Studies On Mechanism Of Embryo Abortion And Genetic Diversity Of Seedlings From Degenerated Seeds By ISSR In Loquat (Eriobotrya Japonica Lindl.)

The present paper studied on,firstly and systematically,pollinating characteristics, pollen viability,stigma receptivity,the pollen germination and pollen tube growth of self-pollination,cross-pollination and open pollination,anatomical structure of embryo development,changes of endogenous hormones and embryo development in 'Dawuxing', through which the embryology data and changes of endogenous hormones for embryo abortion and degenerated seeds origin were proved.Based on investigation on botany,the genetic diversity of fifty-nine degenerated seedlings and three maternal plants('Dawuxing', 'Longquan No.1' and 'Longquan No.5') were analyzed by ISSR,which was to estimate the genetic diversity of the degenerated seed derived seedlings.The main results were as follows:1.Examination on fertility of male and female of"Dawuxing'The results showed(1) The anthers of 'Dawuxing' were big and plump through the observation with stereomicroscope,most of which could disseminate lots of pollens in cracked chamber by longitudinal crack on sunny day.The best time was 2～4 d after flowering with strong stigma receptivity,meanwhile the pollen vitality was high with a large number of pollen,which was benefit to setting in open pollination.(2)The period of pollination and pollen germination were concentrated on the 2～4d after flowering,and about 90%styles were fertilized 5d after flowering,the stigma receptivity could last about 7 days in open pollination.And the pollination and pollen germination time varied,most intervals were 2～3days in a flower.(3) Most pollen tubes developed to upper of styles, only about 30%pollen tubes could reach the base of styles.Pollen tubes were found in about 85%tested ovules,but it was a very general phenomenon that the pollen tubes grew along the integument or became bending,which was indicative of pre-fertilization barrier in 'Dawuxing'.(4) Pollen germination on stigma in cross-pollination was better than self-pollination in 'Dawuxing'.Pollen tubes reaching upper style,middle style and bottom style in cross-pollination were significantly more than those in self-pollination,so was the rate of germination pollen.Pollen tubes reaching upper style were significantly more than those reaching middle style and bottom style in self-pollination,meanwhile there was no obvious difference in cross-pollination.The percentage of ovules in which pollen tube growth was normal in cross-pollination was higher obviously than that in self-pollination, but there was no obvious difference between cross-pollination and self-pollination.Thus it could be concluded that 'Dawuxing' was self-incompatible to some extent,and the key parts were the middle of style and micropyle.2.Embryo development of 'Dawuxing'Results of anatomical structure of 'Dawuxing' showed that(1) The embryo belongs to anatropous ovule,polygonum type embryo-sac,nuclear endosperm in 'Dawuxing'.(2) Most embryos were fertilized and developed to globular embryo and heart-shape embryo stage,partial embryos developed to cotyledon-shape embryo pass by torpedo embryo.The endosperm was formed in big globular embryo,disaggregated in little torpedo embryo.(3) The zygote,2-cell proembryo,T-proembryo,multicellular proembryo,small globular embryo and endosperm nucleus diffusion to micropyle were observed in succession.Most embryos developed to big globular embryo 29～48d after flowering,endosperm appeared, and the early fertilized embryos developed to small heart-shape embryo,moreover the female genitalia of unfertilized embryos disaggregated to empty capsule.The period was the embryo vigorous development stage after 53～64d,and the big globular embryo, torpedo embryo,small cotyledon-shape embryo and mature embryo were observed.(4) The young fruit developed to early enlargement period(1.667cm～2.209cm in longitudinal diameter,,1.432cm～1.904cm in transverse diameter ),3 or 4 cotyledon-shape embryos continued growing to mature satiety seeds,finally about 3 embryos developed to completely degenerated seeds owing to the disaggregating of nucellus and embryo-sac because of fertilization failure.And other 3 fertilized embryos could not develop after developing to globular embryo,heart-shape embryo or torpedo embryo for the disaggregating beforehand of endosperm and nucellus,and finally formed high-degenerated seeds in mature fruit.Meanwhlie some little cotyledon-shape embryos developed to mature embryos(degenerated seeds) continually,and formed middle-degenerated seeds with smaller and thinner cotyledon than normal embryos owing to disadvantage on competition for nutrients and hormones.It was decisive whether the fertilized embryos developed continually 64～90d after flowering.3.Changes of endogenous hormones in flesh and embryo during the early stages of fruit development in 'Dawuxing'.The changes of five endogenous hormones in the flesh and embryo during the early stages of fruit development in 'Dawuxing' were studied.The results showed that(1) the high contents and a peak of IAA,ZR,iPA and GA₃ appeared within 20d after flowering,which was beneficial to pollination,fertilization and fruit setting.The content of IAA,ZR and GA₃ as well as the ratio of IAA/ABA,ZR/ABA and GA₃/ABA in embryo were higher than in pulp,propitious to the growth of young fruit at early development stage.(2) There were two high peaks of the ratios of IAA/GA₃ and IAA/(ZR+iPA) 35d and 60d after flowering respectively,and it had no significant changes and kept on low level during other periods.Moreover the ratio of(IAA+GA₃+ZR+iPA)/ABA kept on high level all long during young fruit development.Therefore,the balance of hormones,rather than a single hormone,was the key factor in young fruit development at early stage.(3) The ratio of IAA/GA₃ and IAA/(ZR+iPA) kept on high level within 20d after flowering in embryo,but it kept low level 35d-80d after flowering in big embryo.However the ratio of IAA/GA₃ and IAA/(ZR+iPA) increased slowly,and it was higher in small embryos than that in big embryos and middle embryos 60d after flowering.Meanwhile,the content of ABA in small embryos was significantly higher than in big embryos and middle embryos.Thus it could be concluded that IAA was the primary hormone influencing fertilization and proembry development(within 20d after flowering);GA₃,ZR and iPA were the primary hormones to promote constant embryonic cell division and differentiation to mature embryo;embryo development could be restrained when the ratios of IAA/GA₃ and IAA/(ZR+iPA) were high to some threshold value,and high content and ratio of ABA played a leading role in embryo abortion.4.Genetic diversity of 62 loquat accessions analyzed by ISSRGenetic diversity of maternal plants 'Dawuxing','Longquan No.1' and 'Longquan No.5' and 59 degenerated seed derived seedlings was analyzed by ISSR markers.18 ISSR primers were applied to the amplification,producing 324 bands,in which 310 bands (95.7%) were polymorphic.The average value of effective number of alleles,Nei's gene diversity(â…¡) and Shannon's information index were 1.5654,0.3249 and 0.5283, respectively.The genetic difference between these cultivars and degenerated seed derived plants was revealed based on similarity coefficients and cluster analysis.The results showed that the degenerated seed derived plants had genetic stability as well as diversity to some extent,which provided abundant germplasm resources for breeding of loquat.