. The affinity decreased with the increase of callose accumulation of stigma papilla cells. The affinity can be identified by the number of germinating pollen grains, initiation time, pollen tube growth rate and callose accumulation in stigma papilla.