Diversity Analysis Of Mini-Seed Derived Plants, Excellent Germplasm Selection And Seed Degeneration Mechanism Research In Loquat

The loquat breeding group of Sichuan Agricultural University had developed a protocol for culture of mini-seeds (middle-degraded seeds) and production of plants called "mini-seed derived plant", providing the valuable material for germplasm resource development and innovation. In the present study, a series of experiments were carried out to investigate the genetic diversity of mini-seed derived plants, excellent germplasm selection and seed degeneration mechanism in loquat. In part I, the genetic diversity and breeding value of mini-seed plants were studied dased on cytology and morphology research, in order to selecte excellent germplasm. In part II, the seed degeneration mechanism was stuied by pollen mother cell meiosis and the unnormal phenomenon of pollen and ovule in morphocytology; meanwhile, the relationship between pistil structure and seed abortion was clarified for the first time in loquat, aiming at providing data for degenerated seed origin. The main results were as follows.1Diversity of mini-seed plants based on leaf morphology and chromosome number of group I identified There was abundant genetic diversity in leaf morphology (D=0.785). The values obtained varied broadly with coefficients of variation ranging from9.8%to40.1%. Analysis of the79accessions divided them into6groups by the UPGMA method. The leaves of group Ⅵ were light green, small, thin and soft with slender petioles; while the leaves of group I were dark green, large, hard and thick with long and thick petioles. These two groups were important excellent germplasm. And1#,115#,137#and249#in group I were triploids (2n=3x=51), which provided precious materials for breeding seedless varieties. The triploid plants had the typical morphological appearances with large leaves, big flowers, big and bell-shaped fruits and orange yellow fruit peel.2Diversity in florescence, fruit and seed morphology of mini-seed derived plants and excellent germplasm selectionThere was abundant genetic diversity in florescence in the mini-seed derived plants and2lines were selected with special traits, early flowering (212#) and late flowering (6060#). There was also abundant diversity in fruit and seed morphology in the mini-seed derived plants (D=0.7482) and the coefficient of variation was27.92%. Considerating the goal of breeding new cultivars in loquat,3lines were selected from the mini-seed derived plants with special traits, big fruit (203#), few seeds (C1#) and white flesh (4-1-5#),respectively.3Investigation on the hardiness and preliminary screening for cold resistant material from mini-seed derived plantsThere were abundant genetic diversity in mini-seed plants in response to the frost, and the range of embryo injury rate and defoliation rate were65.4%and90%, respectively; there was no correlation between the rate of embryo injury and defoliation rate (R=0.2911); a significant negative correlation was found between the rate of embryo injury and blooming time, the rate of embryo injury decreased with the blooming delay; the average frost index of mini-seed derived plants was25.34, and cold-resistant6-3-10and C2were preliminary selected based on the rate of embryo injury as the main index combined with defoliation rate of the biennial autumn shoots. 4Pollen mother cell meiosis and the unnormal phenomenon of pollen and ovule in morphocytologyAbout3.9%of pollen mother cells (material cv. Zaozhong6) showed nondivision or nonsynchronous division at meiosis, and gave rise to dyads (0.3%) triads (0.5%) and hexads (0.1%) after anaphase Ⅱ, indicating a frequency of unreduced gametes of0.28%. There were about21.6%unnormal pollen cells including cavity, with no nucleolus and distorted microspores, which all appeared at unicleate stage. The anatomise observation (material cv. Dawuxing) showed that structure of13.3%ovules was abnormal, which included small embryo sac, female genitalia degeneration, embryo sac degeneration and ovule degeneration. Unnormal meiosis and pollen would result in pollen abortion, and fertilization and embryo development could not be satisfied in abnormal ovules, which all resulted in seed degeneration.5Morphological analysis of styles and stigmas and their relation to pollen germination and pollen-tube growthThe length of styles significantly differed in a flower in loquat (material cv. Dawuxing, Zaozhong6, Longquan1); and there were significant differences in matoid cell characteristics, stigma receptivity, mucus secretion, in situ pollen germination, and pollen tube growth in different lengh styles in a flower. Pollen tube quantity and pollen germination were remarkably different, depending on style length: pollen germination in the stigma of long and middle sized styles was greater than that of short styles, and the number of pollen tubes growing in the short styles was significantly smaller than in the long and middle styles. The results indicated that the long and middle styles were more receptive to pollen germination and pollen tube growth than short styles.6The pistil structure and its relation to seed set and degenerationBased on investigating pistil structure through free-hand section and observation on seed set under manual control-pollination, the results, for the first time, demonstrated that the5styles and5lobules had a one-to-one relationship in both the morphylogical and reproductive characteristics in loquat. Obturator originates from the carpellary margins, formation of the septum in the ovary, extends radiating in5lobules; styles5, free at base, each one leading through the placenta to one of the5locules. After hand-pollination with removal of4styles (only one style left), there are seeds only in one lobule per fruit with two types of seed set patterns:76.7%fruits had two normal seeds, which were in accordance with pattern I;23.3%fruits had only one normal seed and one degenerative seed, which were in accordance with pattern II, while all the8ovules in other4lobules were unfertilized and wilted. Seed degradation rate was11.7%when only one style was remained. The results demonstrated that the5styles and5lobules had a one-to-one relationship in reproductive characteristics in loquat. Moreover, the degenerative seed beside the normal seed in the same lobule was possibly resulted from the stimulation by plant growth substances secreted by the normal embryo in the same locule. In the in vitro culture of the seeds, a haploid (n=x=17) and an aneuploid (n-3=14) were obtained.