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.     

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.     

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.     

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.     

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.     

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.     

Fire blight resistance and genetic trueness-to-type of four somaclonal variants from the apple cultivar Greensleeves

Four somaclonal variants regenerated from adventitious buds of the apple variety Greensleeves were preselected on the basis of their reduced fire blight susceptibility. The present study aimed at assessing precisely their level of fire blight resistance through various inoculation techniques (on in vitro leaves and microcuttings, on greenhouse plants and in field conditions). Overall results of these tests indicated that one clone (R 46/3) was clearly less susceptible than the control. This clone was also characterized as a ‘spur’ variant, with a reduced growth which can explain its limited susceptibility to fire blight. A second clone (R 20/63) was slightly less susceptible than the control in greenhouse and field tests, but this low level of resistance was overcome by high concentrations of inoculum. The absence of variation in chromosome number and isozyme patterns confirmed the genetic trueness-to-type of these four somaclones. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimizing selection for rough rice yield, head rice, and total milled rice

High rough rice yields are important for the profitable production of rice (Oryza sativa L.). The value of rough rice is determined primarily by head rice (whole milled grains) and broken rice, together referred to as total milled rice. The objective of this study was to optimize selection for rough rice yield, head rice, and total milled rice using path-coefficient analysis and selection indices. Thirty-seven long grain pure lines were grown in an experiment at three Arkansas locations during 1993 and 1994. In another test, twenty-eight F₁ hybrids obtained from an eight-parent half diallel of long-grain rices were grown at two locations in Arkansas during 1991. For the pure lines, path analysis revealed that rough rice yield was approximately twice as important in determining total milled rice/ha than head rice. Path analysis for the hybrids revealed that rough rice yield was an even greater component in determining total milled rice/ha. Based on yield components for hybrid rice, panicle density had the largest direct effect determining total milled rice/ha. Filled grain/panicle, grain weight, and head rice, in that order, were secondary, but positive, factors determining total milled rice/ha. A selection index for pure lines based only on selection for head rice was 19.2 percent as effective as selection for total milled rice/ha directly. When selection for total milled rice/ha was based solely on rough rice yield, selection was 82.7 percent as effective as selecting for total milled rice/ha directly. Selection to improve total milled rice/ha should concentrate on increasing both rough rice yield and head rice. Rough rice yield should receive greater priority than head rice in selection strategies. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Variations of the vascular bundle system in Asian rice cultivars

Summary The capacity of vascular bundle system transporting assimilates from source to sink is one of the limiting factors for rice (Oryza sativa L.) yield. Genetic variation in vascular bundle system should be investigated for improvement of transporting efficiency. A total of 531 Asian rice cultivars were studied for vascular bundles in the peduncle and its relation to spike morphology. A few difference was detected for the number of primary rachis branches of the spike among the cultivars from various countries. However, the number of vascular bundles significantly differed among the cultivars; those from Nepal, Bangladesh and India bore more vascular bundles than those from Japan. Therefore, the ratio of vascular bundles to rachis branches (V/R ratio) was nearly 1.0 in cultivars from Japan, while those from Nepal, Bangladesh and India ranged from 1.3 to 2.2. Chinese and Indonesian cultivars incuded two types of high and low V/R ratios. Cultivars with high V/R ratio showed positive reactions to the phenol solution, while those with low V/R ratio were negative reactions, suggesting that the former cultivars are of Indica type and the latter of Japonica type. It was concluded that the relationship between vascular bundles and rachis branches differentiated among the ecotypes of Asian cultivated rice.     

Field performance of bermudagrass germplasm expressing somaclonal variation selected for divergent responses to fall armyworm

Fall armyworm, Spodoptera frugiperda (J.E. Smith), is the major insect pest of bermudagrass, Cynodon dactylon (L.) Pers., in the Southeastern USA. Somaclonal variation in bermudagrass has produced distinctly divergent susceptibilities to fall armyworm under controlled conditions. Plant persistence, productivity, forage quality characteristics, and response to fall armyworm were assessed in field plots at Rosepine, Louisiana from 1995 through 1999. Forage quality characteristics were assessed each year, and natural infestations of fall armyworms were monitored in 1998 and 1999. Most of the somaclonal lines were similar to their parent cultivar in forage yield and quality, and none was consistently superior. Differences in forage quality characteristics among the bermudagrass lines were detected in 1996 and 1997, with little difference among lines in 1998 and 1999. All lines assessed were infested and sustained damage from natural infestations of fall armyworm larvae. Thus despite acceptable forage productivity and quality of most lines, expression of fall armyworm resistance under these field conditions was not detected. This lack of difference under field conditions, despite distinct resistance of some lines under controlled conditions, may primarily be due to either insufficient production of compounds deterring the insects for detectable effects under field conditions or loss of expression of the somaclonally acquired traits even with vegetative reproduction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Feasibility of selection for traits associated with cold tolerance in rice under rapid generation advance method

Summary Variability of the hybrid population can be preserved through generations during the process of rapid generation advance (RGA). Effective selection can be done in an early generation for those traits having high heritability value like growth duration and plant height. But selection is not advantageous for traits having low heritability. Thus, selection for panicle exsertion, cold tolerance at seedling stage and panicle length would not be wise during the early generations. However, in the low temperature areas growth duration and plant height are two important factors which determine varietal suitability. As selection of these two characters are possible through RGA, it should be a standard parctice during RGA to eliminate the plants with long growth duration and select plants with optimum plant height.     

Improving participatory varietal selection processes: participatory varietal selection and the role of informal seed diffusion mechanisms for upland rice in Ghana

This paper considers the process of Participatory Varietal Selection (PVS) and presents approaches and ideas based on PVS activities conducted on upland rice throughout Ghana between 1997 and 2003. In particular the role of informal seed systems in PVS is investigated and implications for PVS design are identified. PVS programmes were conducted in two main agroecological zones, Forest and Savannah, with 1,578 and 1,143 mm of annual rainfall, respectively, and between 40 and 100 varieties tested at each site. In the Savannah zone IR12979-24-1 was officially released and in the Forest zone IDSA 85 was widely accepted by farmers. Two surveys were conducted in an area of the Forest zone to study mechanisms of spread. Here small amounts (1–2 kg) of seed of selected varieties had been given to 94 farmers. In 2002, 37% of 2,289 farmers in communities surveyed had already grown a PVS variety and had obtained seed via informal mechanisms from other farmers, i.e. through gift, exchange or purchase. A modified approach for PVS is presented which enables important issues identified in the paper to be accommodated. These issues include: utilising existing seed spread mechanisms; facilitating formal release of acceptable varieties; assessing post-harvest traits, and; the need for PVS to be an ongoing and sustainable process.     

Field evaluation of somaclonal variation in sunflower (Helianthus annuus L) and its application for crop improvement

Immature zygotic embryos from the American fertility restorer line RHA-857 were used as donor material for induction of direct organogenesis in sunflower (Helianthusannuus L.). The range of spontaneous somaclonal variation among the progenies of regenerants was studied. The genetic modifications observed in regenerants included agronomic traits such as oil content in seed, 1000 seed weight, plant height, leaf width, leaf length, petiole length, internode length, head diameter, number of branches, length of branches, number of ray florets, seed width, seed length, and seed thickness. RAPD molecular analysis carried out on sunflower materials in the R-11 generation showed the absence of a specific 358 bp band in somaclonal line 11/2/51 R. This line showed a modified architecture, full resistance to Phomopsis helianthiand higher oil content in seed in comparison to the standard RHA-857. Line31/3/53 R was with modified architecture and higher 1000 seed weight. Hybrid No. 144 produced with the participation of somaclonal line 20/5/52 R demonstrated high production capacity, shorter vegetation period and reduced height. The combination of these favourable changes is desirable in breeding work on sunflower. Somaclonal variation through director ganogenesis has facilitated the creation of genetically heritable variation in sunflower, which can be used with great success for hybrid seed production of highly productive hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparing farmers and breeders rankings in varietal selection for low-input environments: A case study of rainfed rice in eastern India

A number of breeding institutions developed a project to assess importance of participatory plant breeding approaches for rainfed rice improvement in eastern India. The results of the first two years of participatory varietal selection are reported here. The objective was to evaluate the respective effects of participation of farmers in varietal evaluation and decentralization of varietal testing from breeding stations to farmers' fields on varietal ranking. Fields representing various hydrological situations were chosen in two to three villages at four rainfed lowland sites and one upland site. Sets of 15 to 25 varieties were tested both in farmers' fields and on-station in 1997 and 1998 and ranked by both farmers and breeders. The effect of participation was judged by comparing the rankings attributed by farmers and breeders to a given set of material in a given trial. The effect of decentralization was determined through comparisons between individual breeders' rankings across trials. Farmers' rankings were not randomly allocated, but agreement within the farmers' group was not always very strong. Except at one site, concordance among breeders' ranking was high, but, because of the limited number of breeders involved, it was seldom significant. In about two-thirds of the trials, there was a good agreement between farmers' and breeders' mean rankings. The consensus was particularly strong when severe constraints induced contrasting behavior in the genotypes. The decentralization effect appeared to be moderate, but variations due to a breeder effect were recorded. The part of genotype by environment interactions for grain yield due to location within one site and year was evaluated through various methods, showing more effect of G × E interactions at some sites than at others. Crossover interactions inducing changes in ranks represented a limited part of the yearly G × E interactions at all sites. Both farmer participation and decentralization of varietal testing in farmers' field would help in best matching the varieties to the needs, although their combined contribution would be more useful in some sites than in others. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of selection indices to screen lowland rice for drought resistance

Summary Although many selection indices have been used to screen rices (Oryza sativa L.) for drought resistance, there has been little comparison of the relative merits of these indices. Research was conducted to compare drought resistance as estimated from grain yields, canopy-temperature-based stress indices, visual scoring, and uprooting force for 30 rice genotypes grown in the field with a puddled Maahas clay (Typic Tropaquept) and to evaluate traits related to drought resistance from nonstressed plants grown in the field and in aeroponic culture. Water deficit was imposed in the field by withholding irrigation from 45 to 75 days after transplanting compared to a continuously flooded control. Grain yields in the stress treatment were most strongly correlated with visual assessment of drought stress symptoms according to a standard evaluation system (r = 0.66). Canopy-temperature-based indices were also significantly correlated with grain yields of the stress treatment (r from –0.55 to –0.63). No trait of aeroponically grown plants was correlated with traits of stressed plants in the field. We conclude that visual scoring of stressed plants was the best method of screening for drought resistance, but if controlled water deficit cannot be imposed, then drought resistance may be estimated by measuring both uprooting force and grain yield.     

Identification of two types of differentiation in cultivated rice (Oryza sativa L.) detected by polymorphism of isozymes and hybrid sterility

Magnitudes of genetic variation within each of major varietal groups of cultivated rice were surveyed in terms of isozyme polymorphism and allelic differentiation of hybrid sterility loci, both of which are considered to have litt le selective value. Allelic differences for 20 isozyme loci were examined in a total of 337 accessions, including Indica and Japonica rices. Aus cultivars from India, Javanica cultivars and both landraces and cultivars from China. Eleven out of the 20 isozyme loci were polymorphic. The Aus cultivars contained more alleles per locus than others. The hybrid sterilities in the crosses among Chinese cultivars, Indica and Japonica cultivars were mainly controlled by locus S-5, whereas the hybrid sterilities of Aus cultivars, when they were crossed to Indica, Japonica or Javanica cultivars, were found to be controlled by allelic interaction at hybrid sterility loci S-5, S-7, S-9 and S-15. Also in terms of the number of alleles at S-5 and S-7, Aus cultivars contained more alleles than others. While the Aus group showed an extreme diversity for both hybrid sterility alleles and isozymes, modern cultivars from the south of YangZi River in China were classified into Indica type and those from the north were into Japonica, which were almost the same as those in Japan. On the basis of the measured polymorphism the Indica-Japonica differentiation was explained by founder effects, i.e., through selection and distribution of two original sources each with a unique set of markers. Contrastingly, the continuous and pronounced diversity in the Aus group was attributed to the absence of such a process. The intermediate groups in Yunnan province and Tai-hu Lake region of China are considered to be isolated from such founder effects, retaining an intermediate diversity in terms of isozymes and hybrid sterility genes. Since hybrid sterility hampers the exchange of genes between cultivars of different groups, the understanding of its genetic basis will be important in rice breeding, particularly in hybrid rice breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative Field Performance of Tissue Culture-Derived Lines and Breeder Lines of HY320 Spring Wheat

This study examines the performance of somaclonal spring wheat (Triticum aestivum L.) lines developed through culture of somatic embryos. Twenty-nine breeder lines of the spring wheat cv. ‘HY320’ were compared to 51 somaclonal lines of the same cultivar. Somaclonal lines were derived from 27 individual embryos (with up to four lines in each “family”). Somaclonal and breeder lines were evaluated in five field experiments in western Canada. Somaclonal lines were more variable than breeder lines for most agronomic, yield component and quality characters, suggesting that variability among somaclonal lines resulted partly from the tissue culture process. Somaclonal lines yielded, on average, 11 % less than breeder lines. Somaclonal lines had 3.8 % fewer spikelets per spike, 6.5 % fewer kernels per spike and kernels which were 2.7 % lighter. Somaclonal lines had greater test weight, protein concentration, and sedimentation values, and harder kernels.     

Characterization of rice (Oryza sativa L.) F3 populations selected for salt resistance. I. Physiological behaviour during vegetative growth

Plants belonging to a somaclonal family (R₃-1-23, obtained from a salt-resistant callus of the salt-sensitive cultivar I Kong Pao (IKP, salt-sensitive)) were crossed with this intital cultivar IKP or with plants of the elite breeding line IR31785 (IR31, extremely salt-sensitive). Two other crosses were also performed: IR31 × IKP and IKP × Aiwu, where Aiwu is a moderatly salt-resistant cultivar. The physiological behaviour of F₃ populations was analysed after 21 and 42 days of exposure to 0 or 30 mM NaCl and compared to the behaviour of plants selected within parental populations. A clear improvement in terms of survival percentages was demonstrated for plants issued from IR31 × 1-23 and IR31 × IKP. These F₃ populations exhibited lower Na accumulation in shoots, higher Ca accumulation in roots, higher ratios of shoot P/root P than the parents after 42 days of salt exposure. Salt stress induced a decrease of shoot K, an increase of shoot Cl and proline and a decrease of osmotic potential in all populations. Correlation analysis indicated that no physiological parameter recorded in the absence of salt stress may be used as a reliable selection marker to predict the plant behaviour in salt stress conditions. K/Na ratio recorded in the stressed plants was significantly correlated with surviving percentages recorded after 90 days of salt stress, suggesting that this physiological parameter in stress conditions appears to be the most reliable selection criterion for salt resistance in rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison of phenotypic versus marker-assisted background selection for the SUB1 QTL during backcrossing in rice

Marker assisted backcrossing has been used effectively to transfer the submergence tolerance gene SUB1 into popular rice varieties, but the approach can be costly. The selection strategy comprising foreground marker and phenotypic selection was investigated as an alternative. The non-significant correlation coefficients between ranking of phenotypic selection and ranking of background marker selection in BC₂F₁, BC₃F₁ and BC₃F₂ generations indicated inefficiency of phenotypic selection compared to marker-assisted background selection with respect to recovery of the recipient genome. In addition, the introgression size of the chromosome fragment containing SUB1 was approximately 17 Mb, showing the effects of linkage drag. The significant correlation coefficient between rankings of phenotypic selection with the percentage of recipient alleles in the BC₁F₁ generation suggested that background selection could be avoided in this generation to minimize the genotyping cost. The phenotypically selected best plant of the BC₃F₁ generation was selfed and backcross recombinant lines were selected in the resulting BC₃F₄ generation. The selection strategy could be appropriate for the introgression of SUB1 QTL in countries that lack access to high-throughput genotyping facilities.