【作者】 李旭新；

【导师】 路丙社；

【作者基本信息】 河北农业大学 ， 林木遗传育种， 2013， 博士

【摘要】 本研究以阿月浑子和黄连木为材料，从形态解剖学、孢粉学和生理生化特性等方面系统开展了阿月浑子花期物候、雌雄配子体发育、花粉形态及萌发特性的研究，为进一步开展远缘杂交奠定了科学依据；以阿月浑子和黄连木杂交组合为研究对象，利用荧光显微观察和离体胚培养技术，探讨了远缘杂交授粉受精过程的特点、远缘杂交亲和性以及杂种胚败育进程，明确了杂种胚败育的关键时期和特点，建立了阿月浑子和黄连木远缘杂交的杂种胚挽救技术体系，对杂种F1代适应性进行了初步评价，并对阿月浑子和黄连木幼苗的耐盐性进行了系统研究。研究结果如下：1.阿月浑子雄株T-491和黄连木花序于3月20日左右开始发育，花序生长期持续约15d，而雌株Kerman的花序生长较晚，始于3月28日左右，花序生长期持续约20d。雌株Kerman初花期为4月9～10日，而阿月浑子雄株T-491的散粉物候期与雌株Kerman的花期在4月10～12日重叠3d。雄株黄连木散粉物候期与Kerman的花期在4月10～14日重叠5d。2.利用常规石蜡切片技术，对阿月浑子的大小孢子发生及雌雄配子体发育过程进行了观察，探讨在其发育过程中是否存在生殖障碍。结果表明：（1）花药壁由外向内分别为表皮、药室内壁、中层和绒毡层。绒毡层类型为腺质绒毡层。（2）小孢子母细胞减数分裂过程中的胞质分裂方式为同时型，小孢子四分体排列方式为左右对称型。（3）成熟花粉粒为2-细胞型，具有3个萌发孔。（4）子房具有1个胚珠，基生胎座，双珠被，厚珠心，倒生型；胚囊发育类型为蓼型。（5）雌配子体发育过程存在部分胚珠退化现象。3. T-11花粉为圆球形，其余为长球形。网状纹饰，除T-11网眼为穴状外，其余均为圆形或近圆形；T-491花粉表面着稀疏白色刺状颗粒，其余5个品种均密着白色刺状颗粒；T-491、黄连木、UCB-1和甘谷的花粉萌发孔为椭圆形，Peter花粉萌发孔为圆形，T-11花粉萌发孔为沟状；花粉粒大小顺序依次为：Peter＞黄连木＞T-491＞T-11＞甘谷＞UCB-1。4.阿月浑子和黄连木花粉离体萌发适宜培养基为：蔗糖浓度为15%，硼酸浓度为100mg/L，适宜的培养温度25℃。最适宜阿月浑子和黄连木花粉萌发和花粉管生长的赤霉素（GA3）和2,4-D浓度分别为25mg/L和5mg/L，而萘乙酸（NAA）浓度为0.5mg/L适宜两种花粉萌发。当温度或浓度升高或降低时，花粉萌发率和花粉管长度在一定程度上均会受到抑制。贮藏后花粉内SOD、POD、CAT酶活性呈现先升高后降低的变化趋势。5.利用荧光显微镜观察发现，授粉后杂交组合柱头附着和萌发花粉数比对照组合少，且花粉萌发明显滞后，到达花柱和子房的花粉管数少于对照组合。杂交组合的柱头和花柱中有大量不规则胼胝质沉积，花粉管出现弯曲、膨大变粗等现象，授粉后24h，极少数花粉管进入子房，72h完成受精过程。杂交组合花粉管生长异常、胼胝质形成和沉积是引起杂交不亲和的主要原因。6.对阿月浑子和黄连木远缘杂交杂种胚败育时期进行了调查，建立了阿月浑子远缘杂种胚培养（抢救）技术体系。结果表明：杂交组合果实存在胚败育现象。果实内存在未完全退化的珠被，且珠柄上有褐变斑出现，坏死部分扩展到胚囊。阿月浑子杂种胚挽救的适宜胚龄为授粉后100～120d，杂种胚的发育始于授粉后80d，80d至100d是杂种胚败育的关键时期。适宜杂种胚培苗增殖的培养基为：DKW+2.0mg/L6-BA+0.05g/L IBA+0.3g/L LH+1.5g/L PVP，增殖系数为3.47，苗高为3.32cm。适宜杂种胚培苗生根的培养基为：1/2WPM+2.0mg/L IBA+0.05mg/L NAA，生根率为80%。7.对父本黄连木、母本阿月浑子和杂种F1代适应性初步分析认为杂种F1代抗病性和抗寒性优于两亲本。杂种F1代净光合速率日变化规律为明显的双峰曲线，有明显的午休现象，净光合速率、气孔导度、胞间CO2浓度和蒸腾速率及Fo、Fv/Fm和Fv/Fo均高于两亲本。人工低温处理过程中，三种树木枝条失水速率呈现逐渐升高的变化趋势；SOD、POD活性呈现先升高后降低的变化趋势，而MDA含量呈现升高的变化趋势；可溶性糖含量随处理温度的降低呈现升高的变化趋势。8.对阿月浑子和黄连木幼苗耐盐性的研究可知：(1)随着NaCl胁迫强度的增加，阿月浑子和黄连木叶片细胞膜透性、SOD活性呈逐渐升高的趋势；MDA含量呈先降低后升高的变化，而POD活性呈降低趋势；黄连木叶片中APX活性和阿月浑子叶片中GR活性呈先升高后降低趋势。（2）NaCl胁迫降低了叶绿素a、b含量，提高了阿月浑子a/b比值，而黄连木叶绿素a/b比值虽有下降，仍显著高于对照；净光合速率(Pn)、气孔导度（Gs）逐渐降低，NaCl浓度小于0.3%时，叶片净光合速率下降主要受气孔限制；NaCl浓度大于0.3%时，叶片净光合速率下降主要由非气孔因素限制。（3）随着NaCl浓度的升高，量子产额或能量分配比率(φPo、φo和φEo)、单位面积捕获的光能（TRO/CSO）、电子传递的量子产额（ETO/CSo）、单位面积的反应中心的数量（RC/CSO）逐渐降低，而单位面积光合机构的比活性参数（ABS/CSO）、热耗散的量子比率（φDo）逐渐增加。（4）随着盐胁迫程度的加重，阿月浑子和黄连木叶片中Na+含量、Na+/K+均呈升高趋势，而K+含量呈先升高后降低的变化。叶片中Ca、Mg、Fe、Zn含量均随着NaCl浓度的升高呈现逐渐降低的变化趋势。（5）NaCl浓度大于0.3%时，胞外电阻率r e、弛豫时间τ和弛豫时间分布系数均持续减小。更多还原

【Abstract】 The flower bud differentiation，male and female gametophyte development male andthe optimal conditions of pollen germination for Pistachio (Pistacia vera L.)and PistaciaChinensis Bunge were studied by using of anatomy, palynology and morphological，physiological and biochemical analysis. In order to understand the the key period of hybridembryo abortion characteristic and establishing hybrid embryo rescue technology system,the process of “Pistacia vera and Pistacia chinensis” cross, the hybridization compatibility，the fertilization and the embryo abortion were observed by using fluorescence microscopyand in vitro embryo technology. The adaption of F1hybrid such as salt tolerance and coldresistance ablity were also evaluated in the paper. The results are as follows:1. The discipline of pistachio male plants of T-491and Pistacia Chinensis Bungeinflorescence growth earlier，since March20，inflorescence growing season lasts about15d，and the female plants Kerman late inflorescence growth begins on about March28，while the later on about20d. Early flowering female plants Kerman begins about on April9to10，and the phenophase of Kerman and male plant T-491overlap in the April10to12，only3d. The phenophase of Pistacia Chinensis Bunge and Kerman flowering overlap inthe April10to14，about5d.2. The microsporogenesis，megasporogenesis，and development of the male and femalgametophytes of Pistachio were observed by paraffin section technique to test whetherthere is any obstacle of reproduction in the process of sexual reproduction.The resultsshowed:(1) The wall of anther consists of epidermis endothecium，middle layers andtapetum from out to inside，tapetum cell is of glandular tapetum.(2) Cytokinesis duringmeiosis of the microspore mother cell was simultaneous type，and tetrads were isobilateral.(3) Mature pollen grain was2-cell type with one or three germ pores (4) There is an ovarythat contains one ovule，ovules occur at the base of ovary. The ovule was anatropous，bitegmic and crassinucellate. The development of embryo sacs belonged to polygonumtype.（5）Some of female gametophyte was abnormal.3. The pollen grains were long-globous existing except with T’11pollen. The exineornamentation were all reticulate sculpture. The shape of T-11’s mesh was foveolae whilethe others’ were round or approximately round. The exterior of the T-491’pollen has a fewwhite grains and that of the other five breeds had a lot of white grains. The shape of T-11’sgerminal aperture was furrowed，that of Peter was round and that of the T-491，the PistaciaChinensis Bunge，UCB-1and Gan Gu were oval. The order of pollen size is: Peter＞thePistacia Chinensis Bunge＞T-491＞T-11＞Gan Gu＞UCB-1.4.The bourgeon pollen of Pistachio and Pistacia Chinensis Bunge suitable medium was composed of15%sucrose，100mg/L boric acid，cultivated at25℃；The suitableconcentrations of GA3，2,4-D for the pollen germination and tube growth of Pistachio andPistacia Chinensis Bunge were25mg/L and5mg/L which was the pollen germination rateand the length of pollen tube reached to the largest. The concentration of NAA was0.5mg/L，pollen germination rate reached to the largest.When the temperature or theconcentration were rised or reduced，the pollen germination rate and the length of pollentube would be restrained in a certain degree. Pollen activity of SOD，POD and CATactivity after storage present first increased and then decreased.5. Using fluorescence microscopy observed that the the hybrid combinations stigmaattached after pollination and germination of pollen count is less than the contrast，pollengermination has lagged far behind，and the number of pollen tubes reach the style andovary less than that of the contrast. Intergeneric Cross of stigma and style in a largenumber of irregular callose deposition，pollen tube bending，swelling thicker phenomenon，24h after pollination，a handful of pollen tube enters the ovary at24h，and finishedfertilization at72h after hybridization. Abnormal pollen tube growth and callose depositingafter pollination may be caused by the incompatibility of intergeneric cross betweenPistachio (Pistacia vera L.)and Pistacia Chinensis Bunge.6. Embryo abortion stage and rescue system of hybrids were studied in the distanthybridization between Pistachio(Pistacia vera L.) and Pistacia Chinensis Bunge. Theresults showed: Intergeneric cross has embryo abortion phenomenon after pollination.notcompletely degraded integument and funicle browning spots，necrotic part of the extensionto the embryo sac in fruit. Pistachio hybrid embryo rescue embryo appropriate age100to120days after pollination，the development of hybrid embryo began from80days afterpollination，and the critical period for the hybrid embryo abortion was from the80to100days.The suitable multiplication medium for immature embryo seedlings was DKW+2.0mg/L6-BA+0.05mg/L IBA+0.3g/L LH+1.5g/L PVP，with which the multiplicationcoefficient of3.47and the seedling height of3.32cm were obtained. The suitable rootingmedium was1/2WPM+2.0mg/L IBA+0.05mg/LNAA with a rooting rate of80%.7. The adaptive initial analysis showed that the hybrid F1generation of resistancedisease and cold hardiness better than the two parents. The curve of diurnal variation of Pnin Hybrid F1showed two peaks.with obvious midday depression of photosynthesis，netphotosynthetic rate,stomatal conductance，intercellular CO2concentration and transpirationrate and Fo，Fv/Fm and Fv/Fo were higher than that of two parents. Artificial lowtemperature process，the three tree twigs water loss rate has the trend gradually increased；SOD，POD activity first increased and then decreased trend and MDA content showedincreased；with the treatment temperature the reducting，the soluble sugar content hasincreased trend. 8. Research on salt tolerance of the Pistacia vera L.and Pistacia chinensis BungeSeedlings show that（:1）With the increase of NaCl stress concentration，the cell membranepermeability，SOD activity gradually increased；MDA content has first decreased thenincrease in Pistacia vera L.and Pistacia Chinensis Bunge leaves，while POD activitydecreased;APX activity in Pistacia Chinensis Bunge leaves and GR activity in Pistaciavera L.has first increased and then decreased trend.（2）the content of Chl·a and Chl·b inPistacia vera L.and Pistacia Chinensis Bunge leaves were decreased while the Chla/b ratiowas enhanced of Pistacia vera L. by NaCl stress. The Chla/b ratio of Pistacia ChinensisBunge leaves has decreased，still significantly higher than the control; net photosyntheticrate(Pn)，stomatal conductance(Gs) of leaves significantly decreased.The decreased of Pnwas mainly attributed to stomatal limitation when the concentration of NaCl was lowerthan0.3%and to non-stomatal limitation when the concentration was higher than0.3%（3）With the increase of NaCl stress concentration，Trapped energy flux per RC（TRO/CSO），Electron transport flux per RC（ETO/CSo），Density of RCs（RC/CSO），Yields or fluxratios(φPo，φo和φEo) decreased，and Absorption flux per CS(ABS/CSO)， quantum yieldfor energy dissipation (φDo) increased.(4) With the increase of NaCl stress concentration,the content of Na+，Na+/K+ratio were increased，the content of K+were increased firstlyand then decreased in Pistacia vera L.and Pistacia Chinensis Bunge leaves.And with theincrease of NaCl concentration，the content of Ca，Mg，Fe，Zn content graduallydecreased.(5) When the concentration was higher than0.3%，the extracellular resistivity,relaxation time and distribution coefficient of relaxation time decreased consistently.更多还原