Abnormal Structure of Embryo Sac in Autotetraploid Rice

The structures of mature embryo sacs in 13 genetic stock lines of autotetraploid rice （Oryza sativa L.）, including indica, japonica and javanica, were studied by using the whole-mount stain-clearing laser scanning confocal microscopy （WCLSM）. Among the 13 autotetraploid rice, the majority of ovaries possess normal polygonum-type embryo sacs, while a few ovaries were characterized by abnormal embryo sacs. The abnormalities of embryo sacs could be classified into six categories, i. e. no female germ unit, abnormal polar nuclei, embryo sac degeneration, no egg apparatus, small embryo sac and ＇double set＇ of embryo sacs. The frequency of abnormal embryo sac in japonica （26.6%） was higher than that in indica （19.34%）. In addition, the major abnormalities in each autotetraploid line varied, suggesting that the abnormalities may be related to the genotypes of the varieties.

Endosperm Development in Autotetraploid Rice

By using the laser scanning confocal microscope and plastic （Leica 7022 historesin embedding kit） semi-thin sectioning technique, comparative studies on the development of endosperm were carried out between autotetraploid and diploid rices. About one third of the ovaries in the autotetraploid showed normal endosperm development as those in the diploid. In these ovaries, one of the polar nuclei would fuse with the sperm nucleus, and the primary endosperm nucleus formed and underwent the first division in 4 hours after pollination; the anticlinal wall began to grow centripetally between the free nuclei starting from the wall ingrowths of the embryo sac near the micropylar end, and some of the phragmoplasts formed transformed into periclinal walls. In addition, some of the cell wall situated in the middle of the endosperm appeared to originate from phragmoplasts, whereas others seemed to develop randomly without the obvious formation of phragmoplasts. Cellulose began to accumulate in the wall of aleurone cell layer at 6 days after pollination. The cellulose wall of the cells of the aleurone cell layer appeared to have completely formed within 7 to 8 days after pollination. On the other hand, about two thirds of the ovaries in the autotetraploid showed abnormality in endosperm development with various types, such as non-fertilization, abnormal fertilization, endosperm development-delay and non-synchronization in the development of cellulose wall of cells of the aleurone layer. These abnormalities usually resulted in decreased seed setting in autotetraploid rice.

Biological Effects of Low Energy N^+ Beams Implantation on Calluses of Autotetraploid Rice

Calluses of two autotetraploid rice （DPR （4） and Zijing （4）） were implanted with 30 KeV N＋beams at doses ranging from 1.0×1015 to 9.0×1015 ions cm-2. The differentiation rates of calluses, the concentration of malondialdehyde （MDA）, the activities of superoxide dismutase （SOD）, catalase （CAT） and peroxidase （POD）, and the change of esterase （EST） spectrum were measured and analyzed on the calluses stage. The results showed that differentiation rates of calluses under 3.0×1015 ions cm-2 （（46.12±2.01）%） were higher than the other treatment groups in DPR （4）, and those of Zijing （4） were similar under the doses of 1.0×1015, 3.0×1015 and 5.0×1015 ions cm-2 （（40.55±2.74）, （37.77±3.04） and （34.56±2.65）%） and were higher than that of 7.0×1015 and 9.0×1015 ions cm-2. When the doses were 3.0×1015 and 5.0×1015 ions cm-2, the activities of SOD, POD and CAT were notably enhanced, and the accumulation of MDA content was markedly alleviated in the implanted calluses of two materials, as well as the activity of EST was increased by the additional isforms DPR （4）. Whereas the anti-oxidative systems and enzymes spectrum of EST were destroyed seriously, the accumulation of MDA was signiifcantly aggravated, also the differentiation rate of calluses reduced sharply and almost closed to zero when the implantation doses were 7.0×1015 and 9.0×1015 ions cm-2. It suggested that the proper dose of ion implantation effectively maintained the normal membrane structure and metabolism, and it would be also a feasible approach for autotetraploid rice to improve its genetic characteristics at calluses stage. Additionally, the differences of the radiation resistance between DPR （4） and Zijing （4） could be related to the different genotypes.

Effect of Ar^+ Implantation and Maize Genome DNA on Autotetraploid Rice

The effect of Ar^＋ beam implantation and maize genome DNA on autotetraploid rice is studied. Better mutation types and higher mutation rates were discovered in M2 of T3 with ion implantation and immersion in maize genome DNA. In the five agronomic categories investigated, the mutation rate of the seed setting rate was 9.1%, and the total mutation rate was 14.8% in the T3. However, the total mutation rate was 2.1% with the treatment of only ion implantation and 1.3% with the treatment of only immersion in maize genome DNA. Mutant FA36（4） was discovered in M1 with ion beam implantation and immersion in maize genome DNA. Its RuBPCase activity, PEPCase activity and seed setting rate were 32%, 153%, and 36.79%, respectively, higher than its parent IR36（4）. Rapid analysis of polymorphicDNA （RAPD） analysis of three M2 plants of FA36（4） （FMI, FM2, FM3） and two controls （purple maize and IR36（4）） were also conducted with 40 random primers. S5-3 was RAPD fragment amplified with a template of purple maize, FM2 and FM3 genome DNA using primer S5. There was no S5-3 in the RAPD pattern of IR36（4） or FMI.

Study of Genetics and Embryology of Polyembryonic Mutant of Autotetraploid Rice Induced by N^+ Beam Implantation

In the present study autotetraploid rice IR36-4X was treated by an ion implantation technique with nitrogen ion beams. A polyembryonic mutant （named IR36-Shuang） was identified in the M2 generation. The mutant line and its offspring were systematically investigated in regard to their major agronomic properties and the rate of polyembryonic seedling in the M3-M6 generation. The abnormal phenomena in the embryo sac development and the cytological mechanism of the initiation of additional embryo in IR36-Shuang were observed by Laser Scanning Confocal Microscopy. The results were as follows. 1） The plant height, the panicle length and 1000 grain weight of IRB6-Shuang were lower than that of its control by 35.41%, 5.08% and 15.72% respectively, Moreover, the setting percentage decreased 12.39% compared with that in normal IRB6-4X plants. 2） The polyembryonic trait of IRB6-Shuang was genetically stable and the frequency of the polyembryonic seedlings in the IR36-Shuang line was also relatively stable. 3） The rate of abnormal embryo sacs in IR36-Shuang was significantly higher than that in the control IR36-4X. 4） The additional embryo in IR36-Shuang might arise from the double Jet of embryo sacs in a single owry, antipodal cells or endosperm cells. These results suggest that IRB6-Shuang is a polyembryonic mutant and a new apomixis rice line induced by low energy ion implantation. The prospects for the application in production of the IR36-Shuang line are also discussed. The present study may provide a basis for future investigations of apomixis rice breeding via the ion implantation biotechnology.

同源四倍体水稻:低育性机理、改良与育种展望

同源四倍体水稻具有籽粒增大、营养成分增加和抗性增强等特点,但其育性普遍偏低,影响产量,无法直接应用。高育性四倍体水稻的成功创制解决了同源四倍体水稻育性偏低的瓶颈问题,然而该类型多倍体水稻能否在生产上推广应用需要进一步探讨。本文总结了同源四倍体水稻及其杂种F1育性偏低的细胞和分子遗传学机理研究的概况,重点介绍了高育性四倍体水稻的主要类型及最新的研究进展,最后提出未来利用新型四倍体水稻开展多代杂种优势等研究的设想,以期为水稻多倍体育种提供参考。

利用激光扫描共聚焦显微镜观察同源四倍体水稻胚囊的形成与发育

激光扫描共聚焦显微镜是研究植物胚囊的形成与发育过程以及胚囊变异的理想工具。同源四倍体水稻是二倍体水稻染色体加倍形成的。本文利用激光扫描共聚焦显微镜直接观察经透明处理的水稻子房,研究加倍的基因剂量对于四倍体水稻的胚囊形成与发育的影响。结果表明,广陆矮4号四倍体大部分的胚囊形成与发育的过程基本是正常的,一部分胚囊出现异常现象,在成熟胚囊时期异常频率为10 61%;L202四倍体的异常频率较高,成熟胚囊时期异常频率达到45 65%。

同源四倍体水稻花粉的发育特征

利用激光扫描共聚焦显微技术观察了同源四倍体水稻紫粳-4x花粉的发育过程及其败育时期和方式。在成熟花粉中正常花粉只占45.5%,还有54.5%的花粉在发育过程中发生败育。在小孢子母细胞减数分裂过程中出现了一些异常现象,如染色体不能正常配对、形成异常二分体及二分体分裂不同步等现象。绝大部分花粉败育发生在单核小孢子期和二胞花粉晚期。在败育花粉中典败花粉粒占21.4%,圆败花粉粒占5.0%,染败花粉粒占28.1%。导致紫粳-4x花粉败育的原因可能是由于其染色体数目加倍,在减数分裂过程中有些染色体不能正常配对,从而进行异常分裂造成的。

同源四倍体水稻花粉母细胞减数分裂期间微管骨架组织和结构变化

应用间接免疫荧光标记-激光扫描共聚焦显微术,观察了同源四倍体水稻花粉母细胞减数分裂过程的微管骨架和染色体行为变化。结果表明:同源四倍体水稻花粉母细胞微管组织形态变化与二倍体水稻的基本一致,但出现一些不同,如核周微管较长,纺锤体微管数量较多;并呈现许多异常现象。前期I细线期,微管数量少,分布不均匀;偶线期,呈现不规则网络状结构,未见明显的极性分布;粗线期,核仁解体,呈现混乱的网络状结构;双线期,微管提早解聚,不形成环绕状微管;终变期,微管数量稀少,核周未见明显的微管聚集;中期I纺锤体形态出现多种异常现象;末期I成膜体形状异常,出现点状微管;末期II-四分体时期,二分体细胞两端不分开;出现三分体细胞,其内微管混乱。以上各时期的染色体行为也出现不同程度的异常。综合认为,微管组织的异常可能与染色体的行为异常存在一定的关联,两者共同作用影响花粉发育,导致育性偏低。

同源四倍体水稻与假稻杂交结籽的胚胎学机理研究

利用激光扫描共聚焦显微技术对同源四倍体水稻与假稻杂交给籽的特殊生殖现象进行了研究。研究结果表明 ,假稻的花粉粒不能在二倍体水稻的柱头上荫发 ,因而二倍体水稻与假稻的生殖隔离很严格。然而 ,假稻的花粉粒在同源四倍体水稻的柱头上能萌发 ,前者的花粉管能在后者的花柱中伸长并能将雄配子送人胚囊内。在同源四倍体水稻与假稻的杂交中受精作用比较特殊 ,其中包括精细胞与卵细胞的单受精作用 ,也包括精细胞与次生极核的单受精作用 ,还包括正常的双受精作用。总受精率和总成胚率分别为 2 2 .89%和 2 0 .82 %。

同源四倍体水稻亚种间杂种胚囊的结构及受精特点

利用激光扫描共聚焦显微术对11份同源四倍体水稻亚种间杂种的胚囊结构和双受精特点进行研究，结果表明，所有四倍体杂种均存在一定频率的败育胚囊，败育频率因组合而异，最高的败育率为23．8％，最低为5．5％，平均14．3％。败育胚囊主要有胚囊退化、异常小胚囊、无卵器、无雌性生殖单位和极核位置异常等5种类型。杂种在双受精过程中也存在一定频率的异常，包括胚囊结构正常但未能受精和受精停滞等，其中未能受精的频率为3．5％～11．9％。平均7．9％，受精停滞的频率为1．1％～6．2％，平均3．7％。另外，还存在低频率（平均4．7％）的胚乳发育受阻。认为部分胚囊败育和受精异常（包括未能受精和受精停滞）是导致同源四倍体水稻亚种间杂种结实率降低的主要原因。

同源四倍体水稻雌雄配子体多态性的研究

同源四倍体水稻（２ｎ＝４ｘ＝４８）由于其细胞内染色体数的倍增而导致其生殖性状发生了明显的变化。在其雄配子体中存在着比较少的正常花粉粒（１３．３５％～１７．４９％）和比较多的败育花粉粒（８２．５１％～８６．６５％）。在败育花粉粒中包括典败、园败和染败花粉粒。在同源四倍体水稻ＡＰＩＶ（４）的成熟胚囊内存在着正常蓼型胚囊（４３．０％）、退化型胚囊（３６．０％）和变异型胚囊（２１．０％）。在变异型胚囊中包括双卵卵器胚囊（７１．４％）、三卵卵器胚囊（１７．５％）和无助细胞胚囊（１１．１％）。在不同季节条件下ＡＰＩＶ（４）颖花内的多卵频率存在着明显差异，在同一季节条件下强势颖花的多卵频率要明显地高于弱势颖花的多卵频率。由此认为，同源四倍体水稻的雌雄配子体表现出异常发育状态，其有性生殖能力已明显变弱。

低能氮离子注入同源四倍体水稻的生物学效应

以低能氮离子束为诱变源,对同源四倍体水稻进行注入处理后,对其生物学效应进行了研究。研究结果表明,同源四倍体水稻比二倍体水稻对低能氮离子束注入处理更敏感。在注入离子剂量为3×1017N+/cm2时,同源四倍体水稻所受到的损伤比二倍体水稻所受到的损伤更严重。4份同源四倍体水稻(即IR36(4)、IR28(4)、紫血稻(4)、明恢63(4))的成苗率分别是2.0%、3.5%、3.0%和4.0%,致死率非常高。在同样条件下,4份相应的二倍体水稻(即IR36(2)、IR28(2)、紫血稻(2)、明恢63(2))的成苗率分别是33.0%、31.5%、29.0%和24.5%,致死率也比较高。在经过注入处理后的当代群体内,4份同源四倍体水稻的平均变异频率为32.5%,而4份相应的二倍体水稻的平均变异频率为9.5%。在同源四倍体水稻IR36(4)和IR28(4)的变异群体内分别发现22株和14株结实率均达到75.0%以上的单株,其中在IR36(4)群体内有1单株的结实率高达91.89%;在紫血稻(4)的变异群体内发现2株具有双胚苗性状的单株;在二倍体水稻明恢63(2)的变异群体内发现1株具有红心米性状的单株。在第二代群体中,除了叶鞘变异和米质变异这两个变异性状能稳定遗传之外,其它变异性状在群体内都发生了明显的性状分离现象。同源四倍体水稻的高结实率特性和双胚苗特性表现出一定的可遗传性。

同源四倍体水稻胚乳发育:糊粉层细胞壁纤维素物质发育、胚乳淀粉积累及胼胝质“套”的形成

利用荧光显微术观察 ,发现同源四倍体水稻与其二倍体原种的糊粉层细胞壁纤维素物质都存在一个“充实”的过程。授粉后 5～ 6d,糊粉层开始分化形成 ,此时其细胞壁上未观察到纤维素物质 ;授粉后 6～8d ,糊粉层细胞壁上开始积累纤维素物质 ;授粉后 8～ 9d ,糊粉层细胞壁积累了大量的纤维素物质。胚乳淀粉的积累存在一个变化的过程 :初生胚乳核周围可见一些淀粉体 ,随着胚乳游离核的增多 ,游离核周围淀粉体的数量逐渐减少 ,胚乳细胞化后 ,胚乳细胞内开始大量形成淀粉体。利用苯胺蓝染色连续切片荧光观察 ,发现同源四倍体水稻及其二倍体原种胚乳发育过程中 ,在珠心表皮和内珠被之间存在一层胼胝质物质 ,围绕着胚囊并包裹住珠心呈“套”状。这个结构的形成变化过程为 :授粉后 30min在珠孔端珠心表皮细胞与内珠被之间开始积累少量的胼胝质 ,之后胼胝质逐渐增多并向合点端扩展延伸 ;授粉后 1～ 2d包裹着整个胚囊外的珠心而仅在维管束处断开 ;授粉后 9～ 13d,胼胝质“套”消失。该结构可能与胚和胚乳在发育期间的水分和养分的调节等有密切关系。同源四倍体水稻还存在糊粉层细胞壁发育不同步、糊粉层细胞壁结构异常以及胼胝质“套”不消失等异常现象。这些现象有可能会影响同源四倍体水稻的结实率。

同源四倍体水稻胚囊内反足细胞团的多态性

利用激光扫描共聚焦显微技术对 6份同源四倍体水稻在生殖发育过程中胚囊内反足细胞或反足细胞团的多态性进行了研究。结果表明 ,在同源四倍体水稻的雌配子体发育过程中已经存在着一些不正常的发育现象。在同源四倍体水稻的胚囊内反足细胞或反足细胞团的多态性比在二倍体水稻的胚囊内表现得更明显。反足细胞或反足细胞团的多态性主要表现为在胚囊内形态的多样性和位置的多样性 。

同源四倍体水稻胚乳发育:极核融合和胚乳细胞化

利用激光扫描共聚焦显微术和塑料包埋半薄切片技术观察同源四倍体水稻极核融合及游离核进一步发育的过程 ,发现同源四倍体水稻约有 1 /3子房极核融合过程和游离核进一步发育与其二倍体原种的基本一致 ;2 /3子房出现各种异常现象 ,如极核未受精、受精异常和胚乳游离核发育滞后等。应用荧光增白剂染色对胚乳细胞化过程进行观察 ,直接观察到胚乳初生壁的出现和形成过程以及不同部位的胚乳细胞壁的形成机理。表明同源四倍体水稻与二倍体水稻胚乳的细胞化过程基本一致 ,胚乳细胞初始垂周壁来源于珠孔端胚囊壁内突向心游离生长 ,初始平周壁来源于游离核有丝分裂产生的成膜体 ,而中间胚乳细胞壁则有些来源于游离核有丝分裂产生的成膜体 ,有些是自由生长而来。同源四倍体水稻胚乳细胞化过程也出现相当比例的异常现象 ,如无细胞壁形成、细胞化不同步和产生分枝状的细胞壁等。同源四倍体水稻胚乳发育过程中出现的极核融合。

利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察

应用改进的整体染色透明激光扫描共聚焦显微术(WCLSM),对同源四倍体水稻PDER-2B-4x胚囊的形成与发育过程进行观察。发现其胚囊的形成发育过程与二倍体的一致,可以清楚地划分为8个发育时期,即孢原细胞形成期、大孢子母细胞形成期、大孢子母细胞减数分裂期、功能大孢子形成期、单核胚囊形成期、胚囊有丝分裂期、八核胚囊发育期和成熟胚囊期。除正常发育的过程外,大孢子发育的各个过程均出现一些异常现象,包括:细胞退化、核位置异常、核数目异常和细胞分化异常等。这些异常可能最终导致多种结构异常成熟胚囊的形成。

同源四倍体水稻亲本材料的结实研究

研究了2份二倍体水稻和29份同源四倍体水稻(2n=4x=48)亲本(其中4份低代材料、25份高代材料)在2004年和2005年的结实情况.与二倍体对照相比,同源四倍体材料的花粉育性、结实率均不同程度下降,尤其低代材料的花粉育性(59.6%～65.3%)和结实率(26.8%～33.5%)大幅度降低;但经过选择改良,大多数材料的花粉育性和结实率均不同程度提高.F检验表明,同源四倍体不同品种间各性状均差异显著,说明同源四倍体材料具有很大的改良潜力.从1996年到2005年对部分同源四倍体水稻进行了连续选择改良,并对其结实率进行T检验,结果表明经过9a的选择改良,同源四倍体水稻结实率显著提高.本文还对同源四倍体水稻各性状间的相关性进行了分析,结果表明,结实率与花粉育性、穗着粒数、穗实粒数极显著相关,理论产量与花粉育性、有效穗数、穗着粒数、穗实粒数、结实率及千粒重极显著相关.

同源四倍体水稻恢复系TP-4和D明恢63与保持系D46B的细胞遗传学比较

以高结实率的同源四倍体水稻恢复系TP-4和D明恢63及优良保持系D46B为材料进行细胞遗传学研究。所有四倍体材料的染色体组成均为2n=48,这与有丝分裂的结果一致。恢复系TP-4和D明恢63及保持系D46B的中期Ⅰ单价体和三价体的比例都很低,配对染色体的比率在99%以上,具有优良的细胞学特征。恢复系TP-4和D明恢63在中期Ⅰ四价体频率分别为2.00/PMC和2.26/PMC,而保持系D46B在中期Ⅰ四价体频率为6.00/PMC,极显著地高于恢复系品系因而具有更好的染色体配对性质;后期Ⅰ保持系D46B的染色体滞后频率为10.62%,远低于恢复系材料TP-4的19.44%和D明恢63的23.14%,接近二倍体对照明恢63的7.30%水平;末期Ⅰ保持系D46B具有比恢复系更低频率的微核数而末期ⅡD46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。相关分析表明后期Ⅰ染色体滞后细胞比率同末期Ⅰ异常细胞比率呈极显著的正相关,推测后期Ⅰ染色体分离和末期Ⅰ微核形成可能是由相同的显性单基因或主效基因控制。

离子束生物技术改良同源四倍体水稻的设想

利用离子束注入技术对同源四倍体水稻进行遗传改良 ,有可能为进一步利用水稻强大的杂种优势效应和深入研究水稻无融合生殖现象寻找到新的突破口 .对同源四倍体水稻进行遗传改良的技术思路是 ,瞄准 2个总体目标 (挖掘水稻的产量潜力和简化水稻杂种优势利用的确定 ) ;采用 3种有效技术 (离子束生物技术、组织培养技术和多倍体诱导技术 ) ;确定 4个研究方向 (直接利用同源四倍体水稻的增产潜力、利用同源四倍体水稻的强大杂种优势效应、固定其杂种优势效应和深入研究同源四倍体水稻的生殖发育特性 ) ;达到 5个预期目标 (探索物种形成的途径、研究生物学特性、寻找遗传变异规律、挖掘潜在利用价值、建立新的育种技术体系 ) .