Studies On Embryological Mechanism Of Seed Degeneration And Genetic Diversity Of Seedlings From Middle-Degenerated Seeds In Loquat (Eriobotrya Japonica Lindl.)

In the present study, a series of experiments were carried out of investigation on seed degeneration and embryo abortion in multi-seed loquat cultivars, pollinating characteristics, pollen viability, stigma receptivity, the pollen germination and pollen tube growth in open pollination, anatomical structure of embryo development in 'Dawuxing', aiming at proving the embryology data for degenerated seed origin and seed degeneration in multi-seed loquat. The genetic diversity of fifty-nine degenerated seedlings and three maternal plants ('Dawuxing', 'Longquan No.1' and 'Longquan No.5') was analyzed by ISSR and pollen submicroscopic morphology structure based on investigation of morphology. Meanwhile, the embryology mechanism was stuied in low seediness C1 line and seedless No.115 line by anther structure, pollen submicroscopic morphology, pollen viability, stigma receptivity, pollen germination, pollen tube growth and anatomical structure of embryo development, aiming at offering embryology data to clarify low fruit setting rate and seedless. The mainresults were as follows:1. Investigation on embryo abortion and seed degeneration in loquatThe seed degeneration rate and the embryo abortion rate of 3 cultivars of loquat were investigated. The results showed that there were 6.45～7.19 heavily-degenerated seeds, 0.18～0.22 middle-degenerated seeds; 0.08～0.16 light-degenerated seeds and 2.47～3.29 normal seeds per fruit in 'Dawuxing', 'Longquan No.1' and 'Longquan No.5' loquat.The mean fresh weight of per grain of light-degenerated seeds, middle-degenerated seeds and heavily-degenerated to normal mature seeds were 13.42%～17.04%,4.16%～6.36% and 1.24%～2.24% in the three cultivars respectively. Embryo abortion rates of light-degenerated seeds, middle-degenerated seeds and heavily-degenerated seeds were 33.78%～51.36%, 46.54%～70.59% and 100.0%, respectively.2. Examination on fertility of male and female of 'Dawuxing'The results were as follows: (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, and no abnormality in anther structure. The best time was 2 to 4 days 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～(nd) to 4～(th) day 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'.3. Embryo development of 'Dawuxing'Results of anatomical structure of 'Dawuxing' showed that (1) The ovules belongs to anatropous ovule, polygonum type embryo-sac, nuclear endosperm in 'Dawuxing'. (2) Most ovules 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 cells 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 ovules 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 thefertilized embryos developed continually 64-90d after flowering.4.Genetic diversity of seedlings from degenerated seeds analyzed by pollensubmicroscopic morphologyPollen morphology of 26 seedlings from degenerated seeds and their three maternal plants were examined with scanning electron microscopy. The results showed that (1) The normal pollen granis were prolate (P/E, 1.42～1.73), long oval in equatorial view and 3-lobed circular roundness in polar view, 28.21um×19.22um～33.98umx20.16um in size, and the structure was the typical 3-colporate. (2) The rate of hypogenesis small pollen granis (P. 16.85～24.50, E. 7.54～13.49) and distortion big pollen grains were high, in which the rate of abnormality pollen in six lines was 41.82%～83.65%. (3) The exine ornamentation of these 29 materials was of some similarity, while the diversity was abundant: the exine ornamentation of three maternal plants were striate, striate-perforate and striate-grain-perforate, while the cerebroid-perforate was showed in degenerated seed derived seedlings except their maternal plants with exine ornamentation. Namely some pollen types were common in degenerated seed derived seedlings and maternal plants, meanwhile, the transition, throwback, parallelism and cross type were observed in degenerated seed derived seedlings.5. 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. The results were as follows: 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 (H) 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.6. Embryology mechanism of low rate of fruit setting and low seedness in C1lineInvestigations were carried out of anther structure, pollen viability, pollinating characteristics, stigma receptivity, pollen germination, pollen tube growth and anatomical structure of embryo development in C1 line. The results were as follows: (1) The connective parenchyma cells of C1 line were obviously more than 'Longquan No.5', and the anther chamber was less, the pollen quantity was very small. The rate of hypoplastic pollen was up to 58.94%, which resulted in that the rate of pollen viability and pollen germination was 16.83% and 14.58%, respectively. Thus,Cl line can be defined as low-degree male sterility. (2) The number and length of styles varied in C1 line , the rate of flower with more than 6 or less than 4 styles was up to 35.9%, meanwhile the rate of short style was high comparatively, which resulted in that pollination, pollen germination and pollen tube growth was restrained, then the fertilized embryos were few. (3) The results of anatomise observation showed that the structure of most ovules was normal, and some developed to globular embryo, heart-shape embryo or torpedo embryo stage after fertilization. But there was difference in double fertilization and embryo development in the same ovary, which resulted in that the nutrient and substances were distributed to the early fertilized embryos, the late fertilized embryos stayed in globular stage, ultimately the fruit setting was low, and only 1～2 embryos could develop mature.7. Embryology mechanism of low rate of fruit setting and seedlessness in No.115 lineAnther structure, pollen viability, pollinating characteristics, stigma receptivity, pollen germination, pollen tube growth and ovule structure in No.115 line were investigated. The results showed that (1) The fruit setting rate was very low (less than 0.01%) and the fruit was seedless in No. 115 line. (2) Most anthers were immature , and a few anthers, though mature, colud not dehisce in time. Meanwhile, the connective parenchyma cells were obviously thick, anther chamber was small, the pollen quantity was very small, and most pollen was abnormal (up to 83.65%). Thus, the male was deep-degree sterile No.115 line. (3) The rate of flower with 6 or more than 6 styles was up to 31.25%, the abnormal phenomena were very general in pollen germination and pollen tube growth, which indicated that the pre-fertilization barrier was strong in No.115 line. (4) The anatomise observation showed that structure of most ovules was abnormal, which included small embryo sac, female genitalia degeneration, embryo sac degeneration and ovule degeneration. Thus, the female was deep-sterile in No.115 line. (5) The fertilization and embryo development could not be satisfied in abnormal ovules, which resulted in that the fruit setting was very low in cross-pollination, and fruit could not be produced in open pollination and self-pollination. Even though a few ovules with normal structure were fertilized, the embryo development was abnormal due to the poorly developed sexual reproduction organ and lack of gowth substances for fertilization and embryo development, and consequently the young fruit developed slowly owing to lack of hormones from seeds.