Development and characterization of interspecific hybrids between Oryza sativa and O. latifolia by in situ hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chro- mosome pairing was identified and most of the chromosomes were univalent, which resulted in com- plete male sterility in the hybrid.     

Development and characterization of interspecific hybrids between <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Subscript>O. latifolia</Subscript> by <Subscript>in situ</Subscript> hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization （GISH） and fluorescent in situ hybridization （FISH） analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase Ⅰ of the hybrid pollen mother cell, poor chromosome pairing was identified and most of the chromosomes were univalent, which resulted in complete male sterility in the hybrid.     

花粉愈伤组织群体偏态分离的SSR标记鉴定

利用水稻花粉育性基因S-b的SSR分子标记RMl3，鉴定籼粳稻杂种Fl的花粉愈伤组织的基因型，以确定每块愈伤组织在S-b座位的遗传来源，结果表明该座位的等位基因S^i或S^j在愈伤组织群体中分离比不符合1：1的规律，出现偏态分离现象．选择不同培养基及增加预冷处理不会改变偏态分离的方向，但预冷处理会增大偏分离的趋势．为进一步解释DH群体构建中的某些基因的偏态分离现象提供了依据，同时讨论了将SSR标记引入组织培养体系的可行性．     

中国水仙的胚胎学研究

中国水仙(Narcissustazetta var．chinensis)只开花不结实．以鳞茎球营养繁殖．在花芽发育过程中．减数分裂前的小孢子母细胞发育未显示异常．但在减数分裂过程中．许多细胞中显示出了异常的落后染色体现象．小孢子四分体主要呈左右对称型。细胞质分裂为连续型．四分体时期后。大部分小孢子呈空瘪状态。少部分小孢子含有染色较深的细胞质．约27％的花粉可发育成比较正常的二胞花粉．离体萌发实验中，有极少数花粉可萌发出花粉管。但在授粉的花柱柱头上未观察到花粉管．雌蕊由三心皮构成3室子房．中轴胎座．其上着生了上60～90个胚珠．胚珠倒生．双珠被，薄珠心．胚囊发生属于单孢子型，但大多数胚囊发育中途失败。只有约4％的胚囊可发育成7胞8核的蓼型胚囊．中国水仙雌、雄配子体发育异常和花粉在柱头上不能萌发是它只开花不结实的两个根本原因．     

十里香茶大小孢子发生发育中异常形态的研究

十里香茶在非盛花期(6,11月)所开的花中,花药和胚珠的异常率均最高。胚珠的异常常伴随着花药的异常而发生。绒毡层在各发育时期常出现局部增生;细胞异常肥大;核异常增殖常出现局部增生;细胞异常肥大;核异常增殖和凝集;细胞彼此融合等异常形态,这些异常形态常发生在药室内靠药隔的一侧,而相对一侧的绒毡层却退化甚至解体。绒毡层过早或过迟解体,这些异常都将对小孢子的发育产生极大的影响。花粉母细胞减数分裂时期出现染色体凝聚,核膜溶解推迟,壁形成异常和胼胝质过早或过迟解体等现象。花粉粒时期出现核异常分裂,解体,三核,空瘪等情况。大孢子母细胞在减数分裂期即解体,有的珠被不伸长形成珠孔,使珠孔呈开放状“喇叭口”,导致胚囊部分裸露,其内核解体甚至溢出胚囊外。曾观察到2～3个大孢子母细胞并存和多细胞卵器的现象。对某些异常现象产生的原因及在生产中的意义,胼胝质的功能等问题进行了讨论。     

Advances in the understanding of inter-subspecific hybrid sterility and wide-compatibility in rice

Hybrid sterility is a major form of postzygotic reproductive isolation and frequently occurs in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice （Oryza sativa L.）. It has been a major barrier for utilization of the strong heterosis expressed in hybrids between indica and japonica. A large number of loci for rice inter-subspecific hybrid sterility have been identified by genetic analysis. Cytological studies revealed that male and female gamete abortions and reduced affinity between the uniting gametes all occurred in indica-japonica hybrids, suggesting the complexity of the causes for inter-subspecific hybrid sterility. Two genes conditioning embryo-sac and pollen sterility respectively in indica-japonica hybrids have been cloned recently, providing opportunities for molecular characterization of the indica-japonica hybrid sterility and wide-compatibility. Future studies should aim at cloning more genes for indica-japonica hybrid sterility, characterizing the underlying molecular mechanism, and utilization of the findings for the development of inter-subspecific hybrids to increase rice productivity.     

水稻花药发育的细胞形态学精细分期

本研究通过观察籼型杂交水稻"川优9527"的花粉和药室壁的发育,对幼穗花药从雄蕊原基形成至花粉成熟的全过程进行了分期。选用不同发育时期的水稻幼穗(长度约1.0~10.0 mm),以石蜡切片技术分别制作其花药装片,置于光学显微镜(N ikon DS-R i1-U2)下观察并拍照。根据细胞及组织形态特征的显著变化,将花药的发育进程精细划分为:孢原细胞期、二层药室壁期、三层药室壁期、花粉母细胞形成期(以上4期对应小孢子母细胞形成期)、花粉母细胞减数分裂初期、花粉母细胞减数分裂中期、花粉母细胞二分体时期、花粉母细胞四分体时期(以上4期对应小孢子母细胞减数分裂期)、小孢子早期、小孢子中期、小孢子晚期、二胞花粉时期、成熟花粉时期等13个阶段。     

紫菜苔Ogura细胞质雄性不育系花药发育的细胞形态学研究

通过甘蓝型油菜Ogura细胞质雄性不育材料与紫菜苔的种间杂交和连续回交,获得了稳定的紫菜苔Ogura细胞质雄性不育系,细胞形态学研究表明,该不育系的花药发育兼有无花粉囊型和单核败育型的特征,由于没有造孢细胞的形成或造孢细胞退化,不育系最多只有1－2个花粉囊,而可育系都是4个花粉囊,发育起来的不育系花粉囊里面的小孢子到单核期后不再向前继续发育,而是迅速退化以至解体,开花前彻底败育,在减数分裂前期,不育系绒毡层细胞中已经出现液泡,有肥大趋势,在单核小孢子退化以后绒毡层还很发达,而正常花药绒毡层的发育在减数分裂过程中达到高峰,四分体时出现退化迹象,开花前已完全解体。     

不同授粉方式对祝光苹果受精过程的影响

祝光苹果自花授粉后2小时,花粉粒在柱头上萌发。授粉当日和次日,花粉管在花柱中生长。授粉后第三日,花粉管停止生长,最长花粉管平均长至花柱全长的26％。此时,花粉管先端明显膨大。而后,未见到有抵达花柱基部的花粉管,也未见到受精过程的发生。 异花授粉后4—6小时,大量花粉粒在柱头上萌发。授粉后1—2日,花粉管径直向花柱基部生长。授粉后50小时,花粉管穿过花柱基部,进入子室内。授粉后3—4日,观察到进入胚囊的花粉管将内含物释放到一个已退化的助细胞中。授粉后5—6日,观察到双受精过程。     

红莲型水稻不育系与保持系线粒体DNA酶切分析

为研究红莲型水稻细胞质雄性不育的分子基础，我们提取纯化了红莲型水稻丛广４１Ａ和丛广４１Ｂ的线粒体ＤＮＡ，采用限制性内切酶酶切片段长度多态性（ＲＦＬＰ）方法比较了红莲型不育系和保持系线粒体ＤＮＡ酶切图谱，发现两者之间存在一定的差异，部分支持了细胞质雄性不育与线粒体ＤＮＡ有关的结论。     

Restorable variation of cytoplasmic type of male sterile line in rice by in vitro culture

Rice male sterile (MS) lines, IR66707A and IR69700A, which possess the cytoplasm of Oryza perennis and O. glumaepatula respectively, belong to the cytoplasmic type. Their sterility could be maintained but not be restored. By somatic cell culture of these two MS lines, 47 somaclones with 465 R1 plants were obtained. All of the 465 R1 plants were sterile in the spring season in Guangzhou. According to the expression of the R1 plants and the level of similarity to their donor parents, they could be divided into three types. The plants of type Ⅰ were male sterile. The sterility of some somaclones of this type could be restored by the test crossing varieties or alternated to fertile by changes of some environmental conditions. The hybrid F1 of test cross from the MS somaclones in type Ⅰ was fertile while the hybrid F1 from the donor MS lines was still sterile. The R1 plants of type Ⅱ were similar to the donor parents and also male sterile. The hybrid F1 from all of the plants of type Ⅱ crossed to test variety were still sterile, so they did not possess restorability. For the somaclone of type Ⅲ, all of R1 plants were sterile in both male and female organs. No seed was set in both conditions of self and cross pollination. The fact that the restorable variants obtained in the cytoplasmic type of MS lines of rice by in vitro culture reported here should be the first sample in somaclonal variation in plant kingdom.     

一个药用野生稻异源单体附加系在减数分裂时期的GISH分析鉴定

异源单体附加系是从亲缘关系较远或属间的一个物种单条染色体附加到另一个物种中．栽培稻珍籼97B与药用野生稻Hy18杂交与连续回交，在BC2后代中得到一个药用野生稻单体附加系．生物素标屺的药用野生稻总DNA作为探针，未标记的栽培稻总DNA封阻，对其异源单体系减数分裂染色体进行基因组原位杂交．FISH结果表明，在栽培稻（AA，2n=24）基因组中附加了一条药用野生稻染色体，并鉴定为第8号染色体．研究表明，药用野生稻异源单体附加系的建立为药用野生稻的基因组学和遗传学的研究提供一个新的操作平台．而GISH技术在水稻远缘杂交育种中是最准确有效的染色体鉴定方法．在水稻育种改良中具有重要应用前景．     

Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L. and O. granulata Nees et Arn. ex Watt.

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice,which are the most valuable for study and utilization in China.In this study,the homology and physical locations of three rice resistance genes,Glh,Bph-3 and xa-5 are comparatively analyzed between O.sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH).The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata.By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O.sativa and O.granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O.sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O.sativa and O.granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.     

高效的苜蓿组织培养再生体系的建立

以保定苜蓿为实验材料,从外植体的基因型、培养基的选择,激素(KT)、水解酪蛋白的浓度等方面探讨了影响保定苜蓿组织培养的因素.结果表明:下胚轴比子叶作为外植体获得的效果更好;改良的SH培养基相对于UM培养基对愈伤组织的诱导更有效;在含有0.2mg/L KT和2 g/L水解酪蛋白的UM培养基上,得到了最大的胚状体诱导率;水解酪蛋白的加入提高了胚状体的质量和成熟率.     

麦冬(Ophiopogon japonicus(L.f.)Ker-Gawl)的胚胎学研究——大、小孢子发生,雌、雄配子体形成过程及多糖物质动态

麦冬花药壁层发育为单子叶型,绒毡层细胞消解介于分泌型与变形型间,小孢子母细胞减数分裂时,胞质分裂为连续型,四个小孢子在四分体中排列为左右对称式。成熟花粉粒为二细胞的,具单萌发沟。麦冬为倒生胚珠,双珠被、厚珠心,具珠柄珠孔塞,大孢子母细胞减数分裂后形成线形四分体。胚囊发育为蓼型。成熟阶段,助细胞珠孔端壁上具帚状分枝的丝状器。大、小孢子发生及雌、雄配子体形成中,多糖物质的积累与消失是有规律的。     

苎麻雄性不育系与保持系花药发育的比较观察

 为研究苎麻雄性不育的早期表现和细胞学特征,采用常规石蜡切片,在显微镜下观察苎麻雄性不育系GS14-1及保持系GS13-X₁的雄蕊发育过程.发现保持系GS13-X₁的花药发育正常,形成正常的花粉粒;不育系GS14-1的雄性败育发生在较早阶段,多在雄花被形成后雌雄蕊原基分化期或花药形成初期发生败育,表现为雄蕊原基畸形或退化.但其败育的真正原因尚不清楚,有待进一步研究.     

糜子有性生殖过程中的特异现象

简要报道了糜子有性生殖过程中的一些特殊现象.如花粉败育、小孢子均等分裂、二细胞抱原、双卵卵器、助细胞状卵,双胚囊、三核合子和单受精等.     

NaCl对高蛋白糯稻品系H1701胚培养力的影响

以糯稻Ｈ１７０１为材料,通过胚培养诱导愈伤组织,继代培养并分化,实验结果表明,ＮａＣｌ对胚性愈合伤组织的诱导和生长有不同程度的抑制作用,ＮａＣｌ浓度越高,抑制越明显,转人发化培养基后以后,除含１０ｇ／Ｌ及以上浓度ＮａＣｌ的诱导培养基中愈伤组织分化为零外,其余均能分化出苗,其中,含ＮａＣｌ２ｇ／Ｌ的诱导培养基中诱导出愈伤组织分化率最高。     

寄主植物对不同龄期中华稻蝗若虫酯酶活性的影响

以中华稻蝗为研究对象,以狗尾草、小麦、晋稻8号、晋稻9号、旱稻297号为寄主植物,测定了饲喂不同寄主植物后中华稻蝗1-5龄若虫的酯酶活性.结果表明,饲喂不同寄主植物后,中华稻蝗各龄期若虫酯酶比活性均是狗尾草组最低,晋稻9号组最高,且两者差异显著,晋稻9号组的活性是狗尾草组的1.65-2.22倍,推测晋稻9号可能有某种特殊的物质,对中华稻蝗若虫酯酶的诱导作用较强.研究结果还显示,无论饲喂何种寄主植物,中华稻蝗若虫酯酶比活性均表现为随着龄期的增长,活性逐渐增高的趋势.     

Restorable variation of cytoplasmic type of male sterile line in rice byin vitro culture

Rice male sterile (MS) lines, IR66707A and IR69700A, which possess the cytoplasm of Oryza perennis and O. glumaepatula respectively, belong to the cytoplasmic type. Their sterility could be maintained but not be restored. By somatic cell culture of these two MS lines, 47 somaclones with 465 R₁ plants were obtained. All of the 465 R₁ plants were sterile in the spring season in Guangzhou. According to the expression of the R₁ plants and the level of similarity to their donor parents, they could be divided into three types. The plants of type Ⅰ were male sterile. The sterility of some somaclones of this type could be restored by the test crossing varieties or alternated to fertile by changes of some environmental conditions. The hybrid F₁ of test cross from the MS somaclones in type Ⅰ was fertile while the hybrid F₁ from the donor MS lines was still sterile. The R₁ plants of type Ⅱ were similar to the donor parents and also male sterile. The hybrid F₁ from all of the plants of type Ⅱ crossed to test variety were still sterile, so they did not possess restorability. For the somaclone of type Ⅲ, all of R₁ plants were sterile in both male and female organs. No seed was set in both conditions of self and cross pollination. The fact that the restorable variants obtained in the cytoplasmic type of MS lines of rice by in vitro culture reported here should be the first sample in somaclonal variation in plant kingdom.