【作者】 贺军虎；

【导师】 束怀瑞；

【作者基本信息】 西北农林科技大学 ， 果树学， 2013， 博士

【摘要】 杧果（Mangifera indica L.）又称芒果，是世界著名的热带水果。海南省的三亚市及其周边是我国最典型的热带地区。正常季节下，三亚一带的杧果在每年4～5月上市，应用反季节生产技术后，杧果采收期提前到2～3月份，经济效益显著提高。但是，产生了大量的胚胎败育果实，降低了果实的商品率，已经成为杧果反季节生产中主要的问题之一。本研究以海南省主要的杧果栽培品种‘金煌’为材料，从催花对杧果生理生态的影响入手，研究了反季节生产中花器官的变化，对中途败育的胚胎从细胞学、激素、多胺和分子水平上研究了其与正常胚胎的差异，探索胚胎败育的原因，并采取措施，提高胚胎正常果实的比例。主要研究结果如下：1、通过喷施不同浓度的乙烯利、硝酸钾以及硝酸钾与细胞分裂素的混合物对杧果催花效果的研究表明，高浓度的乙烯利处理降低了叶片中叶绿素的含量，降低了杧果净光合速率。各处理均提高了叶片中ZR的含量，提高了ZR/ABA的比值，启动了成花进程，最终导致叶片中C/N比的上升迅速。高浓度的乙烯利和硝酸钾处理成花明显早于低浓度的。各种处理均降低了两性花比率，降低了胚胎正常果实（大果）的比率。2、反季节杧果生产中，‘金煌’品种雄蕊退化，花粉囊之间发育差异十分显著，花粉囊小的花粉数量少，而花粉囊大的花粉数量多且发育至成熟，仅仅有少部分花粉囊的花粉退化，花粉生活力为76.9%。其雌器官出现子房萎缩变形、胚珠横生现象；胚珠周围的输导组织断裂现象普遍；在胚囊8核期间，在同一或者不同胚珠中出现极核细胞消失或者退化，反足细胞个别退化、助细胞个别或者全部退化的现象，这些都可以影响胚珠受精，引起授粉受精不良。3、针对杧果胚胎中途败育的现象研究表明，败育的胚胎周围没有原形成层的出现，或者原形成层不明显。其超级显微结构显示：类似的形成层及其附近的胚乳细胞呈多角性，体内液泡小而少，胚乳细胞壁出现折曲，嗜锇体沉着物沿细胞壁积累，细胞内液泡大，前质体少，营养物质变少且失去活性，进而影响了胚乳细胞和原形成层的形成。胚乳细胞不能持续地分裂和发育是导致胚胎停止生长并引起败育的重要原因。4、研究了杧果果实内源激素变化与胚胎败育的关系。结果表明：胚胎败育在坐果后30d完成，20～30d为胚发育的关键时期，胚败育果实与胚正常果实大小主要缘于果肉的差异。在果实发育初期，败育胚中IAA、ABA的含量均高于正常胚，GA₃和ZT含量低于正常胚。胚败育果实的果肉中GA₃含量低而ZT含量高于胚正常果实果肉，IAA和ABA的含量在后期也高。胚中高含量的GA₃、ZT和低含量的ABA有利于胚正常发育。胚中ZT的下降和ABA的持续增加以及（GA₃＋IAA＋ZT）/ABA的比值小于其果肉中的比值，是导致胚胎败育的重要因素；在果实发育中，胚胎的败育和胚胎与果肉中（GA₃＋IAA＋ZT）/ABA均低是导致胚败育果实小的重要原因。5、通过对果实中自由态多胺（PAs）含量与胚胎败育关系研究发现：腐胺（Put）是杧果PAs的主要组成部分。败育胚胎的腐胺、亚精胺（Spd）、精胺（Spm）的含量以及（Spd+Spm）/Put、Spm/PAs的比值均小于胚胎发育正常果实，且差异显著。在胚胎败育的关键时期，胚胎正常果肉中Put、Spd的含量升高的幅度大于胚胎败育的，但是（Spd+Spm）/Put、Spm/PAs与胚胎败育果实果肉相比下降，这些因素与胚胎败育有关；果肉中Put含量维持在高水平的时间较长， Spm含量及其Spm/PAs比值在40d后急剧升高为胚胎正常发育的果实在体积上超过败育的起到重要的作用。6、通过了SSH技术探讨杧果胚胎败育过程中的基因表达差异，利用‘金煌’的正常胚胎和败育胚胎建立了两个cDNA文库，得到了1572个表达的tags（ESTs），其中1092个来自于正常胚胎，480个来自败育胚胎。在胚胎正常文库中，这些ESTs由783个非重复序列组成，其中包括147个contigs和636个singletons；在败育文库中有341个非重复序列，其中包括44个contigs和297个singletons。通过半定量PCR得到了不同类别的七个候选基因。其中，5个ESTs（MaAGA、MaSAS、MaAP2、MaERF和MaMADS）在败育胚胎中下调、MaMADS的表达水平下调明显。7、在谢花期，连续两次喷施低浓度的精胺、氯化钙和尿素对于提高大果率均有显著的效果。低浓度的精胺、尿素则在‘金煌’品种上效果显著。更多还原

【Abstract】 Mango （Mangifera indica L.） has been considered a famous tropical fruit in the world.Sanya city of Hainan province and its surrounding areas are located in the typical tropical areawhere mango fruit are usually harvested from April to May every year, but may also beharvested in advance from February to March by application of off-season productiontechnology, which contribute to a high economic benefit. However, the production of highratio of embryo aborted fruit seriously decreased the commodity rate of mango fruit, and ithas become the one of the main problems during the off-season mango cultivation. In thisstudy, the main cultivars ‘Jinhuang’ mango was used as experimental materials to investigatethe effect of the flowering on the physiological ecology in mango, to study on the change ofthe floral organ in off-season production, to study embryo abortion of the difference on themidway with normal embryos from cytology, hormone, polyamines and molecular levels, toexplore the reasons of embryo abortion, and to take measures to improve the ratio of theseed-fruit. The main results are presented as follows:1. The flowering efficacy was evaluated after spraying different concentrations of ethephon,potassium nitrate and its mixure with cytokinin. The results showed that the highconcentrations of ethephon reduced the chlorophyll contentes and the net photosynthetic rate.All treatments enhanced the content of the leaves zeatin and ratio of ZR/ABA, resulting in afaster flowering, which is associated with the increased C/N ratio in the leaves first. On theother hand, the effect of high concentrations of ethephon and potassium nitrate treatment isobviously earlier than that in low concentration induce, the various treatments reduce the ratioof hermaphrodite flower and the ratio of embryo normal fruit （big fruit containing seeds）.2. On the off-seasonal mango production, the stamens of ‘Jinhuang’ cultivar was shownas the degeneration, the significant difference has been observed in development of anther inwhich the bigger anther possessed the more pollen quantity with higher maturity, and only afew sac pollen showed the degeneration, the pollen be in live was still account for76.9%. Inthe female gametophyte, ovule appeared malformation, shrinking deformation and ovaryshowed growth sometimes in flat, there was similarity phenomenon around the conductingtissue cells in the embryo sac nuclear during fracture. When the megaspore developed in stage of eight nucleolus, there was a phenomena that the synergids, polar nuclei or antipodals insame or different ovules degenerated, all those factor might affect the fertilization of theovule.3. Djacent was polygonal, its vacuole was small and cell wall was twist with theosmiophilic deposits accumulated. The central cell bigger than normal, the proplastids wasfew，nutrients loss of activity, which affected the development of embryos and procambiumformed, the endosperm cells cannot be sustained division and development were the reason ofthe embryo stoped developing and induced abortion.4. The relationship between the endogenous hormones level and embryo abortion ‘Jinhuang’ mango fruit at early development stage was investigated. The results showed that thefruit embryo abortion was finished within30d, indicating20～30d could be critical periodfor embryo development. The difference in size between embryo normal and abortion fruitmainly depended on the fruit flesh; the IAA, ABA contents in embryo aborted were higherthan those in the seed at the initial stage, but the lower contents of GA₃and ZT were observed,compared to those in the normal embryo. In contrast to seed fruit, the lower GA₃contentswhile higher ZT, IAA and ABA levels were existed in embryo abortion fruit pulp. Moreover,the higher contents of GA₃and ZT and lower ABA level were favorable to normal embryodevelopment. The decreased ZT, enhanced ABA contents as well as lower（GA₃+IAA+ZT）/ABA ratio in embryo comparing with those in pulp were important factors leading to embryoabortion. During fruit development, with the embryo aborted, the lower ratio of（GA₃+IAA+ZT）/ABA in embryo and pulp were the important reasons resulting in small size in seedlessfruit.5. The relationship between the polyamines and embryo was studied by analyzing the freepolyamine content in mango fruit. The result showed putrescine was the predominantpolyamine in the three fractions, Put, Spd, Spm and （Spd+Spm）/Put were lower in embryoaborted fruit than those in normal embryo, and Spd showed significant difference betweenthem. Put and Spd contents increased to higher values in pulp than those in aborted one,the ratios of （Spd+Spm）/Put and Spm/PAs were lower than those in seedless fruit,all of thefactors involved in the embryo abortion.Put contents in pulp was maintained at higher levelfor a relatively long time, Spm contents and Spm/PAs raised sharply after40days fruit set andmaintained constant increase in embryo normal fruit, which could play an important roles inthe development period of the embryo normal fruit exceed than seedless fruit in size.6. Using subtractive suppression hybridization to investigate the difference in genesexpression involved in the process of embryo abortion. Two cDNA libraries from normal seedand aborted seed embryos of ‘Jinhuang’ mango were established to obtain one thousand five hundred and seventy-two high-quality expressed sequence tags （ESTs）, among which1092were obtained from the normal seed tester library and480from the aborted seed tester library.In normal seed tester library, ESTs were comprised of783unigenes, including147contigsand636singletons in contigs; there were297singletons in gene ontology （GO） indicatedcoverage of a broad range of GO categories in the borted seed tester library. Seven candidategenes from different categories were selected for semi-quantitative PCR analysis. Five ESTs（MaAGA、MaSAS、MaAP2、MaERFand MaMADS） were up-regulated in normal embryotissues compared to aborted embryos among them，and the expression of MaMADS inaborted embryos was significantly down-regulated.7. Spraying with low concentrations of spermine, Calcium chloride and urea alternation10days after bloom significantly improved the ratio of seeded fruit. Low concentrations ofspermine and urea had a significant effect on improving the seeded fruit ratio in ‘Jinhuang’variety.更多还原