DNA,RNA, AND PROTEIN COMPARISONS BETWEEN NODULATED AND NON-NODULATED MALE-STERILE AND MALE-FERTILE GENOTYPES OF SOYBEAN (GLYCINE MAX L.)

Four soybean (Glycine max L. Merr.) lines isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁) were developed to study the effects of reproductive development and nitrogen source on the nucleic acid and protein levels within the leaves. Changes in DNA, RNA, protein, and cellular viability were measured from flowering (77 days after emergence) until maturity (147 days after emergence) in leaves of nodulated and non-nodulated male-sterile and fertile soybean genotypes. Leaf nuclei from the sterile genotypes yielded DNA amounts that were significantly higher than those from the fertile lines. The average DNA values for the nodulated sterile and nodulated fertile lines at 147 days after emergence were 7.01 and 2.45 picograms, respectively. The average 2C DNA amount as determined from dividing root-tip nuclei was 2.83 picograms, which indicated occurrence of endopolyploid mechanisms in the sterile lines and age-related loss of DNA in fertile lines. Similar to DNA findings, the RNA and protein values in the sterile lines were significantly higher than those values observed in the fertile lines, suggesting an increased capacity to synthesize protein. The soybean leaf nuclear DNA declined, especially in the fertile lines in terms of the percent endopolyploid nuclei as well as the average DNA content during maturation. The DNA decline in leaves of fertile genotypes suggests that the leaves may be exporting nucleosides and phosphates to the seeds during embryo formation. In the sterile lines, due to the reduced pod-set, these ready reserves of nucleosides and phosphates tended to accumulate in the chromatin of the leaf nucleus as manifested by the DNA specific Feulgen stain. By the end of the study (147 days after emergence), the nodulated fertile genotypes had experienced a dramatic loss in DNA, RNA, and protein. The nodulated sterile genotypes, however, indicated 65% more DNA, 59% more RNA, and 53% more protein as compared to the nodulated fertile genotypes at 147 days after emergence. The sterile lines also indicated the slowest increase in the death of cells, while the fertile lines indicated the fastest increase in nonviable cells, as shown by trypan blue staining. The fertile lines displayed normal monocarpic senescence throughout the study. The reproductive structures of fertile plants utilized the molecules in seed production, whereas in the sterile lines, these accumulated in leaf cells.     

小麦T型细胞质雄性不育系、保持系蛋白质双向电泳比较研究

以小麦T型细胞质雄性不育系为材料,利用双向电泳技术,对苗期、分蘖期、拔节期和孕穗期叶片和花粉母细胞减数分裂期、单核小孢子期、二—三核小孢子期蛋白质变化作了分析。在细胞质雄性不育系小麦拔节期、孕穗期叶片中,有一个33KD/PI6.3蛋白组分存在,保持系中没有发现这个蛋白组分。在花粉败育的关键时期二—三核小孢子期,小麦细胞质雄性不育系有53KD/PI5.5、50KD/PI5.7、48KD/PI5.6和20KD/PI7.5四种蛋白组分存在,而保持系中也没有存在。小麦细胞质雄性不育系叶片和小孢子发育过程中存在的这五种特异蛋白可能参与育性调控,与细胞质雄性不育特性的形成有关。     

小麦T型细胞质雄性不育系、保持系蛋白质双向电泳比较研究

以小麦T型细胞质雄性不育系为材料,利用双向电泳技术,对苗期、分蘖期、拔节期和孕穗期叶片和花粉母细胞减数分裂期、单核小孢子期、二—三核小孢子期蛋白质变化作了分析。在细胞质雄性不育系小麦拔节期、孕穗期叶片中,有一个33KD／P16．3蛋白组分存在,保持系中没有发现这个蛋白组分。在花粉败育的关键时期二—三核小孢子期,小麦细胞质雄性不育系有53KD／P15．5、50KD／P15．7、48KD／P15．6和20KD／P17．5四种蛋白组分存在,而保持系中也没有存在。小麦细胞质雄性不育系叶片和小孢子发育过程中存在的这五种特异蛋白可能参与育性调控,与细胞质雄性不育特性的形成有关。     

Microsporogenesis Abortion in Cytoplasmic Male Sterile Plants from H. petiolaris or H. petiolaris fallax Crossed by Sunflower (Helianthus annuus)

The microsporogenesis of two sunflower lines carrying two differentcytoplasmic male sterilities (CMS) from H. petiolaris (PL) andfrom H. petiolaris fallax (PF) has been studied and comparedto the normal process in male fertile lines. The first signsof abortion are, respectively, the vacuolation of the endoplasmicreticulum in PL during the first division of meiosis and theabnormal deposition of the endexine layer in PF during the vacuolationof the microspore. The genotype of the male line seems to modulate the stage ofpollen abortion, Furthermore, two ways of abortion have beenobserved for a single male sterile plant. Consequently, we suggestthat the ultrastructural observations reflect only the mainconsequences of a primary unknown event which takes place earlier. Cytoplasmic male-sterility, meiosis, Helianthus annuus L, H. petiolaris Nutt, microsporogenesis, electron microscopy     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Mechanism of male sterility in Petunia

Summary The free amino acid contents in the anthers of male fertile, cytoplasmic male sterile (cms) and genic male sterile (gms) petunia lines were compared at different developmental stages of the male gametophyte. Quantitative differences in the amounts of free amino acids were found between the fertile and male sterile lines and between the cms and gms lines. The differences between the sterile lines were correlated with the different developmental stages at which the breakdown in microsporogenesis occurred. In the Rosy Morn (RM) cms line, where breakdown of microsporogenesis occurred at the end of prophase 1, there was an associated increase in asparagine and decrease in the other amino acids. In the RM gms line, in which breakdown occurred at the tetrad stage, an accumulation of asparagine in the anthers corresponded with an accumulation of glutamine beginning at prophase 1. Compared with fertile anthers, the sterile anthers accumulated much proline at the early meiotic stages, but no -aminobutyric acid. Comparison of the free amino acids of the fertile and the male sterile lines indicates that certain biochemical events leading to breakdown of microsporogenesis precede the observed cytological breakdown. The results from adding asparagine and glutamine to extracts of anthers at different developmental stages suggest that the amino acid balance may contribute to the changes in pH in the fertile and male sterile anthers which we observed previously.Contribution from the Volcani Center, Agricultural Research Organization Bet Dagan, Israel. 1972 Series, No. 2083 E.     

小麦粘类雄性不育系生化标记及小孢子细胞色素氧化酶同工酶研究

对4种同核异质小麦粘类非1BL/1RS雄性不育系、保持系和恢复系的幼苗叶片、乳熟期籽粒以及不育系、恢复系和F1小孢子发育四分体至三核期花药进行了细胞色素氧化酶（COD）同工酶聚丙烯酰胺凝腔电泳（PAGE）分析。结果表明：（1）幼苗叶片COD同工酶谱带可以标记4种不育系和保持系;乳熟期籽粒COD同工酶谱带可以将4种不育系、保持系及恢复系区别开。（2）COD在不育系小孢子败育时或败育之前（单核到二核期）酶量降低,面在三核期酶量升高。（3）相同胞质背景下引入不同核恢复基因或不同胞质背景下引入桢核恢复基因,F1小孢子COD同工酶谱带之间有差异。可以将不同发育时期COD同工酶谱带作为鉴别1种不育系以及不育系、保持系、恢复系（“三系”的可靠生化标记）。     

Leaf Nitrate Reductase, d-Ribulose-1,5-bisphosphate Carboxylase, and Root Nodule Development of Genetic Male-Sterile and Fertile Soybean Isolines

The objectives of this study were to determine the effect of pod and seed development on leaf chlorophyll concentration, and on activities of leaf ribulose bisphosphate carboxylase, leaf nitrate reductase, and root nodule acetylene reduction in field-grown soybean (Glycine max [L.] Merr.). Two genetic male-sterile lines and their fertile counterparts (Williams and Clark 63) were compared in both 1978 and 1979. Two additional lines (Wells × Beeson and Wells × Corsoy) were compared in 1979.

The expression of male-sterile character was nearly complete as very little outcrossing due to insect pollinators was observed. Male-sterile plants showed a delayed late season decline in leaf chlorophyll content and ribulose bisphosphate carboxylase activity when compared with fertile plants. A slight delay in the loss of in vivo leaf nitrate reductase activity was also observed for male-sterile plants. Root nodule fresh weight and acetylene reduction activity declined slightly more rapidly for fertile lines than for male-sterile lines in both years with differences significant on the last two to three sampling dates as leaf loss occurred in the control plants.

Seed development was found to increase slightly, the rate of decline of metabolic activity in fertile lines compared with that of male-sterile lines. However, pod development was not an a priori requirement for leaf and root nodule senescence. Male-sterile plants also lost photosynthetic and nitrogen metabolic competence, but at a slower rate. These results support the concept that pod and seed development does not signal monocarpic senescence per se but rather affects the rate at which senescence occurs after flowering.

     

The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.     

Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.)

Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.     

Small mitochondrial DNA molecules of wild abortive cytoplasm in rice are not necessarily associated with CMS

Summary Mitochondrial DNA was isolated from leaf tissue of both the cytoplasmic male sterile line of Indica rice variety V41, which carries wild abortive (WA) cytoplasm, and from the corresponding maintainer line. In addition to the main mitochondrial DNA, four small plasmid-like DNA molecules were detected in both the male sterile and fertile lines. Restriction analysis of total mitochondrial DNA from the male sterile and fertile lines showed DNA fragments unique to each. Our findings suggest that the four small mitochondrial DNA (mtDNA) molecules are conserved when WA cytoplasm is transferred into different nuclear backgrounds. However, there is no simple correlation between the presence/ absence of small mitochondrial DNA molecules and the expression of WA cytoplasmic male sterility (CMS).     

主要抗蚜小麦品种(系)的抗性类型及其生化抗性机制

通过对10个抗麦蚜品种(系)室内苗期生命参数、抗性类型和抗蚜次生物质的研究,明确了不同抗性级别的品种对麦蚜种群控制力及部分生化抗蚜机制。实验结果表明,参试的抗蚜品种(系)中30%左右为不选择性：表现为爬行频繁,定殖率低,但是定殖个体生长发育良好;70%为抗生性;表现为使麦长管蚜Sitobion avenae(F.)和禾谷缢管蚜只Rhopalosiphum padi(L.)的发育历期分别延长2.1%～28.2%和3.7%～13.9%,若蚜死亡率增加1.0～3.6倍和1.0～2.25倍,平均寿命缩短10.2%～96.5%和37.5～97,1%,繁殖力下降3.4%～72.8%和25%～97.2%。苗期生化测定结果表明：不同抗源的单宁和总酚含量明显高于感蚜品种,其总酚含量与抗麦长管蚜级别呈显著负相关,以抗生性为主的品种其总酚含量亦与麦长管蚜的内禀增长力(r_m)呈显著负相关(P<0.05),表明总酚是小麦抗长管蚜的关键因子之一,而与禾谷缢管蚜抗性水平无关;单宁含量与麦蚜抗性关系不密切。     

光照强度对温光敏细胞核雄性不育小麦育性的影响

以具有低温不育、高温可育特性的温光敏细胞核雄性不育小麦C412S和C404S为材料,以其回交转育亲本、育性正常的C412和C404为对照,用人工气候箱研究了光照强度对温光敏细胞核雄性不育小麦育性表达的影响。结果表明,在低温条件下(日温8℃/夜温6℃),C412S和C404S在不同光照强度(160μmol/m2.s和300μmol/m.2s)下自交结实率都为0,表现为完全不育。在较高温度条件下(日温18℃/夜温14℃),从花粉母细胞形成期到开花期的光照处理,C412S在160μmol/m.2s弱光照下的自交结实率仅为5.4%,表现为高不育,在300μmol/m2.s较强光照下的自交结实率高达65.0%,表现为高度可育;而另一不育系C404S在2种光照强度下的自交结实率分别为69.9%和73.2%,都达到了高度可育水平。表明光照强度对温光敏细胞核雄性不育小麦的雄性育性表达具有重要影响,但不同材料对光照强度的响应程度有所差异。     

玉米雄性不育系及其同型可育系根系的时空变化

采用土柱栽培试验,比较了玉米细胞质雄性不育系（CMS）及其同型可育系根系特性的差异.结果表明：CMS的地上部分物质积累能力较强,其籽粒产量显著高于可育系（P<0.05）;其根系干质量相对较高,根冠比变化趋势与根系干质量类似.CMS在0～20 cm和20～40 cm土层平均根量高于其可育系,在40～80 cm土层优势更为明显.CMS在0～20 cm和20～40 cm土层的平均根系活力显著高于其可育系（P<0.05）;0～20 cm和20～40 cm土层根系的SOD活性和可溶性蛋白含量与可育系差异不显著, 40～80 cm土层显著高于其可育系（P<0.05）;在生育后期,CMS的根系活力、SOD活性和可溶性蛋白含量优势更明显.表明玉米不育植株具有深层根量多、生育后期根系活性高的特点,从而使其根系功能期延长,植株衰老进程减缓.这可能是雄性不育玉米增产的重要原因之一.     

Growth Pattern of Tall and Short Lines of Rice and their Response to Gibberellin

The growth pattern of two types of dwarfness in rice was studied.The difference in height of the tall and short Peta plants wasapparent even at the seedling stage. In the dwarf and in thenormal Century Patna 231 the difference in height was apparentonly after panicle initiation. The four lines had the same numberof elongated internodes, with the taller lines having longerinternodes. The application of gibberellin resulted in increased plant height,longer internodes, blades and leaf sheaths, and decreased tillernumber. Century Patna 231-dwarf showed the least, and shortPeta the greatest, response to gibberellin application.