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.     

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.     

Somaclonal variation and improvement in chilling tolerance in rice: changes in chilling-induced electrolyte leakage

Excised leaves of four rice varieties, ‘Facagro 57’, ‘Facagro 76’, ‘Kirundo 3’, ‘Kirundo 9’, were chilled for six days at 10/5°C day/night, along with 11 somaclonal families arising from three of these varieties, which had been selected over four generations for improved chilling tolerance. At the end of the chilling treatment, electrolyte leakage from leaf pieces was followed for 12 h and compared with an unchilled control. With unchilled leaves, no varietal differences nor a rise in leakage over time were observed. Chilling induced higher leakages in all varieties, except in ‘Kirundo 3’ at the time of first measurement, and a continuous rise over time was found in all varieties. Varietal differences were detected, ‘Kirundo 3’ being the least affected, followed by ‘Kirundo 9’ and ‘Facagro 76’, and finally ‘Facagro 57’, which coincides with their ranking for chilling tolerance established through chilling survival tests. The leakages were generally lower in the somaclonal families than in the variety they originated from in ‘Kirundo 9’ and ‘Facagro 76’, while the opposite was observed in ‘Kirundo 3’. The significance and usefulness of electrolyte leakage measurement for chilling tolerance screening, and the potentials of somaclonal variation for chilling tolerance improvement are discussed.     

Field evaluation and selection of rice somaclonal variants at different altitudes

A field experiment was designed to compare control with tissue culture-derived plants of rice, and to study the effects of natural selection pressure applied to the first generation (R0) of in vitro plants on the performance of their progeny. Control- and R0 in vitro plants of four rice varieties from middle altitude in Burundi, i.e. Facagro 57′, Facagro 76′, ‘Kirundo 3’ and ‘Kirundo 9’, were cultivated at four different altitudes (800, 1380, 1650 and 1900 m). Several parameters concerning tillering capacity, plant height development and seed production were measured. The means for most measured parameters were higher in control plants than in the in vitro plants, while the higher variation coefficients and most extreme values were usually found in the in vitro plants. For each variety, the 20 control plants and 20 in vitro plants having the highest production were selected at each altitude of 1380 and 1650 m, and their progenies were cultivated on the same site at 1580 m. Among the plants arising from 1650 m, most maximum values, higher variation coefficients and, in some cases, higher means were found in the in vitro plants. In contrast, among the plants arising from 1380 m, higher means most usually corresponded to control plants, while the tendency for maximum values and variation coefficients were unclear. These results indicate the positive effect of natural selection pressure applied in the first generation to plants rising from tissue culture. They also demonstrate that considerable variation may be generated in vitro. The interest for plant breeding purposes is discussed.     

Culture selected somaclonal variation in five Triticum aestivum L. genotypes

Somaclones (R₃ and R₄ generations) regenerated from five winter wheat (Triticum aestivum L.) genotypes were evaluated for variation in agronomic and morphological characters. Immature embryos were used as initial explant material. Comparisons for plant height, top internode length, spike length, number of seeds per spike and 100 seed weight were made between the somaclones and their parents. Some morphological variations of stem and spike characteristics were registered which demonstrate that plant height and spike length can be changed by using immature embryo culture. The results obtained may be considered a biotechnological contribution to wheat plant improvement. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inter simple sequence repeat polymerase chain reaction for the detection of somaclonal variation

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) analysis is quick, simple and generates complex bind profiles. The technique has been used widely for DNA fingerprinting and genetic distance analysis. In this study, the potential of ISSR-PCR is evaluated for the detection of somaclonal variation in potalo tissue culture regenerants. Four primers were used to screen 40 regenerants of Solanum tuberosum cv. ‘Skirma’ for genetic change arising from callus culture. Two regenerants were found to have altered hand profiles. The use of an additional 13 primers on these variant clones failed to reveal any further evidence of genetic change. The potential and limitations of the approach for assessing the incidence of somaclonal variation is discussed.     

Tissue culture-induced morphological somaclonal variation in St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze]

Somaclonal variation has been observed in many plant species and is an alternative way to create variants and expand the germplasm pool. A large scale tissue culture experiment was conducted with St. Augustinegrass, an important turfgrass species for the southern USA, to induce somaclonal variation to enlarge the germplasm pool for breeding efforts. Using an improved protocol, approximately 7900 St. Augustinegrass plants were regenerated from cv. 'Raleigh', and 119 morphological variants were identified. Among the variants, 115 had a semi-dwarf growth habit with shorter and narrower leaves, and shortened internodes and stolons. However, 100 of them showed little vigour, which either grew very slowly or did not survive. The remaining 15 showed reasonable growth vigour and were further investigated in the field. Among them, 13 were semi-dwarf and 2 had longer leaves. In addition, 2 other variants, with variegated (yellow striping) leaves, or significantly thicker stems were also observed and characterized. The altered traits in the variant lines were stable during vegetative propagation and when grown in different environments.     

An assessment of somaclonal variation as a breeding tool for generating herbicide tolerant genotypes in wheat (Triticum aestivum L.)

The present work was initiated to assess tissue culture techniques as a means of generating and selecting herbicide tolerant genotypes of wheat through exploiting somaclonal variation. Callus was initiated from immature embryos of genetically defined varieties and subcultured onto selective media containing 5 μm, 10 μm and 50 μm concentrations of difenzoquat and atrazine. Plants were regenerated from all the selective media except that media containing the highest herbicide concentration. The progenies of the regenerated plants were tested as whole plants for their response to spray application of the herbicides. For difenzoquat, variation in response from extreme susceptibility to tolerance was observed. However, genetic characterization by progeny testing tolerant lines revealed that the induced variation was not heritable. No plants tolerant to atrazine were obtained. Overall, no clear evidence of heritable mutations was obtained. Alternative strategies to obtain herbicide tolerant genotypes using cell culture techniques are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of chromosome-doubling agents on somaclonal variation in the progeny of doubled haploids of maize

This study was carried out to determine whether the treatment of anther-culture-derived haploid callus of maize (Zea mays) with chromosome-doubling agents, such as colchicine or the herbicides pronamide and amiprophos-methyl (APM), induces higher than normal levels of somaclonal variation. A total of 79 R₁ families produced by diploid regenerated plants resulting from chromosome-doubling treatments were evaluated in the field in comparison with the three parental inbreds. Four qualitative variant phenotypes — male sterility, chlorophyll deficiency, earless plants, and short plants with narrow leaves and thin stalks —– were observed. The last phenotype (narrow leaves and thin stalks) was also found in the inbreds FR16 and H99 grown from seed, so it may not be directly related to the tissue-culture conditions or the anti-microtubule-agent treatments. The frequency of R₁ families segregating for the other three mutations was 3.8%, which is no higher than the somaclonal variation frequencies observed previously in tissue-culture-derived maize plants. Observations of three quantitative traits—–days to anthesis, days to silk emergence, and plant height—– also failed to detect any extra variation that could be related to the treatments with anti-microtubule agents. These studies indicate that the anti-microtubule agents APM, pronamide and colchicine can be used to induce chromosome doubling of anther-culture-derived callus to produce a high proportion of doubled haploid plants without causing increased rates of mutation (somaclonal variation).     

Transmission of somaclonal variation in wheat

Summary Transmission of somaclonal variation was studied in callus derived (SC1) plants of wheat, Triticum aestivum L. Second (SC2), third (SC3) and fourth (SC4) generations were compared with those obtained from embryo culture (E1-E4). SC2 generation had significantly lower grain yield (–37.3%), reduced thousand kernel weight (–7.8%) and shorter height (–2.1%) than that of E2, and included 5.7% seed-sterile and 15.0% partially seed-sterile plants. SC3 generation showed reduced yield (–12.6%), lower thousand kernel weight (–4.9%) and shorter plant height (–1.9%) than E3 generation. SC4 progeny of a short height, partially sterile SC1 variant included aberrant plants: a haploid, few aneuploids, mixoploids, some showing multiploidy, spindle abnormalities, multivalents, bridges and fragments, and tillers having supernumerary spikes with branched rachis. Somaclonal variation resulted from genome instability in callus, and likely involved transposable elements.     

Is somaclonal variation a reliable tool for spring wheat improvement?

Summary Seed progeny of tissue culture regenerants of a spring wheat (Triticum aestivum L. cv. HY320) was evaluated for key agronomic traits for three years under field conditions. Initially, 27 regenerant families were tested in hill plots. Among-family and within-family variation was generally highly significant (p < 0.01) and nonsignificant, respectively. The variation observed among regenerants on the basis of hill plot testing was not duplicated in subsequent four-row plot experiments. On average, regenerant families yielded 28 and 5% less than the control in dryland and irrigated tests, respectively. Low yielding regenerants tended to produce fewer, lighter kernels per spike. Higher grain protein levels among regenerants were associated with low yields (r=0.85). This study demonstrated that putative somaclonal variation arising from tissue culture failed to produce genotypes agronomically superior to the parental cultivar, HY 320.NRCC Publication No. 33533     

植物体细胞无性系变异的研究进展

植物体细胞无性系变异是组织培养过程中的普遍现象。本文综述了有关植物体细胞无性系变异的影响因素、类型、机理等方面的研究成果。     

DNA markers for eating quality of indica rice in Indonesia

Rice eating quality is considered to be one of the top priorities in determining the agronomical value of rice; thus, the rapid evaluation of eating quality at early breeding generations in breeding programmes for better eating quality is of great importance. In an attempt to develop DNA markers associated with eating quality of indica rice, we used multiple regression analysis to test 54 markers, which were preselected for their possible association with eating quality, using 24 indica varieties with different palatability scores. Of these markers, eighteen markers were found to be significantly associated with palatability according to sensory evaluation. Accordingly, a marker set in the model regression equation with a high R² (0.997) was formulated to estimate indica rice palatability. Validation suggests that markers and the statistical parameters formulated by the equation could be a potential tool to predict the palatability of cooked Indonesian indica rice and could be reliable in developing country‐dependent model equations for eating quality.     

Cell selection as a possible reason for the specificity of somaclonal variation in pea

A study of regenerants obtained from long-term callus cultures of different pea (Pisum sativum L.) genotypes revealed specificity of in vitro mutagenesis. The specificity was displayed in the preferential generation of the somaclonal variations affecting quantitative and developmental characters. About 60% of regenerated lines obtained from the cultivar Ranny Zeleny carried mutations in the Lf and Sn loci, which control initiation of flowering. An in vitro study of isogenic lines differing at the Lf and Sn loci illustrates the growth advantages of mutant genotypes in tissue culture. These experiments suggest that mutations affecting developmental characters (e.g. mutations in loci which control flowering behaviour) may be expressed in tissue culture and may cause increased adaptation in mutant cells to the in vitro conditions. Rapid propagation of mutant cells during in vitro culture may lead to a higher proportion of quantitative mutations among the regenerated plants.     

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.     

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.     

杂交籼稻恢复系稻米淀粉RVA谱特征相关及聚类分析

测定早造种植收获的42个恢复系和7个不同蒸煮及食味对照稻米淀粉RVA谱特征值,对其进行了相关分析和系统聚类分析。结果表明如下：（1）恢复系RVA谱特征值变异系数最大是SBV,依次是BDV,CSV,CPV,HPV,最小是PKV。（2）各RVA谱特征值表现较好相关性。RVA谱特征值3个基本粘度间,只有HPV与CPV表现极显著正相关。PKV与BDV呈极显著正相关,与SBV和CSV均分别呈极显著负相关;HPV、CPV与BDV分别呈显著、极显著负相关,HPV、CPV分别与SBV和CSV均呈极显著正相关。BDV、SBV和CSV均分别呈极显著负相关,而SBV和CSV呈极显著正相关。（3）RVA谱特征值可分为3大类型,三类型区分主要依据SBV,Ⅰ类型SBV最高(37.4RVU～156.8RVU),Ⅱ类型SBV最低(-141.2RVU～-86.0RVU),而Ⅲ类型SBV介于两者之间(-83.6RVU～23.7RVU);在两极端类型Ⅰ类型和Ⅱ类型之间区分还可依据BDV和CSV指标。Ⅰ类型亚类间区分主要依据PKV和CPV,Ⅱ类型亚类区分依据PKV,Ⅲ类型亚类区分则比较复杂,依据PKV、HPV、BDV、SBV和CSV指标组合。     

Genetic variation of germination cold tolerance in Japanese rice germplasm

Low temperatures at the initial stages of rice development prevent fast germination and seedling establishment and may cause significant productivity losses. In order to develop rice cultivars exhibiting cold tolerance, it is necessary to investigate genetic resources, providing basic knowledge to allow the introduction of genes involved in low temperature germination ability from accessions into elite cultivars. Japanese rice accessions were evaluated at the germination under two conditions: 13°C for 28 days (cold stress) and 28°C for seven days (optimal temperature). The traits studied were coleoptile and radicle length under optimal temperature, coleoptile and radicle length under cold and percentage of the reduction in coleptile and radicle length due to low temperature. Among the accessions studied, genetic variation for traits related to germination under low temperatures was observed and accessions exhibiting adequate performance for all investigated traits were identified. The use of multivariate analysis allowed the identification of the genotypes displaying cold tolerance by smaller reductions in coleoptile and radicle lenght in the presence of cold and high vigour, by higher coleoptile and radicle growth under cold.     

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.     

Genetic analysis for protein content in indica rice

Analysis of genetic main effects and GE interaction effects for protein content (PC) and protein index (PI) of indica rice (Oryza sativa L.) was conducted for two year experimental data by using a genetic model for quantitative traits of seeds in cereal crops. Nine cytoplasmic male sterile lines or maintainers as female parents and five restorer lines as male parents were used for a NC II mating design in 1995 and 1996. The results indicated that PC and PI traits were simultaneously controlled by genetic main effects as well as GE interaction effects. For PC and PI of rice, the embryo interaction effects (embryo additive and dominance interaction effects) were important as effects of triploid endosperm, cytoplasm and diploid maternal plant. The estimates of narrow-sense heritability for PC and PI of rice were 85.3% and 77.9%, respectively. The interaction heritability was found to be larger than the general heritability for PC and PI of rice. Significant relationships for components of different genetic effects between PC and PI were found. The GE interaction covariances tended to be positive. This revised version was published online in July 2006 with corrections to the Cover Date.