Methods for Selection of Low-Temperature Tolerant Mutants of Chrysanthemum morifolium Ramat. Using Irradiated Cell Suspension Cultures II. Preselection in vitro under Low-Temperature Stress

The effect of in vitro cold treatment (+6°C, 90 days) on selection of low-temperature tolerant (LTT) mutants was investigated using X-irradiated (15 Gy and 20 Gy) and non-irradiated cell suspensions of Chrysanthemum morifolium cv. ‘Parliament’. Low temperature reduced significantly the number of regenerating calli, especially in the irradiated cultures. After the end of the cold stress 684, 197 and 54 plants were obtained from the 0 Gy, 15 Gy and 20 Gy treated cultures, respectively. Testing these plants in the greenhouse (+12 °C, winter low-light) most of the early flowering variants were selected from the 15 Gy population.     

Methods for the Selection of Low-Temperature Tolerant Mutants of Chrysanthemum morifolium Ramat. by Using Irradiated Cell Suspension Cultures

In order to improve mutant selection procedures, 5000 plantlets were regenerated in three different harvests (flush A, B and C) from X-irradiated (15 and 20 Gy) and non-irradiated cell suspensions of Chrysanthemum morifolium cv. ‘Parliament’. After screening under suboptimal energy condition (12°C greenhouse temperature, winter low-light), a total of 95 early flowering plants were selected. These plants flowered at least as early as a low-temperature tolerant (LTT) mutant E₂, used as a reference. The percentage of early flowering variants was highest among plants obtained from calli, X-irradiated with 15 Gy and regenerating relatively late (flush B). It appeared that the applied selection method (e.q. harvest) had a large impact on the number of early plants obtained.     

Radiation-induced low-temperature tolerant solid mutants of Chrysanthemum morifolium Ram.

Summary From irradiated explants of the chrysanthemum cultivar Parliament solid non-chimeric low-temperature tolerant mutants were obtained. Some of the mutants flowered approx. 2 weeks earlier than control plants at a temperature of 12°C D/N and looked otherwise identical to the original genotype. The significance of it is discussed, a.o. in relation to attempts in developing selection methods at cell level for such characteristics.     

Use of somaclonal variation and in vitro selection for chilling tolerance improvement in rice

Embryo-derived calli of four rice varieties cultivated at high altitude in Burundi — Facagro 57, Facagro 76, Kirundo 3 and Kirundo 9 — were submitted to different temperature regimes. The percentage of regenerating calli greatly varied depending on variety, length of culture and callus temperature treatment. The reduction of regeneration percentages induced by low temperature was more pronounced in the more sensitive varieties. Regenerated plants (R0) and their progenies in R1, R2 and R3 were cold-screened together with control plants. In all varieties, significantly higher survival rates were obtained in R3 with in vitro plants than with control plants. Such chilling tolerance improvement was not obtained following a massal selection applied during 3 successive generations onto the control plants. In vitro plants regenerated from calli cultivated either at 25 °C, either at 4 °C, were cultivated at different altitudes in Burundi during two successive generations. For most observed traits, the in vitro plants were characterized by lower means, larger variation and higher maximum values than the control plants. The most chilling-tolerant somaclonal families were most usually characterized by extensive differences in fatty acid composition, chilling-induced electrolyte leakage and chlorophyll fluorescence, compared to the varieties they derived from. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation of flower color and shape mutations by gamma radiation of Chrysanthemum morifolium Ramat cv. Youka

We investigated the effect of radiation damage on in vitro mutation induction in chrysanthemum. White petals of chrysanthemum (Chrysanthemum morifolium Ramat cv. Youka) were selected to induce mutation by gamma radiation. Calli produced were irradiated with gamma rays at 0, 10, 15 and 20 Gy. We found that the plants from the irradiated calli were different from control plants in number of leaves, leaf length & width, number of flowers, flower diameter, petiole diameter and petiole length after transplanting into the greenhouse for almost 70 days. Three mutants in flower color and shape were found in 15 Gy-treated plants. First type of mutant (M.1) has tubular petals. The second (M.2) and third (M.3) ones both have yellow flowers, while one of them has spooned shaped ray florets similar to the original cultivar and the other one has flat shaped florets. Semi-quantitative RT-PCR showed that most of carotenoid-biosynthesis related genes, except for violaxanthin deepoxidase (VDE) and lycopene ε-cyclase (LCYE), showed similar expression patterns in petals of the original ‘Youka’ and its mutants (M2 & M3). VDE and LCYE results showed high expression levels in M3 and M2 & M3 respectively, comparing with the control. On the other hand, expression patterns for VDE were similar in control and M2. These yellow mutants were maintained vegetatively and proved to be true-to-type in one successive generation. It can be concluded that gamma radiation with 15 Gy dose can be used for in vitro induction of flower color and shape mutations of chrysanthemum cv. Youka.     

Pear mutagenesis: In vitro treatment with gamma-rays and field selection for vegetative form traits

In vitro shoots of four pear (Pyrus communis L.) cultivars, 'Conference', 'Doyenné d'Hiver', 'Passe Crassane' and 'Bartlett', were irradiated with gamma rays (3.5 Gy). Around 1000 microcuttings of each cultivar were treated. After three subcultures, microcuttings from both the irradiated shoots and additional non-irradiated shoots were rooted; plants obtained were used to establish a survey orchard in Ostellato (Ferrara), Italy. For the first 2 years in the field, growth and survival were observed, but no data were collected. Subsequently, field surveys were conducted to characterize the population and identify mutants for vegetative traits. Trees that showed phenotypic evidence of vegetative growth characteristics more desirable than those shown by the overall population and by control plants were selected and measured for three years. Traits used for selection were small tree size, wide branch angle and short internodes. Frequencies of variants with compact habit varied with cultivars from 0.5% to 2.7% of irradiated trees. Effect of mutagenic treatment and efficacy of the selection methods are discussed.     

Selection of Al-resistant plants from a sensitive rice cultivar, using somaclonal variation, in vitro and hydroponic cultures

A selection procedure is described that isolated Al-resistant individuals from a sensitive rice (Oryza sativa L.) genotype. Somaclonal variation was used as the only source of variability and selection pressures were applied at both callus and regenerated plant levels. When 10 week-old, embryo-derived calli were submitted to different Al stresses for a period of 20 weeks. After this in vitro stress period, selected calli were grown during 18 weeks, without selection pressure, on regeneration media. Selection pressures were applied on regenerated plants (R0), and 9 R0 plants which produced seeds were selected. The transmission of the Al-resistant character to the R1, R2, R3 and R4 generations was then investigated, and three plant lines which had an increased percentage of Al-resistant plants till the fourth generation of self-pollination, were identified. One was from a callus maintained on an Al-free medium. The efficiency of in vitro selection pressures was therefore debated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selection and characterization of a 5-methyltryptophan resistant mutant in Zea mays L.

Summary A maize plant resistant to 5-methyltrytophan (5MT) was selected from M₂ seeds (Zea mays L. Danggin, inbred line) originating from ears treated with ethylmethane sulfonate (0.2%) at 6 hr after self-pollination. Genetic analysis of the progeny of plants selected from a medium containing 50 ppm 5MT showed that 5MT resistance was inherited as a single dominant nuclear gene. This resistance was also expressed in callus and seedling. Analysis of the free amino acids in kernels and calli showed that homozygous resistant plants (MR1) contained higher levels of total free amino acids than sensitive plants and calli. In particular, the their kernels the levels of tryptophan, threonine and serine were, respectively, 4.5, 5.9 and 6.3 times higher than those of the sensitive plants. From the results, it may be expected that mutants resistant to amino acid anologs will be useful not only for studying amino acid biosynthesis but also for improving the nutritional quality of maize.Abbreviations EMS Ethylmethane sulfonate - 5MT 5-methyltryptophan - 2,4-D 2-4-dichlorophenoxyacetic acid     

抗草甘膦基因aroA转化陆地棉胚性愈伤组织的研究

为了建立以草甘膦为选择标记的农杆菌介导的棉花胚性愈伤组织转化体系,本研究将草甘膦抗性基因(aroA)转化陆地棉WC和YZ-1的胚性愈伤组织,分别以5 mg·L-1和10 mg·L-1的草甘膦浓度作为选择压,经过2~3轮筛选,获得了抗性胚性愈伤组织,诱导出胚状体并发育成苗。对T0再生植株进行PCR及草甘膦抗性检测,以WC和YZ-1为受体的转抗草甘膦基因(aroA)分别获得了33株和10株阳性植株,转化效率分别为82.5%和62.5%。该体系为棉花遗传转化提供了实验方法,获得的抗草甘膦棉花为棉花育种提供种质资源;同时抗除草剂基因还可以作为筛选标记与其它基因串联在一起转化棉花,进行基因功能验证。     

Chilling Tolerance, Cold Hardening and Freezing Tolerance of Low-temperature-tolerant Mutants of Dendranthema × grandiflorum (Ramat.) Kitamura

Two low-temperature-tolerant mutants of the Dendranthema × grandiflorum (Ramat.) Kitamura cultivar ‘Puck’ derived from an x-radiated tissue culture system were compared, as vegetatively growing plants, to ‘Puck’ ssfor several physiological properties under low-temperature conditions. So far, the low-temperature tolerance of the mutants has been defined as the capacity to flower earlier under low temperature conditions (8—15°C) the wild type. All three lines were not chilling sensitive in the vegetative state and were able to acclimatize and to develop freezing tolerance. Differences between ‘Puck’ and the mutants were found but could not be related to the low-temperature tolerance of the mutants in the generative phase. Levels of the amino acid proline increased during the hardening process, reaching the highest level in the cultivar ‘Puck’. No evidence could be obtained that proline is causally involved in the cold-hardening process. Freezing-induced membrane injury was found to decrease with cold hardening (0—28 days) but the two mutants did not show a greater capacity to acclimatize and cold harden than the original cultivar ‘Puck’.     

In vitro Selection for Tolerance to Fusarium in F1 Microspore Populations of Wheat

Microspore populations of eight Fhybrids of winter wheat (Triticum aestivum L.) whose parents had different levels of resistance to Fusarium were screened in vitro, using phytotoxins of Fusarium as biochemical probe. Two selection methods were compared for the in vitro selection: either embryoids and calli were first initiated from anthers in toxin-free medium and then grown on medium with 0.3—0.9 %Fusarium toxin; or anthers were immediately cultured in modified liquid potato-2 medium in the presence of 1.5, 2.0 and 2.5ml Fusarium toxin per liter culture medium, a concentration which reduced the number of calli and embryoids to about 10 % compared to the toxin-free controls. Microspores from donor hybrids which were produced from very susceptible cultivars were killed by lower toxin concentrations than micro-spores from hybrids of less susceptible parents. From surviving calli and embryoids, originally initiated from 242,000 anthers in both procedures a total of 375 green lines could be regenerated. The results indicate that it is possible to enrich the fraction of regenerating microspores by those which contain the gene complex responsible for reduced susceptibility to Fusarium by the use of a pathotoxin.     

Anther culture of tropical japonica × indica hybrids of rice (Oryza sativa L.)

Summary Nine japonica × indica F1 hybrids of rice involving 6 indica and 3 japonica tropical varieties, were large scale anther cultured. The frequency of callusing anthers averaged 18.7%. The microspore-derived calli produced green plants with a mean frequency of 8.7%. Albino plants represented 61% of the shoot forming calli. Monitoring of the green and albino plant regenerating capabilities of calli arising between week 4 and week 8 of incubation of the anthers showed no increase of the albino/green ratio and a slow decrease of the shoot forming ability of the transferred calli after the sixth week of culture. Spontaneous doubled haploids (SDH) represented 46% of the regenerated green plants in 4 hybrids. However, a high frequency of partially sterile regenerants was noticed among 132 SDH plants generated from a hybrid.     

Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion

Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R₃. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R₀) than the non-selected ones. Among 18 R₁ lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R₃ lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R₃ selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.     

Mutation induction with ion beam irradiation of lateral buds of chrysanthemum and analysis of chimeric structure of induced mutants

We compared the effects of ion beam and gamma ray irradiation on mutation induction in axillary buds of chrysanthemum, and analyzed the chimeric structure of the resulting mutants. Axillary buds were irradiated with carbon ions at 2 Gy (mean linear energy transfer 122 keV/μm), helium ions at 10 Gy (mean 9 keV/μm), and gamma rays at 80 Gy, all of which have the similar effects on survival. The lower five nodes of the shoots elongating from the irradiated buds were cut one by one, and new shoots were allowed to grow from the axillary buds. This procedure was repeated twice, and flower color mutation was investigated. Chimeric structure was analyzed by comparing the flower color of mutants to that of plants regenerated from the roots. Flower color mutants emerged at a high frequency (17.4–28.8%), and there were no significant differences in the mutation frequency between the treatments. All the flower color mutants induced with gamma rays were periclinal chimeras. In contrast, some mutants obtained with ion beams had the same flower color as that of the plants derived from the roots. This suggests that they were solid mutants, where both LI and LIII tissues were derived from the same mutated cell. Solid mutants were also obtained when irradiated with 5 Gy of helium ions, which had less effect on survival and mutation than other treatments. Factors for obtaining solid mutants only with ion beams are also discussed.     

Vitamin C,B5, and B6 contents of segregating potato populations detected by GC‐MS: a method facilitating breeding potatoes with improved vitamin content

Vitamin C, B5, and B6 contents of potato tubers were measured by gas chromatography‐mass spectrometry (GC‐MS). Two diploid potato populations derived from two different crosses were analyzed. Plants were grown at two locations under different environmental conditions. Measurements of vitamin content of tubers showed high variation in both populations. A weak correlation was found between the vitamin concentrations of tubers harvested at the two locations. Correlations between the mean values of vitamins in field‐grown tubers and in different tissues derived from in vitro or greenhouse‐grown potato plants were also determined. A very high correlation between the vitamin content of field‐ and greenhouse‐grown tubers, and between field‐grown tubers and sink leaves of greenhouse‐grown plants was detected. This finding can facilitate breeding by preselection of individuals with improved vitamin content under greenhouse conditions at their early developmental stage.     

水稻体细胞无性系雄性不育突变

对5个类型的水稻不育系进行了幼穗培养, 在红源A、 包源A和W6154s中, 共获得了10例雄 性不育变异株, 水稻花粉败育可分为无花粉、 典败、 圆败和染败四种类型。 发现了不育 花粉败育类型之间可以相互转换现象。 对雄性不育变异株用一批现有CMS不育系的保持系和恢复系进行测交和回交, 有的变异株其 恢保关系发生了变化,     

Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars

A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies on somaclonal variants for resistance to scab in bread wheat (Triticum aestivum L.) through in vitro selection for tolerance to deoxynivalenol

This study was conducted to develop an efficient in vitro selection system for scab resistance by using in vitro screening for tolerance to deoxynivalenol (DON). Immature embryos of two wheat varieties, a scab-resistant variety Sumai 3 and a susceptible variety Mianyang 11, and their reciprocal F₁ hybrids were cultured on MS medium supplemented with 2,4-D 2 mg/l and 0.6 × 10^-4 M DON for callus induction. The responses of callus induction and plant regeneration to 0.6 × 10^-4 M DON differed significantly between resistant and susceptible varieties, according to observed scab resistance levels at the plant level in the field. The percentage of callus formation of resistant variety Sumai 3 on induction medium containing DON was higher than that of susceptible variety Mianyang 11. Regeneration of DON-tolerant calli on DON-containing differentiation medium differed significantly between Sumai 3 and Mianyang 11. Averaged across the DON-tolerant calli of two varieties and their reciprocals, regeneration of DON-tolerant calli was decreased 3-fold on DON-containing medium. By an inoculation test with conidiospores of Fusarium graminearum Schw, we obtained several resistant lines from progenies of regenerated plants from DON-tolerant calli. These somaclonal lines had lower disease scoring (reaction index, infected spikelets and disease incidence), shorter plants and better yield components than Sumai 3, a famous Chinese resistant variety. This revised version was published online in July 2006 with corrections to the Cover Date.     

以EMS诱变创制软质小麦宁麦9号高分子量谷蛋白亚基突变体

本研究旨在创制遗传背景一致的不同高分子量谷蛋白亚基(HMW-GS)缺失系, 为小麦品质研究和育种提供材料。将软质小麦品种宁麦9号, 用0.4% EMS溶液处理10 000粒种子, 获得3781个M1代单株, 采用“半粒法”对每个株系进行SDS-PAGE鉴定, 从中筛选出299个(7.91%) HMW-GS突变株, 包括HMW-GS缺失和分子量突变两种类型。其中, HMW-GS缺失突变176株, 突变频率为4.65%, 缺失亚基涉及Ax1、Bx7、By8、Dx2和Dy12, 突变频率为0.24%~3.28%; 分子量突变130株, 突变频率为3.44%。将突变体的具胚端种子于温室中繁殖获得M2代, 再次鉴定各株系的HMW-GS, 并经M3代验证, 最终获得Ax1、Dx2、Bx7、By8和Dy12缺失突变体, 及Ax1+By8双缺失突变体。用高效液相色谱(HPLC)分析这些突变体的谷蛋白大聚体(GMP)和谷蛋白/醇溶蛋白(GLU/GLI)比值, 发现不同缺失突变体的GMP含量都有不同程度的降低, 尤以Bx7亚基缺失突变体中GMP含量降幅最大, 高达42%。另外, 不同缺失突变体的谷蛋白总量和GLU/GLI比值也低于对照, Ax1+By8双缺失突变体的GLU/GLI比值最小。