Observation of pollen tubes behavior and early embryogenesis following self and cross-pollination in Chinese jujube

Abstract: 【Objective】Ziziphus is an important genus within the family Rhamnaceae. This genus includes several important fruit tree species that are widely planted in China and India, such as the Chinese jujube(Ziziphus jujuba Mill.), the wild jujube (Z. acido jujuba), and the Indian jujube(Z. maur-itiana). Chinese jujube, native to China and domesticated from wild jujube, is one of the most important and widely dried fruit crops in China. But wide spread of blossom and fruit drop, difficulty in hand emasculation, low fruit set, low seed kernel and self-incompatibility seriously restricted the germplasm innovation and hybrid breeding of jujube, and there is an urgent need to promote crossbreeding in production. Therefore, to investigate difference in the growth behavior of pollen tube in the pistil and early embryogenesis of Chinese jujube after self-and cross-pollination, to identify the types and characteristics of self-incompatibility of the plant and to complement the current knowledge of Chinese jujube in reproductive biology. Further to analyze the problems of low kernel content in terms of self-incompatibility and also to provide a theoretical basis for reproduction and cross-breeding.【Methods】The pollination materials are five to eight years general Zizyphus jujube Mill.‘Dongzao',‘Fengmiguan',‘Xiangfenyuanzao',‘Huizao',‘Yongjihamazao',‘Yanchuanlaoyasuanzao',‘Jinzao'. Designing self-pollinators of‘Dongzao',‘Huizao'and cross-pollinators include‘Huizao'בJinzao',‘Fengmiguan'בXiagfenyuanzao',‘Xiangfenyuanzao'× 'Yongjihamazao',‘Fengmiguan'בYanchuanlaoyasuanzao'. According to the experimental principles of observation of the pollen tube under a fluorescence microscope as well as the characteristics of the pistil, the growth process of pollen tube in the pistil and early embryogenesis were observed using fluorescence microscopy combined with the modified softening method, and the routine paraffin method, to characterize the anatomic properties of the self-incompatibility of Chinese jujube. Operation is expressed in the following way: the pollinated flowers tagged and recovered immediately with paper bags, pistils collected at 3 h, 6 h, 24 h, 48 h, 72 h,96 h after pollination and immersed in FFA fixative solution, fixed 24 h after vacuum pumping for 20 minutes, then flushed with distilled water and transferred into 70% ethanol solution, stored at 4 ℃.Hand-pollinated pistils were hydrated with an ethanol series(70%, 50% and 30% ethanol) for 30 min,washed with distilled water, and boiled with 2 mol · L-1 of sodium hydroxide solution for 30 min to allow softening, washed with distilled water again, immersed in buffer solution(Glacial acetic acid and sodium hydroxide configuration, pH 5.5 to 6.5) for 30 min and stained overnight with 0.1%(m/V) aniline blue solution containing 108 mmol · L-1 K3 PO4 at pH 11 and 2% glycerol after washing with distilled water. Pistil was sucked with plastic drop pipette, and dropped on glass slides, covered with cover glass lightly without pressing. The section under the OlympusBX-51 fluorescence microscope was used to observe the pollen germination and pollen tube growth in the style, and take pictures. According to the results of pollen tube growth, flowers were selected for paraffin section, processes as follows: fixation, dehydration, clearing, wax-filling, embedding and trimming, sectioning, adhering to slides, deparaffinization and staining, mounting, slice thickness 6 to 8 μm which was stained with Safranine and Fast Green.Olympus microscope imaging system was used to observe the structure and formation of the embryo.【Results】In self and cross-pollination of different jujube cultivars, Pollen grains begun to germinated on stigma, 3 h AFP; Pollen tube penetrated into stigma and grew into stylar canal, 6 h AFP. pollen tubes penetrated at 1/3 of stylar canal, 24 h after pollination; pollen tubes penetrated at middle of stylar canal,48 h after pollination; pollen tube that stopped growing while penetrated at middle of stylar canal, 96 and 129 h after pollination as was observed in the pollination combination(‘Dongzao'בDongzao',‘Huizao'בHuizao'). In pollination combination(‘Huizao'בJinzao',‘fengmiguan'בXiangfenyuanzao',‘Xiangfenyuanzao'בYongjihamazao'), pollen tubes penetrated at 1/2 of stylar canal, 24 h after pollination; pollen tubes grew to the lowest part of stylar canal, 48 h after pollination; pollen tubes penetrated the stylar base and reached the ovule, 72 h after pollination. 13 days after pollination, microscopic observation showed that in the ovary of pollination(‘fengmiguan'בXiangfenyuanzao'), one ovule degenerated and the other kept on developing. Globular stage gradually formed after the transverse fissure of zygote, free nucleus of endosperm gradually increased. Embryo abortion caused by poor pollination and fertilization was observed in pollination(‘Huizao'בHuizao').【Conclusion】Based on the anatomical characteristics, we ascertained that Chinese jujube was a self-incompatible plant and belonged to the prezygotic incompatibility type, which occurred in the stylar base. The current study revealed that the Chinese jujube with very low kernel content and fruit set are caused firstly by self-incompatibility and followed by abortion due to hypoplasia of embryo, and this finding also provides insight into crossbreeding and parental choice for the jujube.