The multiple-cluster mutation complex in mutagenesis with higher plants

Summary After treatment of dry and pre-soaked seeds of barley with gamma-rays, EMS, NEU and EI, the frequency of multiple mutations (multimutations) was higher with EMS and NEU treatment, while cluster mutations appeared in greater numbers following treatment with gamma rays and NEU. Pre-soaking the seeds led to a reduction in the frequency of total mutations, cluster mutations and multimutations. This has been explained as a result of the application of lower doses and the induction of mutations at a relatively later stage in ontogenetic development in the case of pre-soaked seeds.Some new mutation types in barley have been described and some of the old types have been given names representing the mutation characters more precisely.The compound mutation frequency of different seedling mutation types, when taken separately, was found to be independent of the mutagen employed and the stage of treatment. The size of mutated chimeras in M ₁ plants, as indicated by the segregation ratio of mutants in M ₂, was largest in albina, xantha, chlorina, albina-tigrina, chl-terminalis and eceriferum, and lowest in viridis, viridoalbina etc. This could be expected if the unstable premutations induced by mutagenic treatment are resolved into mutations at different intervals after their initiation, or it can be explained by the induction of dominant mutations, or lethal changes together with visible mutations.     

TA29-barnase基因转化菜心

利用根癌农杆菌导入法, 以菜心带柄子叶为外植体, 对TA29-barnase基因转化菜心进行研究。获得转化植株,进行PCR、Southern blotting杂交和半定量RT-PCR检测, 表明目的基因已经整合到转化植株中, 并且目的基因在转基因植株花蕾中得到表达, 但是表达水平在不同转基因植株间存在差别; 转基因植株开花后, 均表现雄性不育, 不能产生花粉或产生没有活力的少量花粉, 自交不能结实; 用未转化植株正常花粉对雄性不育植株进行授粉, 能够正常结实; 保持系(未转化植株)与不育株杂交后代中雄性不育株与可育株的比例为1:1, 在杂交后代植株子叶期, 喷洒10 mg/L的PPT可以完全杀死可育株; 利用其他菜心品种为父本与不育株进行杂交, 获得的F1植株在生长势和产量方面表现优势, 表明开展菜心优势育种具有一定的潜力。     

TA29-barnase基因转化菜心

利用根癌农杆菌导入法, 以菜心带柄子叶为外植体, 对TA29-barnase基因转化菜心进行研究。获得转化植株,进行PCR、Southern blotting杂交和半定量RT-PCR检测, 表明目的基因已经整合到转化植株中, 并且目的基因在转基因植株花蕾中得到表达, 但是表达水平在不同转基因植株间存在差别; 转基因植株开花后, 均表现雄性不育, 不能产生花粉或产生没有活力的少量花粉, 自交不能结实; 用未转化植株正常花粉对雄性不育植株进行授粉, 能够正常结实; 保持系(未转化植株)与不育株杂交后代中雄性不育株与可育株的比例为1:1, 在杂交后代植株子叶期, 喷洒10 mg/L的PPT可以完全杀死可育株; 利用其他菜心品种为父本与不育株进行杂交, 获得的F1植株在生长势和产量方面表现优势, 表明开展菜心优势育种具有一定的潜力。     

Mutagenic action of dimethyl sulfate and N-nitrosoethylurea on the mulberry silkworm

Mutagenic effect of chemical mutagens, dimethalsulphate (DMS) and N-nitrosoethyl urea (NEU), on silkworm is studied. The effect of these mutagens have never been studied on silkworm before. DMS is found to be completely insufficient as mutagen for the silkworm, because its low mutability is accompanied with strong cytotoxic effect. NEU appeared to be of "milder" effect. Injection of male-moth proved to be very efficient. The majority of exuded mutants showed mosaic colouring of the graine.     

Evaluation of the effect of pollen irradiation on karyotype variability in M2 cotton plants

The karyotypes of biomorphologically abnormal cotton (Gossypium hirsutum L.) plants obtained in M2 after pollination with pollen irradiated at dose rates 10, 15, 20, and 25 Gy were studied. Various genomic and chromosomal mutations were detected in 57 M2 families. The primary monosomics isolated in M2 were found to be cytologically more stable and more viable, since they had higher meiotic indices, pollen fertility, and seed formation. In M2, a decrease in the number of plants with multiple karyotype aberrations and interchromosomal exchanges with high frequency of multivalent formation was observed. The multivalents had diverse patterns and types of chromosome segregation and translocation complexes. Their pollen fertility was higher than in translocants found in M1. Desynapsis often occurred in M2, including plants with chromosome deficiency or rearrangements. The variation in the number of univalents in various cells was found to result from different expression of synaptic genes. The results indicate stabilization of karyotypes, increase in cytologic stability and viability, and the absence of sterility in aberrant plants.     

Fertility of Triticum aestivum plants derived from anther culture

The fertility of plants from wheat anther culture was studied. It was found that one half of the 36 plants with diploid root tips didn't set seeds at all, and that 41 of the 42 plants with haploid root tips were completely sterile. It was surprising that sterility was so widely distributed even among the plants with diploid root tips.     

Somaclonal variation in plants regenerated from cultures of soybean

Plants were regenerated from embryogenic and organogenic cultures derived from immature embryos of nine soybean (Glycine max L. Merr.) genotypes and extensive qualitative variation was noted in different regenerated families. Three lethal sectoral albinos were seen in the regenerated plants (R₀). Variants observed in later selfed generations included twin seeds, multiple shoots, dwarfs, abnormal leaf morphology, abnormal leaflet number, wrinkled leaves, chlorophyll deficiency, partial sterility and complete sterility. The frequency of possible mutations ranged from 0 to 4% in R plants as determined by studies of corresponding R₁, R₂, R₃ and R₄ families. No significant differences were seen in the frequencies of possible mutations for embryogenic as compared to organogenic culture derived plants. Chlorophyll deficiency, sterility and wrinkled leaf traits were followed in two or more generations and showed that these traits were inherited stably. The known traits of this nature are controlled by single recessive nuclear genes. Other traits occurred more randomly and not in all generations. The genetic basis of the random variation is not known at the present time. This study indicates that heritable somaclonal variation does occur in tissue culture derived plants of soybean.Abbreviations R₀ Original regenerated plant - R₁ Selfed seeds of R₀ plants - R₂ Selfed seeds of R₁ plants - R₃ Selfed seeds of R₂ plants This research was supported by funds from the Illinois Agricultural Experiment Station and Agrigenetics Inc.     

Photosynthetic pigments and photosynthetic products of endophyte-infected and endophyte-free Lolium perenne L. under drought stress conditions

Endophyte-infected (EI) seeds of Lolium perenne L. were used to attain endophyte-free (EF) population by heating the seeds at 43°C for 15 min and then 57°C for 25 min. Relative water content (RWC), chlorophyll, soluble sugar and starch content of EI and EF populations under normal and drought stress conditions were compared to investigate the effect of endophyte infection on the host plant. Under severe stress, RWC of EI leaf was significantly higher than that of EF leaf, i.e. EI plants took more advantages over EF plants in water-holding ability. Under mild stress, endophyte could enhance soluble sugars in host plants to improve their osmotic ability. With stress intensification, the improvement of endophyte no longer existed, and more photosynthetic products (such as starch) accumulated in EI plants to survive through the undesirable conditions. In the next spring, EI populations will recover more rapidly than EF populations. The biomass of a population is closely related to its photosynthesis. Under severe stress, EI population significantly accumulated more biomass than EF population. As far as photosynthetic pigments were concerned, contents of Chla, Chlb and Car of EI plants were close to those of EF plants, which suggested that endophyte infection didn’t alleviate photosynthetic pigments from being destroyed by drought stress, and endophyte might improve photosynthesis ability of its host plant in other ways. __________ Translated from Acta Ecologica Sinica, 2005, 25 (2) [译自: 生态学报, 2005, 25(2)]     

In-situ seed production after pollination with in-vitro-matured,isolated pollen

Immature tobacco (Nicotiana tabacum L.) pollen has been isolated from anthers in three distinct stages of development, including the microspore stage. In in-vitro cultures, fully functional, mature pollen was obtained. In a germination medium, this pollen produced pollen tubes. After application to stigmas in situ, the in-vitro-matured pollen fertilized ovules, and seeds were produced. Genetic tests with seedlings obtained from pollinations with in-vitro-matured pollen from a transgenic plant revealed normal Mendelian segregation of two marker genes, the neomycin-phosphotransferase II gene and the nopaline-synthase gene. These results are of interest with respect to the control of self-incompatibility, cytoplasmic male sterility and pollen-allergen formation, and it offers an alternative route for gene transfer in those plants which cannot be regenerated in vitro.Abbreviations cms cytoplasmic male sterility; AMGLU, MS, M2S, MR26 - GK culture media, see Material and methods     

Estimation of efficiency of seed irradiation by thermal neutrons for inducing chromosomal aberration in M2 of cotton Gossipium hirsutum L

Cytogenetic analysis of M2 plants after irradiation of cotton by thermal neutrons was performed in 56 families. In 40 plants of 27 M2 families, different abnormalities of chromosome pairing were found. These abnormalities were caused by primary monosomy, chromosomal interchange, and desynapsis. The presence of chromosome aberrations in some cases decreased meiotic index and pollen fertility. Comparison of the results of cytogenetics analysis, performed in M1 and M2 after irradiation, showed a nearly two-fold decrease in the number of plants with chromosomal aberrations in M2, as well as narrowing of the spectrum of these aberrations. The latter result is explained by the fact that some mutations are impossible to detect in subsequent generations because of complete or partial sterility of aberrant M1 plants. It was established that the most efficient radiation doses for inducing chromosomal aberrations in the present study were 15 and 25 Gy, since they affected survival and fertility of altered plant to a lesser extent.     

Photosynthetic pigments and photosynthetic products of endophyte-infected and endophyte-free Lolium perenne L. under drought stress conditions

Endophyte-infected (EI) seeds of Lolium perenne L. were used to attain endophyte-free (EF) population by heating the seeds at 43°C for 15 min and then 57°C for 25 min. Relative water content (RWC), chlorophyll, soluble sugar and starch content of EI and EF populations under normal and drought stress conditions were compared to investigate the effect of endophyte infection on the host plant. Under severe stress, RWC of EI leaf was significantly higher than that of EF leaf, i.e. EI plants took more advantages over EF plants in water-holding ability. Under mild stress, endophyte could enhance soluble sugars in host plants to improve their osmotic ability. With stress intensification, the improvement of endophyte no longer existed, and more photosynthetic products (such as starch) accumulated in EI plants to survive through the undesirable conditions. In the next spring, EI populations will recover more rapidly than EF populations. The biomass of a population is closely related to its photosynthesis. Under severe stress, EI population significantly accumulated more biomass than EF population. As far as photosynthetic pigments were concerned, contents of Chla, Chlb and Car of EI plants were close to those of EF plants, which suggested that endophyte infection didn’t alleviate photosynthetic pigments from being destroyed by drought stress, and endophyte might improve photosynthesis ability of its host plant in other ways.     

Intergeneric hybrids between Brassica napus and Orychophragmus violaceus containing traits of agronomic importance for oilseed rape breeding

Protoplast fusions between Brassica napus and Orychophragmus violaceus for transfer of valuable traits to oilseed rape resulted in 257 somatic hybrid plants. Hybridity was confirmed by morphological, cytological and molecular means. Symmetric fusions gave rise to 131 plants. Fifty eight of these plants had an intermediate morphology and contained nuclear DNA corresponding to the sum of the parental species. All 131 plants were sterile with no pollen grains observed upon flowering. Another 126 plants were derived from asymmetric fusions in which protoplasts of the donor parent O. violaceus were irradiated by 100 or 200-Gy X-rays prior to fusion. Morphologically these plants showed a larger variation compared to the plants regenerated from symmetric fusion experiments. In contrast to plants obtained from symmetric fusions, fertile hybrids were recovered among regenerants from the asymmetric fusions. Twenty four of these plants released viable pollen grains and 14 of the determined 17 plants set seeds after either selfing or backcrossing to B. napus. Fourteen male-sterile plants were identified with female fertility. This observed male sterility most-likely originated from alloplasmic recombination and would be of great potential for the development of a new cytoplasmic male sterility system. The fatty acid composition of the fertile hybrids and their progenies showed a biased distribution towards the B. napus parent, which has a high erucic acid-content type. However, increased levels of palmitic and linoleic acids compared to B. napus were found in subsequent generations, as well as a reduced level of erucic acid.     

Transgene insertion induced dominant male sterility and rescue of male fertility using round spermatid injection

Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFR alpha 2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.     

Bisexual sterility conferred by the differential expression of Barnase and Barstar: a simple and efficient method of transgene containment

To establish a simple and an efficient system to minimize the environmental risk of genetically modified plants, we tested the applicability of the barnase/barstar system in conferring bisexual sterility; that is, in preventing plants setting seeds by self-fertilization and out-crossing. Transgenic tobacco plants were generated to express barnase, a cell death inducing ribonuclease, under the control of the gamete-specific AtDMC1 promoter, and barstar, a specific inhibitor of barnase, under the control of the ACT2 promoter, which is constitutively active in almost all tissues except gametes. In contrast to control plants harboring the barstar expression unit only, which set seeds normally with self-pollination, all transformants harboring both barnase and barstar were bisexually sterile. They produced aberrant anthers containing no detectable pollen and failed to set seeds even after pollination with wild-type tobacco pollen.An erratum to this article can be found at     

Inducing male sterility of tomato using two component system

Production of hybrid seeds and pursuing heterosis breeding of many crops have been accomplished using male sterile lines. However, not all crops have valuable male sterile lines due to instability of male sterility and absence of a restorer system. In this study, male sterile lines have been induced using a two-component system. The extracellular ribonuclease Barnase was cleaves into two inactive yet complementary fragments, designated as ??Bn-5?? and ??Bn-3??. Both components were controlled by a TA29 promoter. They were transferred into the tomato inbred line ??Yellow tomato?? by Agrobacterium method. Southern blotting identified that 11 transgenic Bn-5 plants (T₀) and 10 transgenic Bn-3 plants (T₀) were obtained. The vegetative phenotypes of all T₀ plants were similar to wild-type, and they were capable of producing viable pollen grains and normal fruit with seeds, indicating that Barnase had lost its function after it being split two partial fragments. After self-pollination, homozygous progenies (T₁) of transgenic Bn-5 and Bn-3 plants were chosen to cross each other, Barnase could be reconstituted and co-expressed in the same cell, which caused the hybrid plants to produce collapsed pollen grains with no viability and thus100?% male sterile plants were obtained. Stamens of male sterile plants were shorter than those of the wild type plants. PCR detection demonstrated that all male sterile plants contained Barnase, but male fertile plants did not. The male sterile plants were crossed with the male fertile inbred lines, and the result showed that hybrid (F₁) plants were capable of producing normal fruit with seeds, and their pollen grain fertility was restored. The co-segregation ratio of Bn-5 and Bn-3 fragments showed 1:1 among hybrid plants. In conclusion, the results verified that the male sterility could be generated by two component system and be used in hybrid seed production. The F₁ between the male sterile plant and the inbred line showed heterotic comparing to both parents. This system needs not breed restoration line.     

A cytonuclear incompatibility causes anther sterility in Mimulus hybrids

Multilocus interactions (also known as Dobzhansky-Muller incompatibilities) are thought to be the major source of hybrid inviability and sterility. Because cytoplasmic and nuclear genomes have conflicting evolutionary interests and are often highly coevolved, cytonuclear incompatibilities may be among the first to develop in incipient species. Here, we report the discovery of cytoplasm-dependent anther sterility in hybrids between closely related Mimulus species, outcrossing M. guttatus and selfing M. nasutus. A novel pollenless anther phenotype was observed in F2 hybrids with the M. guttatus cytoplasm (F2G) but not in the reciprocal F2N hybrids, F1 hybrids or parental genotypes. The pattern of phenotypic segregation in the F2G hybrids and two backcross populations fit a Mendelian single-locus recessive model, allowing us to map the underlying nuclear locus to a small region on LG7 of the Mimulus linkage map. Anther sterility was associated with a 20% reduction in flower size in backcross hybrids and we mapped a major cytoplasm-dependent corolla width QTL with its peak at the anther sterility locus. We argue that the cytonuclear anther sterility seen in hybrids reflects the presence of a cryptic cytoplasmic male sterility (CMS) and restorer system within the hermaphroditic M. guttatus population and therefore name the anther sterility locus restorer-of-male-fertility (RMF). The genetic mapping of RMF is a first step toward testing hypotheses about the molecular basis, individual fitness consequences, and ecological context of CMS and restoration in a system without stable CMS-restorer polymorphism (i.e., gynodioecy). The discovery of cryptic CMS in a hermaphroditic wildflower further suggests that selfish cytoplasmic evolution may play an important, but often undetected, role in shaping patterns of hybrid incompatibility and interspecific introgression in plants.     

甜菜属厚皮菜SWTY-1细胞质VNTRs多态性分析

利用数目可变串联重复序列(Variable Number of Tandem Repeats,VNTRs)微卫星标记方法,对重庆厚皮菜甜菜材料SWTY-1群体中100个单株的细胞质线粒体DNA片段中TR2位点VNTRs片段多态性进行分析。结果显示97个单株线粒体TR2位点微卫星串联重复序列均为3拷贝,与普通糖甜菜一致;3个单株线粒体TR2位点微卫星串联重复序列为6拷贝,发现甜菜属厚皮菜细胞质TR2位点VNTRs存在多态性,在该群体中发现了不同于甜菜栽培种新的细胞质单株。对该群体材料100个单株的抽薹及结籽进行观测,结果显示微卫星串联重复序列为6拷贝的变异植株中2个单株花期未抽苔开花,1株抽苔晚未形成正常种子;细胞质TR2位点VNTRs片段拷贝数为3的植株中2个单株未能正常抽薹,其他植株均正常抽薹结籽。     

二角型非1B/1R 小麦CMS不育恢复体系的建立

通过对具有二角山羊草细胞质的1B/1R型小麦雄性不育系m s(A e.bicorn is)-5-1回交置换,获得了新型二角山羊草细胞质小麦雄性不育系m s(A e.bicor)-V 9125和m s(A e.bicor)-M 853.利用染色体制片、分子标记、原位杂交和酸性聚丙烯酰胺凝胶电泳对其保持系V 9125和M 853进行分子细胞遗传学检测,并对其育性恢复性机理进行了初步研究.结果表明:(1)V 9125和M 853均为非1B/1R易位系.V 9125为小麦-簇毛麦易位系,m s(A e.bicor)-V 9125是二角山羊草细胞质与普通小麦核背景中簇毛麦外源染色体互作产生的一类新的核质互作不育系.(2)M 853为小麦-滨麦易位系,m s(A e.bicor)-M 853是二角山羊草细胞质与普通小麦核背景中滨麦外源染色体互作产生的一类新的核质互作不育系.(3)利用一些普通小麦品种(系)与这两个不育系杂交,m s(A e.bicor)-V 9125和m s(A e.bicor)-M 853仅与T 6-3杂交育性得到恢复,且恢复度较高,变异小,而与其它大部分普通小麦杂交F1表现雄性不育,且扬花期花药不外露.而m s(A e.bicor)-5-1与包括T 6-3在内的普通小麦品种(系)杂交F1育性得到不同程度的恢复.从而得出结论,二角山羊草细胞质与小麦细胞核的互作存在两个不同的不育-恢复系统.     

A rapid method to increase the number of F(1) plants in pea (Pisum sativum) breeding programs

In breeding programs, a large number of F(2) individuals are required to perform the selection process properly, but often few such plants are available. In order to obtain more F(2) seeds, it is necessary to multiply the F(1) plants. We developed a rapid, efficient and reproducible protocol for in vitro shoot regeneration and rooting of seeds using 6-benzylaminopurine. To optimize shoot regeneration, basic medium contained Murashige and Skoog (MS) salts with or without B5 Gamborg vitamins and different concentrations of 6-benzylaminopurine (25, 50 and 75 μM) using five genotypes. We found that modified MS (B5 vitamins + 25 μM 6-benzylaminopurine) is suitable for in vitro shoot regeneration of pea. Thirty-eight hybrid combinations were transferred onto selected medium to produce shoots that were used for root induction on MS medium supplemented with α-naphthalene-acetic acid. Elongated shoots were developed from all hybrid genotypes. This procedure can be used in pea breeding programs and will allow working with a large number of plants even when the F(1) plants produce few seeds.