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.     

Variants of Digitalis obscura from irradiated shoot tips

Shoot tips excised from genotype T4 of Digitalis obscura were exposed to gamma rays (20–100 Gy). Radiosensitivity was assessed and LD₅₀ determined was about 60 Gy. The effect of the herbicide amitrole on shoot-tip culture was investigated, efficient bleaching was obtained with 10 mg/l amitrole. Shoot tips mutagenized (20–40 Gy) were cultured on several selective amitrole-containing media, but none of them permitted prolonged survival of the green shoots formed. Ploidy level of the plantlets developed was analysed by flow cytometry 8 and 18 months after culture establishment. Variations detected corresponded to aneuploid changes and increased in parallel with the gamma radiation dose. Plantlets developed from irradiated shoot tips presented a high variability in their cardenolide production (878 to 3291 μg/g d.w.), including variants with similar or even higher productivities than the native T4 plant. This revised version was published online in August 2006 with corrections to the Cover Date.     

Preliminary results of using in vitro axillary and adventitious buds in mutation breeding of Chinese gooseberry

Summary Aseptically cultured shoots of Chinese gooseberry exhibited growth disorder and morphological aberrances, and some died after being exposed to sufficient gamma-ray irradiation. The death rate was dose dependant and the LD₅₀ was 80–90 Gy and 50–60 Gy respectively for cv. Hayward and clone 4. All petiole explants irradiated with gamma-ray could form calli as the control, but the rate of differentiation of adventitious shoots of the petiole explants decreased and was dependant on dose. Sensitivity of the shoot or petiole explants to gamma-ray irradiation varied with species. Gamma-ray irradiation did not deter either the 2-node segments from producing axillary shoots M₁, M₂, and M₃ or the advantitious shoots originating in the petiole explants and the M₃ shoots from forming advantitious roots. Therefore, using aseptically cultured axillary or adventitious buds for mutation breeding of Chinese gooseberry is feasible. A bacterium surviving in the explants lessened the efficiency of these two in vitro techniques in mutation breeding of Chinese gooseberry.Abbreviations IAA 3-indole acetic acid - IBA -indole butyric acid - MS Murashige & Skoog (1962)     

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.     

高能重离子辐射对水稻的生物学效应

本文观察了数种高能重离子辐射处理水稻干种子的生物学效应，结果表明；与具低传能线密度的＾Ｃｏ６０－γ射线相比，这些具高ＬＥＴ的重离子能更有效地抑制受照射种子萌发生出的幼苗的生长，诱导根尖细胞和花粉母细胞的染色体畸变和微核形成，降低当代植株的结实率，并发后代出现形态性状及农艺性状的变异，在一定剂量范围内，辐射诱导的上述数种生物学效应与吸收剂量间呈线性关系，根据剂一－效应关系曲线，估算了重离子辐射的上述     

Screening of Chinese cabbage mutants produced by 60Co γ‐ray mutagenesis of isolated microspore cultures

Since the release of the Chinese cabbage genome sequence, increasing interest has focused on the functional analysis of unidentified genes in Chinese cabbage. Mutant analysis forms the basis of functional genomics research. To produce a variety of Chinese cabbage mutants in the same genetic background, buds containing late uninucleate spores from a doubled haploid line of the Chinese cabbage variety ‘Fukuda 50’ were irradiated with ⁶⁰Co γ‐rays at doses of 20, 40 and 60 Gy. Then, the treated microspores were isolated and cultured. A total of 492 putative M₀ mutants were isolated from 1483 regenerated plants. Of these, six M₁ mutants were verified; the mutant frequency was 0.41%. These mutants comprise a mutant library that includes one plant shape mutant, two flower mutants and three male sterile mutants. Pollen viability detection and DNA flow cytometry were used to determine the ploidy of the regenerated plants. Some of the mutants isolated in this study may be useful for Chinese cabbage breeding and functional genomics research.     

Selection of a mutant from adventitious shoots formed in X ray treated cherry leaves and differentiation of standard and mutant with RAPDs

Summary An obstacle when using scions orin vitro shoots for mutation induction is the occurrence of chimeras. When adventitious shoots are formed from irradiated material these usually are derived from single cells, this leading to homohistont mutants. SincePrunus avium regenerates adventitious shoots from leaves at a low rate only (Yang & Schmidt, 1992), leaves of the interspecific cherry rootstock ‘209/1’ (P. cerasus ×P. canescens) were irradiated. ‘209/1’ regenerates adventitious shoots readily. Dosages applied were 5, 10, 20 and 40 Gy. Shoot production following 5 Gy irradiation was similar to the control. The application of 40 Gy resulted in strong damage with only few leaves regenerating. Among the adventitious shoots from leaves irradiated with 20 Gy one shoot was evident alreadyin vitro with thicker and smaller leaves having a serrate margin. It was cloned as ‘209/1-20m’. The clone stayed stable since 1990in vitro, in the greenhouse and the field. Compared with standard ‘209/1’ the mutant is very dwarf. Research to differentiate between standard ‘209/1’ and ‘209/1-20m’ was done using RAPDs. Among the decamer primer kits D, J, and T from Operon Technologies, Calif. Only primer OPJ05 (5‘CTCCATGGGG3’) differentiated between ‘209/1’ and ‘209/1-20m’. Rootstock ‘209/1’ showed one band of 2 kb additionally. This band is missing in the mutant.     

Pear mutagenesis: In vitro treatment with gamma-rays and field selection for productivity and fruit traits

In vitro shoots of six pear (Pyrus communis L.) cultivars, ‘Conference’, ‘Doyennéd'Hiver’, ‘Passe Crassane’, ‘Bartlett’, ‘AbbéFetel’ and ‘Butirra Precoce Morettini’ were irradiated with gamma rays (3.5 Gy). After three subcultures, microcuttings from the irradiated shoots and from additional non-irradiated microcuttings were rooted to establish plants for survey orchards. All trees were individually observed for variation in fruit traits and for productivity. Trees were selected for improved characters related to production such as early bearing and consistent productivity. Variations observed in fruit appearance concerned degree of russeting, fruit shape and size. The frequencies of the observed variations in fruit traits depended on the cultivar, ranging from 0.81% in ‘Doyennéd'Hiver’ to 3.64% in ‘Passe Crassane’. Of the 97 variants selected, only two showed chimeral behavior. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microspore Embryogenesis Induced by Low Gamma Dose Irradiation in Apple

Flower buds and extracted anthers of two apple genotypes, ‘Golden Delicious’— a previously known non-androgenic genotype — and ‘Topred’— a known androgenic genotype —, were irradiated by gamma-rays at doses ranging from 5 to 20 Gy, after 0 to 5 weeks of cold pre-treatment (3 °C). When the extracted anthers were irradiated, both microspore embryogenesis and callogenesis were limited for the two genotypes. For ‘Golden Delicious’, androgenic embryogenesis was induced from the flower buds irradiated at 10 to 20 Gy and after 3 to 5 weeks of cold pre-treatment. For ‘Topred’, androgenic embryogenesis was slightly improved after flower bud irradiation at 5 to 10 Gy following cold pre-treatment from 0 to 3 weeks; it was limited after 5 weeks of cold pre-treatment.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

离子束对甘薯胚性悬浮细胞团的辐射诱变效应

以甘薯品种栗子香为材料,用0～100 Gy 12C5+和0～200 Gy 4 He2+离子束分别照射其胚性悬浮细胞团,结果表明栗子香胚性细胞团适宜的12C5+和4He2+辐射剂量分别为30～50 Gy和50～70 Gy。对辐照后的材料进行培养,通过体细胞胚胎发生途径获得大量再生植株。将获得的再生植株移栽到大田,对其地上部和地下部特征进行分析,发现离     

A tissue culture technique for vegetative propagation and low temperature preservation of Beta vulgaris

Summary A tissue culture technique for clonal propagation of Beta vulgaris is described. Flower buds or flower bud clusters formed adventitious shoots on half-strength Murashige & Skoog's medium supplemented with 10 mol/l benzyladenine (BA). The shoots were multiplied by axillary bud proliferation on a medium with 1 mol/l BA and rooted on a medium with 10 mol/l indolebutyric acid. The plants were vegetative. No mutations were observed. Genotypic variation was found in shoot formation, shoot multiplication and rooting. Some genotypes showed leaf malformations which were attributed to BA. Rooted plants in culture tubes could be stored for one year at 2 or 5°C and a low light intensity.     

The induction of mutations in chrysanthemum using X- and gamma radiation

Plants of the Chrysanthemum variety New Princess were irradiated with X- and gamma rays at dose rates varying between 500–2000r X-rays and 1–4 Krad gamma rays. Flower colour changes were induced by both types of radiation and these were accompanied by change in chromosome number and by chromosome fragmentation. Optimum doses were 1000r X-rays and 1 Krad gamma rays. Most of the changes resulted from loss of pigmentation but a number with deeper colours were found. The frequency of mutation was directly proportional to the dose. Other phenotypic changes were also observed. The mutants arise by the formation of chromosomal chimaeras or by the re-arrangement of pre-existing chimaeras.     

理化诱变小豆京农6号突变体的鉴定

选用小豆品种京农6号种子,分别采用甲基磺酸乙酯(EMS)(0.5%、0.9%和1.4%处理12h和24h)、电子束(100、300、600Gy)、60Co-γ(400Gy)诱变处理,将处理后的种子种于大田,鉴定后代植株性状的变异。观察表明,EMS诱变的变异类型最丰富、60Co-γ射线次之、电子束产生的变异类型较单一。EMS处理小豆以浓度0.5%和0.9%处理24h为宜;0.5%EMS处理的粒色和荚色变异突出,有鲜红、黄白、绿白粒色和黑荚、褐荚、黑褐荚变异;0.9%处理的叶形变异突出,有鸡爪叶、剑叶、肾形叶、小密叶等突变类型;电子束诱变后,M2变异率分别为4.09%、3.64%和2.22%。400Gy60Co-γ射线处理种子,后代变异率为7.23%。通过两年的鉴定筛选,获得937个EMS诱变M3代株系,934个60Co-γ射线和电子束诱变M2代株系,已得到株高、叶形、叶色、粒形、粒色、荚色、无分枝、多分枝、叶簇生、分枝簇生、光叶、蔓生、有限结荚习性、株型松散、育性、成熟特性等突变体材料1490份。本研究为小豆基因遗传分析、基因定位与克隆及其进一步的基因功能分析奠定了基础,为小豆育种提供了重要的材料。     

Vegetative propagation of Beta vulgaris by leaf cuttings

Summary Leaf cuttings without axillary buds were made from young vegetative beet plants. Roots were formed at the petiole end. Adventitious bud formation occurred at a very low frequency and could not be stimulated by application of growth regulators. Leaf cuttings with axillary buds formed plants at a higher frequency but development of buds into shoots was slow and irregular. As no vascular connections were found between petiole and axillary bud, the slow growth of the buds was attributed to insufficient supply of assimilates from the leaves. Axillary buds rapidly developed into shoots when petiole explants with buds were placed on culture media. Nearly all cultures, however, were contaminated with bacteria that originated from the inner tissue of the explants.     

X-ray induced mutations in Begonia x hiemalis

Summary A study was made of the occurrence of mutants in plants of four clones of Begonia x hiemalis grown from leaf cuttings irradiated with 0, 1500, 2000 or 2500 rad of X-rays. Irradiation reduced the number and slowed down the development of adventitious shoots, but to a different degree in the four clones used. The percentage of mutated shoots was as high as 18–35% in the non-irradiated shoots. Irradiation raised it to more than 80%. In the control plants, almost exclusively flower colour mutants were found. Irradiation caused a wider range of flower colour mutants and also caused mutations of growth habit, colour and size of the leaves, and size and shape of the flower. Almost all mutants were solid, i.e. non-chimeral.Publication 405, Department of Horticulture, Agricultural University, Wageningen, the Netherlands.     

Genetic variation in mutants of black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper) evaluated by RAPD markers

Genetic variation among four mutants with parent of black gram (high seed protein, tall, bushy, and dwarf mutants) was investigated. The mutants were generated with physical gamma rays and chemical ethyl methane sulphonate. These mutants had some advantages like high protein content, lodging resistance, and high seed yield compared with parent cultivars (cvs.). The objective of the present investigation is to find genetic variation in mutants of black gram with their parents. The genetic variation was evaluated with 20 random primers, generated total 202 fragments scored with 58 polymorphic alleles, and the average was 10.1 alleles per locus and a range of 1–9 alleles. The average polymorphic rates were 38.37 among the mutants and parents through the 20 primers. Primers OPA-14 and OPI-04 revealed 35% of DNA polymorphism in this investigation. The genetic distance (GD) among the genotypes was 0.19 suggesting a significant degree of genetic diversity. The five genotypes were used to construct a dendrogram based on the similarity matrix, revealing a genetic distance varying from 0.600 to 0.725. This variation was due to the mutation induced by gamma rays and ethyl methane sulphonate. Further research is needed to analyze the sequence and expression of these mutated genes and to develop and improve black gram via genetic engineering to evaluate their use.     

Use of gamma rays to induce mutants resistant to cocoa swollen shoot disease in Theobroma cacao L.

Vegetative buds of three cocoa varieties, ‘Amelonado’ (P30), ‘Trinitario’ (K5) and ‘Upper Amazon’ (T85/799) were irradiated with 15, 20 and 25 Gy of γ-rays, respectively, and budded on to rootstocks to generate MV1 shoots. The terminal buds of the shoots were removed to induce the formation of MV2 shoots, from which MV3 shoots were similarly derived. The MV3 plants were screened for resistance to the Cocoa Swollen Shoot Virus (CSSV) by patch-graft inoculation of the root-stocks. Only a few plants from each of the three cocoa varieties were found to be symptomless after indexing. These plants were multiplied by budding to the MV4 and MV5 stages and screened at each stage for CSSV resistance by inoculation using virus-carrying mealybugs. At the MV5 stage, some plants still remained symptomless and this was confirmed with enzyme-linked immunosorbent assay (ELISA). The study reveals that γ-rays can be used to induce genetic variability for resistance to CSSV in cocoa, as well as for other traits such as chlorophyll deficiency.     

Mutation breeding of Achimenes

Summary Freshly cut leaves of Achimenes cv. Paul Arnold were irradiated with X-rays or fast neutrons, or treated with colchicine and then allowed to root and form adventitious buds. As found previously in Saintpaulia and Streptocarpus, almost exclusively solid, non-chimeral mutants or tetraploids were, respectively, obtained by using the adventitious bud technique, demonstrating that the apex of such buds originates from single cells.Although the heterozygosity of the original material apparently was not large, a relatively wide mutation spectrum was observed among the mutants produced.After comparative judging, three mutants were awarded a Certificate of Merit and have been commercialized.Two of these are earlier flowering mutants (Early Arnold and Spring time), the third having a more compact form (Compact Arnold).     

Mutation breeding in seed-coat colors of beans (Phaseolus vulgaris L.)

Summary Seed-coat color is an important agronomic character to determine the marketability of a dry bean variety. People in a location have a specific preference for certain colors of beans. It is known that many black bean varieties are superior in disease resistance and yielding capacity, and because of their seed-coat color, the cultivation is restricted in many bean growing areas.By treating the seeds of black bean varieties with EMS or gamma rays, it is possible to induce a range of seed-coat colors from white, yellow, to grey brown. Coupled with the screening technique described in this paper, it is practicable to isolate the seed-coat color mutants which correlate with green hypocotyl color at a very early stage of seedling development. Genetic tests showed that the induced mutants are due to single genetic locus changes from dominant to recessive. Thus, by using mutation breeding technique, homozygous lines of seed-coat color mutants can be achieved in the second selfed generation. The advantages of using the induced mutation technique for seed-coat color breeding in beans, as compared with the conventional hybridization method, are discussed.This work was supported by the U.S. Atomic Energy Commission under Contract AT (30-1)-2043. Publication No NYO-2043-214.