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.     

离子束介导大豆DNA的小麦变异株系蛋自水解酶谮分析

为研究离子束介导大豆DNA的小麦变异株系在返青期、起身期和拔节期的蛋白水解酶变化,采用复性电泳对新乡9号和筛选出的高蛋白变异株系05-10-1和低蛋白变异株系05-6-1,做不同酸碱条件下的蛋白水解酶分析.结果显示.与对照相比.变异株系05-6-1在返青期酸性条件下少检测到一条29kD酶带:在起身期中性和碱性条件下少检测到一条49kD酶带:在拔节期碱性条件下多检测到一条154kD新酶带.而少检测到158kD的酶带:而变异株系05-10-1在这3个生理时期和对照的蛋白水解酶酶谱带型基本一致.主要在某些酶带的酶活上存在差异.     

Induced changes in growth and flowering of chrysanthemum after irradiation and in vitro culture of pedicels and petal epidermis

Summary To increase the genetic variation for yield in Chrysanthemum morifolium cv. White Spider, pedicel segments and petal epidermis were induced in vitro to regenerate adventitious buds either directly from the original tissue or indirectly via callus. Besides, pedicels were irradiated before in vitro culture with an X-ray dose of 8 Gy. All treatments yielded variation in flower morphology. Changes in flower colour were largely restricted to treatments involving irradiation.Treatments yielding many morphological variants also yielded most production variants. Plants regenerated from petals differed from those originating from pedicels. Clones were found that had retained the morphology of White Spider, but outperformed the controls in flowering time and flower number.     

Regeneration and somaclonal variation in apomictic Paspalum dilatatum Poir

Summary In an attempt to incorporate variation into a uniform obligate apomict, plants of apomictic common dallisgrass, Paspalum dilatatum Poir., were regenerated from callus derived from immature inflorescences. Plants developed through both organogenesis and embryogenesis. A total of 682 regenerants were produced and more than 400 were transplanted into a field nursery and screened for somaclonal variation. Eventually 20 regenerants were selected, increased, and planted into a replicated nursery along with normal common dallisgrass. The characteristics examined were maturity date, plant height, number of racemes per inflorescence, number of spikelets per raceme, pubescence, stigma and anther color, ergot resistance, seed germination, seed set, pollen stainability, method of reproduction, and chromosome number. There were differences among the regenerants and between them and common dallisgrass for all traits except chromosome number, stigma and anther color, and ergot resistance. One of the more important regenerants had significantly higher seed set than common dallisgrass. All regenerants reproduced by aposporous apomixis but some exhibited a high degree of abortion while others had more aposporous embryo sacs per ovule than common dallisgrass. These findings demonstrate that common dallisgrass can be regenerated through tissue culture and that somaclonal variation is expressed in some of the regenerants, even though some of the altered traits are deleterious.     

Towards development of Al-toxicity tolerant lines in indica rice by exploiting somaclonal variation

Somaclonal variations, induced in vitro, were used to enhance tolerance to aluminium (Al) toxicity in rice. Tolerant plants were developed through in vitro screening of embryogenic calli. The calli were derived from mature seed embryos and cultured on medium stressed with different concentrations of Al₂(SO₄)₃⋅18H₂O. Seed germination, callus induction, plantlet regeneration and callus health declined with increased concentration of Al. At higher Al concentrations, callus health deteriorated drastically with partial to total necrosis. Plantlet regeneration varied largely among varieties and treatments. The variety IR72 produced maximum plantlets among all genotypes tested. An amount of 60 ppm or more Al was highly toxic, which greatly reduced plantlet regeneration from callus. R₀ plantlets were grown under glasshouse. Based on the appearance of bronzing symptoms on leaves, the tolerant R₁ plants were selected. R₁ and R₂ lines derived from putative tolerant somaclones, were evaluated in fiberglass tanks filled with Al-toxic soil. R₃ population was evaluated in the field. A few lines derived from IR72 showed high yield and good plant type. The progenies at R₃ showed normal root growth under stressed environment in sand culture. The study revealed that in vitro screening would be an appropriate alternative to conventional breeding in evolving Al-tolerant lines as observed in case of other abiotic stresses. The technique was useful in creating de novo synthesized Al-tolerance character in rice.     

Relationship between somaclonal variation and type of culture in cucumber

Highly inbred B line of cucumber was used to compare the effect of four types of in vitro culture on somaclonal variation. The plants were regenerated from the following types of culture: twelve- and eighteen-month-old liquid culture of meristematic clumps (LMC₁₂₍₁₈₎), ten-month-old embryogenic cytokinin-dependent suspension (CDS), eighteen-month-old embryogenic cytokinin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (CDS 1.7), twelve-month-old embryogenic auxin-dependent suspension (ADS), thirty six-month-old embryogenic auxin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (ADS 1.7) and recurrent leaf callus regeneration (RLC) – repeated 5 times. The differences in the incidence of the following properties were observed: the ploidy of R₀ plants, the segregation of new morphological traits in R₁ and the germination ability of R₁ seeds. R₁ families with the segregation of new phenotypes were most numerous in CDS (62.5%) and LMC₁₈ (57.9%), next in CDS1.7 (35.7%), while the smallest number was found in LMC₁₂ (11.1%) and RLC (3.4%).Tetraploid and mixoploid plants occurred in ADS1.7 and ADS (100%) whereas CDS and RLC were observed to contain only tetraploids, respectively 33.3% and 55.2%. There were no changes of ploidy after LMC₁₂, LMC₁₈ and CDS1.7. Among new phenotypes there were such that have not been described so far in cucumber: ginkgolike leaf (gll), yellow-green chlorophyll mutants (y-gc), serrate margin of corolla in male and female flowers (smc). This revised version was published online in August 2006 with corrections to the Cover Date.     

High frequency regeneration and heritable somaclonal variation in Brassica juncea

Summary Multiple shoot formation in cotyledonary callus of Indian mustard (Brassica juncea cv. Prakash) was induced on modified MS media supplemented with high cytokinin (kinetin or zeatin) and low IAA concentrations. Complete plants were obtained on prolonged incubation of shoots on the same medium. 6-Benzyladenine alone or in combination with IAA or NAA did not support plantlet regeneration. A total of 71 plants were transferred to greenhouse. The seed, however, could be collected from 37 plants only. The seed was sown in the field to evaluate the material for somaclonal variation in R₁ generation. Data were recorded for yield, plant height, number of primary branches, siliqua number, 1,000 seed weight and oil content. Somaclonal lines showed tremendous amount of variation for all the characters studied. A number of plants in this generation showed significantly higher yield and/or other improved agriculturally important characteristics as compared to the control. A line with dwarf plant type was also identified. A number of plants were selected from this generation and carried forward to R₂ generation. Most of these lines bred true in R₂ generation. The material seems to be very promising for future breeding programmes.     

RAPD analysis of sporting and chimerism in chrysanthemum

Summary The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 M for 12 h.Treatments with moderate concentrations of colchicine (3–100 M) produced a small increase in embryo production, while concentrations above 300 M were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine.All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.Abbreviations APM amiprophos methyl - DMSO dimethyl sulfoxide     

In vitro callogenesis and detection of somaclonal variations in Plantago ovata L.

Planago ovata L. is an economically important species in the monotypic genus Plantago. It is a short-stemmed annual herb. The seed husk of this plant is commonly called psyllium or isabgol which is important in pharmaceutical formulation and food industry. In this study, callus induction was optimized using different explants of Plantago ovata. Callus DNA was utilized to access the somaclonal variations using the Random Amplification of Polymorphic DNA (RAPD) markers. The maximum callus growth was observed in Murashige and Skoog (MS) medium containing 4 mg L^?1 2,4-D concentration for shoots, 0.5 mg L^?1 for seeds and 2 mg L^?1 for roots. Moreover, the effect of culture age was considered in assessing genetic variability. Maximum genetic variability was observed in the DNA samples of callus at the concentration of 2 mg L^?1 2,4-D for all explants (roots, shoots, and seeds). Cluster analysis was performed based on 1) similarity coefficient between samples and 2) molecular data using the Numerical Taxonomy and Multivariate Analysis System (NTSYS) PC version 2.01; similarity index was generated by similarity for Quantitative Data (SIMQUAL). Our study indicated that Random Amplified Polymorphic DNAs can successfully be used to explore polymorphism among callus samples at different hormonal concentrations. This study can be useful for the production of callus from Plantago ovata and estimation of genetic variations due to tissue culture conditions. Evaluation of genetic variations can display novel features and manipulate genetic bottlenecks in Plantago ovata. New genetic variations in somaclones can bring vital insight for plant improvement.     

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.     

Assessment of somaclonal variability in hop (Humulus lupulus L.) in vitro meristem cultures and clones by molecular methods

In vitro meristem tissue cultures are used for production of virus-free rootstocks of hop (Humulus lupulus L.). Because use of plant tissue cultures is associated with occurrence of somaclonal variability, we assessed somaclonal variability in hop meristem in vitro cultures before and after thermotherapy by different molecular methods (RFLP, RAPD, STS, ISSR and AFLP) and compared it with existing clonal variability of Osvald's clones 31, 72 and 114. No molecular differences were observed between mother plants and in vitro mericlones by RFLP and STS analyses. Amplified molecular differences were found in RAPD and ISSR products of one from five in vitromericlones cvs. Eroica (E5) and Southern Brewer (SB2), respectively. Similarities with mother plants were 0.965 and 0.913 (JSC), respectively. Specific amplified polymorphic products were found for every mericlone and mother plant in AFLP reactions and variability of DNA sequence ranged from 0.824 to 0.993 (JSC). This variability was very similar to determined intra-clonal variability within Osvald's clones 31, 72 and 114 by AFLP analysis. Inter-clonal variability of DNA sequence was exactly higher than intra-clonal variability of DNA sequence in these clones. The molecular differences between Osvald's clone 72 normal and meristem derived were not verifiable. Thermotherapy increased frequency of molecular changes, since amplified differences were found in 14 from 20 in vitro mericlones of cv. Eroica, in 6 from 11 in vitro mericlones of cv. Yeoman and in 15 from 23 in vitro mericlones of cv. Southern Brewer by RAPD and ISSR analyses.     

A comparison of somaclonal variation in rice plants derived and not derived from protoplasts

To clarify the effect of the type of in vitro culture on the generation of somaclonal variation, protoplast-derived rice plants were compared with rice plants derived from suspension culture or primary calli (not derived from protoplasts). Regenerated plants showing polyploid-like phenotypes appeared at a higher frequency (33–70%) in plants derived from protoplasts than in those not derived from protoplasts (3–6%). In the first progeny (R₁) generation of all regenerated plants, 120 of 368 lines (33%) segregated plants with mutated characters such as albino, dwarf and sterile. In quantitative traits, 62 (21%) and 144 (50%) of 290 Rj lines showed shorter culm and lower seed-fertility, respectively, compared with the control line derived from the selfed seeds of the original cultivar. The frequency of the mutant-possessing R₁ lines among lines derived from protoplasts was not significantly different from those not derived from protoplasts. These results indicate that isolation and culture of protoplasts does not enhance genetic changes other than cytogenetical changes.     

Monitoring genetic fidelity vs somaclonal variation in Norway spruce (Picea abies) somatic embryogenesis by RAPD analysis

Summary Somaclonal variation, which is a welcome source of genetic variation for crop breeding, is unwanted when direct regenerants have to be used in tissue culture mass propagation (eg. in many forest trees), or in the regeneration of genetically transformed plants. Random amplified polymorphic DNA (RAPD) was used to analyse somatic embryos and plants regenerated from embryogenic cell lines in Norway spruce, Picea abies (L.) Karst. RAPD facilitated the identification of clones, as material from the same cell lines shared identical patterns of amplified fragments, whereas regenerants from different cell lines were easily distinguishable by their respective patterns. For comparisons with explant donor genotypes, cell lines were initiated from cotyledons. Some of the seedlings that had parts of their cotyledons removed were grown on as control plants. Somatic embryos regenerated from cotyledon cell lines showed no aberrations in RAPD banding patterns with respect to donor plants. We conclude that gross somaclonal variation is absent in our plant regeneration system.Abbreviations ESM embryogenic suspensor mass - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - (2,4-dichlorophenoxy)acetic acid 2,4-D - 1-naphthaleneacetic acid NAA     

Effect of proton beam irradiation on germination, seedling growth, and pasting properties of starch in rice

This study was carried out to determine the pretreatment effect of proton beam irradiation on germination and pasting properties of starches in two rices. Mature and healthy seeds irradiated with 10 doses (0, 50, 100, 200, 300, 400, 500, 600, 700, and 800 Gy) for determination of LD50 and characteristics of germination were recorded at 14 days after irradiation. The rice seeds irradiated with five doses (0, 50, 100, 200, and 300 Gy) were used to evaluate the irradiation effects of pasting properties of starches. It showed that a lower survival rate in germinated rice seeds was observed above 300 Gy showing 31 and 35% in Ilpum and Hanmaum, respectively. The higher plant height and root length were also recorded in 50 and 100 Gy. Amylose content in proton beam irradiated with 50, 100, 200, and 300 Gy was significantly decreased in two rice cultivars. Peak viscosity, hot peak viscosity, cooling peak viscosity, and setback viscosity decreased with increasing proton beam dose levels. The degree of crystallinity was significantly increased with increasing proton beam dose levels. Consequently, it might be deduced that proton beam irradiation causes changes of starch properties affecting crystalline regions of starch granules, especially at high dosage irradiation.     

电子束辐照对稻粒黑粉菌和禾草腥黑粉菌冬孢子萌发活性的影响

辐照技术作为一种检疫措施受到国际上的认可,同时也是国际上备受关注的一种检疫处理技术.本试验采用电子束照射稻粒黑粉菌和禾草腥黑粉菌病粒,研究不同辐照剂量对病菌冬孢子萌发活性的影响.试验结果表明,随着照射剂量的加大,冬孢子萌发活性明显降低,当照射剂量达到5.31KGy时,未发现萌发的稻粒黑粉菌和禾草腥黑粉菌冬孢子.     

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.     

Mutations in potatoes induced by gamma irradiation

The report deals with experiments in which gamma irradiation was applied to induce somatic mutations in potato clones. The best results are obtained when an irradiation dose of 3200 r is applied.Most of the aberrants recognized were colour mutations or leaf mutations. All these aberrants were inferior to the original clones when tested in yield trials.In the variety Pimpernel occurred a mutant with definitely shorter stolons than the original clone. Unfortunately this character was correlated with a great tendency of the tubers to cracking and the clone will not be released for commercial growing.The possibilities of using artificial induction of mutations in potato breeding are briefly discussed.     

Application of RAPD markers in the characterisation of Chrysanthemum varieties and the assessment of somaclonal variation

Characterisation of fifteen commercial varieties of Chrysanthemum was carried out through RAPD analysis. Varieties could be distinguished from each other and the level of similarity between varieties seemed to be not very high. In vitro cultures were establish from four varieties and were subjected to different proliferation conditions. Five individuals from each variety and treatment were analysed using RAPD at the beginning of the treatment and after a month of culture. Variation was detected at both stages of the culture period. The rate of variation found showed differences between varieties, but no significant difference was found between culture conditions. This revised version was published online in August 2006 with corrections to the Cover Date.