枇杷种子退化的胚胎学机制及中度退化种子株系遗传多样性研究
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摘要
本研究对多核枇杷品种种子退化及胚败育情况进行了调查,并首次从花药结构、花药散粉情况、花粉活力、柱头可受性、自然授粉后花粉原位萌发和花粉管生长特性、胚胎发育解剖观察等方面,系统研究了‘大五星'枇杷的雌雄性器官育性和胚胎发育过程,探明了多核枇杷品种种子退化及中度退化种子产生的胚胎学基础。采用扫描电镜技术和ISSR分子标记技术对‘大五星'、‘龙泉1号'、‘龙泉5号'3个栽培品种及其中度退化种子株系进行花粉亚显微形态和分子遗传多样性分析。同时,在形态学调查的基础上,从花药结构、花粉亚显微形态、花粉活力、柱头可受性、授粉亲和性、胚珠结构及胚胎发育过程等方面对少核株系C1和坐果极低且无核的115号株系进行了系统研究,明确了其少核和结果极少且无核的胚胎学机制。主要研究结果如下:
1.枇杷种子退化和胚败育调查
调查结果表明,‘大五星'、‘龙泉1号'和‘龙泉5号'3个枇杷品种平均每个果实中仅有2.47～3.29个胚珠最终能发育为大粒饱满种子,有0.08～0.16个胚珠发育为轻度退化种子,0.18～0.22个胚珠发育为中度退化种子,而多达6.45～7.19个胚珠发育为高度退化种子。3个品种的轻度退化种子、中度退化种子和高度退化种子平均单粒鲜重分别是正常饱满种子的13.42%～17.04%、4.16%～6.36%和1.24%～2.24%。轻度退化种子胚败育率在33.78%～51.36%,中度退化种子的胚败育率为46.54%～70.59%,而高度退化种子的胚完全败育。
2.‘大五星'枇杷雌雄性器官的育性检测
雌雄性器官的育性检测表明:(1)‘大五星'品种花药较大而饱满,开花当日多数花药能纵裂散粉;花药结构正常,花粉量很多,畸形花少;开花2～4d花粉具较高活力,同时柱头具强可受性。表明‘大五星'枇杷在花期内雌雄性器官的育性强弱具有同步性,利于自然授粉结实。(2)自然授粉后,花粉粒在柱头上萌发主要集中在开花2～4d;开花5d约有90%花柱的柱头上有萌发的花粉粒;柱头具可受性的持续时间长约7d。同一花蕾中不同长度的花柱,柱头花粉原位萌发时间不一致,多数相差2～3d。(3)多数花粉管能生长到花柱上部,仅30%的花粉管能够生长至花柱基部;近85%的胚珠中发现有花粉管,但花粉管在胚珠中弯曲生长或沿珠被生长的现象严重,表明自然授粉条件下,‘大五星'枇杷表现出一定的受精前障碍。
3.‘大五星'枇杷的胚胎发育
解剖观察表明:(1)‘大五星'枇杷胚珠为倒生胚珠,蓼型胚囊和核型胚乳。(2)花蕾中多数胚珠能正常受精发育至球形胚和心形胚阶段,部分胚经鱼雷胚阶段发育为成熟子叶胚。胚乳细胞在大球胚阶段形成,在小鱼雷胚阶段解体。(3)开花14～23d,胚珠中可见合子、2-细胞原胚、T-形原胚、多细胞原胚、小球胚及扩散至珠孔端的胚乳核。开花29～48d,多数胚发育为大球胚和胚乳细胞,早受精的胚已发育为小心形胚阶段;而未受精胚珠的雌性生殖器解体成空胚囊。开花53～64d时,胚胎发育旺盛,胚珠中可见大球胚、鱼雷胚、小子叶胚及成熟种胚。(4)开花90～120d,果实进入膨大初期(纵径为1.667cm～2.209cm、横径为1.432cm～1.904cm),果实中3～4枚成熟子叶胚继续发育为饱满大粒种胚;约3枚胚珠因未受精发生珠心、胚囊解体,最终形成完全退化种子;另约3枚受精胚珠发育至球胚、心形胚及鱼雷胚阶段,但胚囊内胚乳细胞和珠心组织提前解体导致其无法继续发育,至果实成熟时形成高度败育的种子;此期发育至小子叶胚阶段的胚,部分能继续发育为成熟种胚,但因养分及激素竞争处于弱势,子叶长度和厚度均明显小于正常的种胚,形成中度退化种子。开花64～90d,是影响受精胚胎能否继续发育的关键时期。
4.中度退化种子株系的花粉亚显微形态多样性
花粉亚显微形态分析表明:(1)正常的枇杷花粉为长球形(P/E为1.42～1.73),赤道面观为长椭圆形,极面观为三裂圆形;花粉为中等大小(28.21um×19.22um～33.98um×20.16um);萌发孔为典型的三孔沟结构。(2)26份中度退化种子株系中出现发育不良小花粉和变形大花粉的比率较大,其中6个株系花粉畸形率高达41.82%～83.65%。(3)29份枇杷材料的花粉外壁纹饰丰富多样,甚至绝大多数材料的同一花粉3个赤道面上的条纹走向都不一致。‘大五星'、‘龙泉1号'和‘龙泉5号'3个亲本品种花粉外壁雕纹属于条纹状、条纹-穴状(或条纹-穿孔状)和条纹-颗粒-穴状(或条纹-颗粒-穿孔状)。26份中度退化种子株系的花粉除具亲本的纹饰外,还出现脑纹-穿孔状雕纹类型;同时中度退化种子株系既具亲本特征的花粉类型,又分离出其亲本间的过渡类型和原始类型,花粉形态有平行、交叉现象,这与形态学特征表现出的多样性一致。
5.中度退化种子株系的分子遗传多样性
ISSR标记技术对‘大五星'、‘龙泉1号'与‘龙泉5号'3个亲本及59份中度退化种子株系的多样性分析结果表明,18条ISSR引物在62份供试材料中共扩增出324条带,其中310条多态性带,多态性比率为95.7%,18条引物在62份枇杷材料的平均有效等位基因数、平均Nei's基因多样性指数和平均Shannon信息指数分别为1.5654、0.3249和0.5283。ISSR分析结果进一步表明,枇杷中度退化种子株系具有丰富的遗传物质基础以及高度复杂的遗传背景。
6.C1株系坐果率较低、多独核的胚胎学机制
雌雄育性检测及胚胎发育观察表明:(1)C1株系药隔组织较其亲本‘龙泉5号'发达,药室腔较小,花粉粒数量较少,并有58.94%为发育不良的小花粉,使其花粉活力及花粉萌发率均较低(分别为16.83%和14.58%),表现出浅度雄性不育。(2)C1株系花柱数目不为5枚的花蕾比率高达35.92%,同时,花蕾中花柱长短不均,短花柱比例较高。较高比例的异常花柱数和短花柱使其接受花粉、花粉萌发和花粉管生长均受到抑制,导致能受精的胚珠较少。(3)C1株系绝大多数胚珠结构正常,部分胚珠能正常受精并进一步发育为球形、心形、鱼雷形及子叶胚。但同一果实的胚珠间受精和胚发育不同步,导致养分及激素分配偏向早受精的胚,晚受精的胚多停留在球胚阶段,最终导致果实坐果率较低,果实中能发育成熟的种胚仅1～2粒。
7.115号株系结果极少、无核的胚胎学机制
雌雄育性检测及胚珠结构解剖观察表明:(1)115号株系叶大、花大、花期长,但自花授粉坐果率极低,不到0.01%;果实较小、无核。(2)115号株系开花时花药发育不成熟,多数花药仍为具4个小孢子囊的不成熟结构,少数的成熟花药药壁不能及时开裂散粉。同时,药隔组织极厚,药室腔极小,花粉数量极少,并且83.65%为变形花粉粒。花粉活力和花粉萌发率极低(分别仅为3.67%和0.89%)。115号株系雄性器官表现为深度不育。(3)115号株系花柱数目≥6枚的花蕾比率高达31.25%;花粉原位萌发及花粉管生长异常比例高,表现出较强的受精前障碍。(4)115号株系绝大多数胚珠结构异常,包括小胚囊、雌性生殖器退化、胚囊退化和胚珠退化等4类,115号株系雌性器官为重度败育类型。(5)异常的胚珠结构不能提供受精及胚胎发育的场所,最终导致115号株系自然授粉和自花授粉基本不结果,异花授粉坐果率仍很低。即使极少数结构正常的胚珠能受精,但因有性生殖器官先天发育不足,缺少受精和胚胎发育的物质基础,因而胚胎发育受阻,同时,果肉细胞因缺少来自种子的激素刺激而发育缓慢。
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 loquat
The 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 morphology
Pollen 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 ISSR
Genetic 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 C1line
Investigations 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 line
Anther 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.
1.枇杷种子退化和胚败育调查
调查结果表明,‘大五星'、‘龙泉1号'和‘龙泉5号'3个枇杷品种平均每个果实中仅有2.47～3.29个胚珠最终能发育为大粒饱满种子,有0.08～0.16个胚珠发育为轻度退化种子,0.18～0.22个胚珠发育为中度退化种子,而多达6.45～7.19个胚珠发育为高度退化种子。3个品种的轻度退化种子、中度退化种子和高度退化种子平均单粒鲜重分别是正常饱满种子的13.42%～17.04%、4.16%～6.36%和1.24%～2.24%。轻度退化种子胚败育率在33.78%～51.36%,中度退化种子的胚败育率为46.54%～70.59%,而高度退化种子的胚完全败育。
2.‘大五星'枇杷雌雄性器官的育性检测
雌雄性器官的育性检测表明:(1)‘大五星'品种花药较大而饱满,开花当日多数花药能纵裂散粉;花药结构正常,花粉量很多,畸形花少;开花2～4d花粉具较高活力,同时柱头具强可受性。表明‘大五星'枇杷在花期内雌雄性器官的育性强弱具有同步性,利于自然授粉结实。(2)自然授粉后,花粉粒在柱头上萌发主要集中在开花2～4d;开花5d约有90%花柱的柱头上有萌发的花粉粒;柱头具可受性的持续时间长约7d。同一花蕾中不同长度的花柱,柱头花粉原位萌发时间不一致,多数相差2～3d。(3)多数花粉管能生长到花柱上部,仅30%的花粉管能够生长至花柱基部;近85%的胚珠中发现有花粉管,但花粉管在胚珠中弯曲生长或沿珠被生长的现象严重,表明自然授粉条件下,‘大五星'枇杷表现出一定的受精前障碍。
3.‘大五星'枇杷的胚胎发育
解剖观察表明:(1)‘大五星'枇杷胚珠为倒生胚珠,蓼型胚囊和核型胚乳。(2)花蕾中多数胚珠能正常受精发育至球形胚和心形胚阶段,部分胚经鱼雷胚阶段发育为成熟子叶胚。胚乳细胞在大球胚阶段形成,在小鱼雷胚阶段解体。(3)开花14～23d,胚珠中可见合子、2-细胞原胚、T-形原胚、多细胞原胚、小球胚及扩散至珠孔端的胚乳核。开花29～48d,多数胚发育为大球胚和胚乳细胞,早受精的胚已发育为小心形胚阶段;而未受精胚珠的雌性生殖器解体成空胚囊。开花53～64d时,胚胎发育旺盛,胚珠中可见大球胚、鱼雷胚、小子叶胚及成熟种胚。(4)开花90～120d,果实进入膨大初期(纵径为1.667cm～2.209cm、横径为1.432cm～1.904cm),果实中3～4枚成熟子叶胚继续发育为饱满大粒种胚;约3枚胚珠因未受精发生珠心、胚囊解体,最终形成完全退化种子;另约3枚受精胚珠发育至球胚、心形胚及鱼雷胚阶段,但胚囊内胚乳细胞和珠心组织提前解体导致其无法继续发育,至果实成熟时形成高度败育的种子;此期发育至小子叶胚阶段的胚,部分能继续发育为成熟种胚,但因养分及激素竞争处于弱势,子叶长度和厚度均明显小于正常的种胚,形成中度退化种子。开花64～90d,是影响受精胚胎能否继续发育的关键时期。
4.中度退化种子株系的花粉亚显微形态多样性
花粉亚显微形态分析表明:(1)正常的枇杷花粉为长球形(P/E为1.42～1.73),赤道面观为长椭圆形,极面观为三裂圆形;花粉为中等大小(28.21um×19.22um～33.98um×20.16um);萌发孔为典型的三孔沟结构。(2)26份中度退化种子株系中出现发育不良小花粉和变形大花粉的比率较大,其中6个株系花粉畸形率高达41.82%～83.65%。(3)29份枇杷材料的花粉外壁纹饰丰富多样,甚至绝大多数材料的同一花粉3个赤道面上的条纹走向都不一致。‘大五星'、‘龙泉1号'和‘龙泉5号'3个亲本品种花粉外壁雕纹属于条纹状、条纹-穴状(或条纹-穿孔状)和条纹-颗粒-穴状(或条纹-颗粒-穿孔状)。26份中度退化种子株系的花粉除具亲本的纹饰外,还出现脑纹-穿孔状雕纹类型;同时中度退化种子株系既具亲本特征的花粉类型,又分离出其亲本间的过渡类型和原始类型,花粉形态有平行、交叉现象,这与形态学特征表现出的多样性一致。
5.中度退化种子株系的分子遗传多样性
ISSR标记技术对‘大五星'、‘龙泉1号'与‘龙泉5号'3个亲本及59份中度退化种子株系的多样性分析结果表明,18条ISSR引物在62份供试材料中共扩增出324条带,其中310条多态性带,多态性比率为95.7%,18条引物在62份枇杷材料的平均有效等位基因数、平均Nei's基因多样性指数和平均Shannon信息指数分别为1.5654、0.3249和0.5283。ISSR分析结果进一步表明,枇杷中度退化种子株系具有丰富的遗传物质基础以及高度复杂的遗传背景。
6.C1株系坐果率较低、多独核的胚胎学机制
雌雄育性检测及胚胎发育观察表明:(1)C1株系药隔组织较其亲本‘龙泉5号'发达,药室腔较小,花粉粒数量较少,并有58.94%为发育不良的小花粉,使其花粉活力及花粉萌发率均较低(分别为16.83%和14.58%),表现出浅度雄性不育。(2)C1株系花柱数目不为5枚的花蕾比率高达35.92%,同时,花蕾中花柱长短不均,短花柱比例较高。较高比例的异常花柱数和短花柱使其接受花粉、花粉萌发和花粉管生长均受到抑制,导致能受精的胚珠较少。(3)C1株系绝大多数胚珠结构正常,部分胚珠能正常受精并进一步发育为球形、心形、鱼雷形及子叶胚。但同一果实的胚珠间受精和胚发育不同步,导致养分及激素分配偏向早受精的胚,晚受精的胚多停留在球胚阶段,最终导致果实坐果率较低,果实中能发育成熟的种胚仅1～2粒。
7.115号株系结果极少、无核的胚胎学机制
雌雄育性检测及胚珠结构解剖观察表明:(1)115号株系叶大、花大、花期长,但自花授粉坐果率极低,不到0.01%;果实较小、无核。(2)115号株系开花时花药发育不成熟,多数花药仍为具4个小孢子囊的不成熟结构,少数的成熟花药药壁不能及时开裂散粉。同时,药隔组织极厚,药室腔极小,花粉数量极少,并且83.65%为变形花粉粒。花粉活力和花粉萌发率极低(分别仅为3.67%和0.89%)。115号株系雄性器官表现为深度不育。(3)115号株系花柱数目≥6枚的花蕾比率高达31.25%;花粉原位萌发及花粉管生长异常比例高,表现出较强的受精前障碍。(4)115号株系绝大多数胚珠结构异常,包括小胚囊、雌性生殖器退化、胚囊退化和胚珠退化等4类,115号株系雌性器官为重度败育类型。(5)异常的胚珠结构不能提供受精及胚胎发育的场所,最终导致115号株系自然授粉和自花授粉基本不结果,异花授粉坐果率仍很低。即使极少数结构正常的胚珠能受精,但因有性生殖器官先天发育不足,缺少受精和胚胎发育的物质基础,因而胚胎发育受阻,同时,果肉细胞因缺少来自种子的激素刺激而发育缓慢。
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 loquat
The 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 morphology
Pollen 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 ISSR
Genetic 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 C1line
Investigations 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 line
Anther 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.
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