Somaclonal variation as a tool for crop improvement

Summary Somaclonal variation is a tool that can be used by plant breeders. The review examines where this tool can be applied most effectively and the factors that limit or improve its chances of success. The main factors that influence the variation generated from tissue culture are (1) the degree of departure from organised growth, (2) the genotype, (3) growth regulators and (4) tissue source. Despite an increasing understanding of how these factors work it is still not possible to predict the outcome of a somaclonal breeding programme. New varieties have been produced by somaclonal variation, but in a large number of cases improved variants have not been selected because (1) the variation was all negative, (2) positive changes were also altered in negative ways, (3) the changes were not novel, or (4) the changes were not stable after selfing or crossing. Somaclonal variation is cheaper than other methods of genetic manipulation. At the present time, it is also more universally applicable and does not require containment procedures. It has been most successful in crops with limited genetic systems and/or narrow genetic bases, where it can provide a rapid source of variability for crop improvement.     

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).     

Tissue culture-derived variation in crop improvement

Tissue culture generates a wide range of genetic variation in plant species which can be incorporated in plant breeding programmes. By in vitro selection, mutants with useful agronomic traits, e.g. salt or drought tolerance or disease resistance, can be isolated in a short duration. The successful use of somaclonal variation is very much dependent on its genetic stability in the subsequent generations for which molecular markers such as RAPDs, AFLPs, SSRs and others can be helpful. The potential of somaclonal variation has yet to be fully exploited by breeders, even though a few cultivars have been developed in crops such as Brassica juncea, rice and others. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heterosis in crop mutant crosses and production of high yielding lines using doubled haploid systems

Heterosis appearing in crosses between mutants derived from the same parent variety and crosses of mutants with parent varieties has been observed by many authors for many plant species including such important crops as barley, faba bean, maize, pea, pearl millet, rice, sunflower, sesame, sweet clover, triticale and wheat. Mutant heterosis was reported for crosses of spontaneous mutants, mutants obtained after treatment with various mutagens and recently also for somaclonal variants. The heterotic effects are usually related to an increase in some yield components. There is a lack of correlation between mutation of a particular plant character and appearance of heterotic effect. The yielding performance of a mutant was not correlated with its potential to give yield heterosis in crosses with another mutant, often also a very poor one, or with the parent variety. Poor yielding barley mutants from the collection of semi-dwarf forms of the Department of Genetics, Silesian University gave heterosis in crosses with other mutants or parent varieties for such characters as tillering, grain number and weight per plant. In addition to mutants with deleterious mutations of such characters as chlorophyll synthesis or fasciated stem there were also lines mutated for earliness, semi-dwarfness, low glucoside or high protein and lysine content which gave significant heterosis in crosses. One possible explanation of the phenomenon of mutant heterosis is related to the frequency of mutations induced by chemical and physical mutagens. The appearance and the level of heterotic effect of mutated genes will depend on their interaction with other mutated genes or with genes from the parental genotype. High specific combining ability of mutants giving heterotic effect makes hybrid seed production, based on crosses with defined sources of cytoplasmic or genetic male sterility, unfeasible or even impossible. Doubled haploids provide a unique system to attempt the ‘fixing’ of hybrid performance in homozygous lines and to avoid the step of hybrid seed production. The assumption on the ‘fixability’ of hybrid yield in homozygous lines based on reports that large additive genetic variance is responsible for yield heterosis in wheat or barley was proven also for mutant crosses. This revised version was published online in August 2006 with corrections to the Cover Date.     

植物离体诱变及在百合育种中的应用

植物离体诱变技术目前已广泛应用于作物优质、高产、抗病、抗逆等新种质的筛选中。综述了体细胞无性系变异简况、离体诱变育种采用的技术、抗性突变体的筛选技术以及检测方法、应用现状及在百合育种中的应用情况。分析了离体诱变育种的优势及在百合抗病育种中的应用前景。     

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.     

Effects of selection and opportunities for recombination in doubled-haploid populations of barley (Hordeum vulgare L.)

Doubled-haploid breeding systems are typically based on sampling gametes from F₁ plants. However, in the case of repulsion linkages, additional recombination could be advantageous. Pre-selection of gamete donors might also shift progeny performance in a desired direction. The objectives of this study were to quantify the effects of an additional round of recombination and assess the effectiveness of pre-anthesis selection in the production of barley doubled haploids. Assessments were conducted on: 1. 100 F₁-derived lines representing a subset of lines used in previous genome-mapping studies; 2. 100 random F₂-derived lines; and 3. 50 F₂-derived lines from gamete donors selected for early heading. An additional round of recombination had only a modest effect on generating more favourable genotypes. Pre-anthesis selection was ineffective in generating an earlier heading population. According to published quantitative-trait locus (QTL) analyses based on the F₁ -derived population, there are few repulsion linkages between QTL determining the traits measured in this experiment. Any advantages to be gained from postponing the generation of derivation of doubled haploids must be weighed against the delay and additional cost.     

Morphology and Cytology of Tissue Culture Derived Octoploid of Elymus canadensis and its Selfed Progeny

Thirty five octoploids (2n = 56). two aneuploids (2n = 54), and one hexaploid (2n = 42) were obtained from the self-fertilized octoploid which was regenerated from the immature inflorescence culture of Elymus canadensis. The octoploid regenerant showed gigas leaves and stems but reduced tillering and fertility. The selfed octoploid progeny varied from genotype to genotype for all of the characters investigated. Hexaploid was morphologically superior to other ploidy levels. Its dry matter yield was 34 % to 40 % higher than the octoploids and the tetraploids but fertility markedly decreased to 7.4 %. Chromosome pairing at metaphase 1 in the octoploid regenerant and its selfed progeny — octoploid, aneupioid. and hexaploid — were 2.57IV +0.83III + 21.23II + 0.971; 3.06IV + 1.06III + 19.66II + 1.251; 2.23IV + 0.85III + 16.54II + 4.69I; and 0.41IV + 8.34III + 8.25II ? 3.141 per microsporocyte, respectively. High frequency of trivalents in hexaploid indicated that it is a doubled triploid. Unequal chromosome disjunction, laggards, and chromatid bridges were commonly observed at the anaphase stage in the first and second meiotic division.     

Agronomic performance of Coffea canephora P. trees derived from large-scale somatic embryo production in liquid medium

In order to validate the propagation technology of Coffea canephoraPierre var. Robusta via somatic embryogenesis in liquid medium, the clonal fidelity of regenerated trees has been assessed for the first time in large-scale field trials. A total of 5067 trees originating from 5- to 7-month-old embryogenic cell suspension cultures were planted in the Philippines and in Thailand for comparing with control trees derived fromin vitro axillary budding (microcuttings). For the observed morphological traits and the yield characteristics, no significant differences were seen between the somatic seedlings (SS) and the microcutting-derived trees (MC).After three harvests in Thailand, the most productive clones had a cumulative yield higher than 3000 kg of green coffee ha^-1. The only detected ‘off-type’ concerns a low vigorous phenotype (2.3% in the Philippines and 3.8% in Thailand), which is probably the consequence of the planting out process as it is also observed in the control trees. The occurrence of some phenotypic variants difficult to visualize or somaclonal variations at the DNA level cannot be excluded. Nevertheless, this study shows that this propagation method can be used for large-scale commercial applications without any negative unforeseen consequences for the grower. This revised version was published online in July 2006 with corrections to the Cover Date.     

Random amplified polymorphic DNA (RAPDs) markers for genetic analysis in micropropagated plants of Robinia pseudoacacia L.

Four years' old micropropagated plants regenerated by enhanced axillary branching from shoot buds of a single genotype of Robinia pseudoacacia were characterized by RAPDs. Random amplified polymorphic DNA analysis was carried outusing 19 random 10-mer DNA primers and 286 RAPD bands were examined which showed 30% polymorphism. Similarity indices ranged from 0.86 to 0.96 among different plants based on RAPD data. The UPGMA dendrogram was constructed based on similarity indices which showed clustering of different plants into subgroups based on similarity values. Our results suggest that somaclonal DNA sequence variations are present even when organized cultures such as shoot buds were used as explant for micro-propagation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Retrospect on in vitro androgenesis of sorghum (Sorghum bicolor)

Development of sorghum hybrids can immensely benefit from an enhanced anther culture protocol since the resultant doubled haploids would accelerate, as in other major cereal crops, the attainment of homozygosity and fixation of alleles. Production of the hybrids that have got high yielding, stress tolerance and other traits of industrial significance in a shorter time as compared to the current trend can hence be realized. This review presents the advances that have been made in the development of such a protocol, with closer attention to androgenic induction components of pre-treatment, culture media composition and conditions as well as ploidy determination and eventual chromosome doubling. From the findings of this review, it is clear that further work is desired to ensure a protocol, that to a large degree, breaks the genotype dependency trend that has been widely cited to impede the usability of the tested protocols.     

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.     

Comparison of doubled haploid lines and F2 bulks for the improvement of barley in the dry areas of North Syria

Drought stress is the main factor limiting barley yields in West Asia and North Africa. This study compares the utility of doubled haploid lines (DHLs) and conventional F₂ plant-derived bulks (F₂Bs) in improving barley in stress environments. Double crosses were made, DHLs were developed by anther culture from double-cross F₁ plants, and F₂Bs were produced by bulking the offspring of F₂ plants. Field tests were conducted in three drought-stressed environments. No major differences were observed in the mean performance of DHLs and F₂Bs. For most traits, both the genotypic and the genotype × location interaction variances were higher in the DHL group, whereas heritabilities were similar. Higher gains from selection were predicted for the DHL group. Regression analysis of yield stability indicated a lower predictability of the DHL performance. The haploid technique can improve breeding populations from which varieties with stable yields can be developed. The costs involved are determined by the DHL production rate, which needs to be improved in many developing countries.     

Relative efficiency of different Gramineae genera for haploid induction in triticale and triticale x wheat hybrids through the chromosome elimination technique

The study was undertaken to evaluate the relative efficiency of different Gramineae genera for haploid induction in triticale (x Triticosecale) and triticale × wheat (Triticum aestivum) hybrids through the chromosome elimination (wheat × maize, Zea mays) system. Eight intergenotypic triticale and 15 triticale x wheat crosses were subjected to hybridization with nine different Gramineae genera viz., Z. mays, Sorghum bicolor, Pennisetum americanum, Setaria italica, Festuca arundinacea, Imperata cylindrica, Cynodon dactylon, Lolium temulentum and Phalaris minor in two separate experiments. This was followed by in vivo auxin treatment of the crossed spikes and subsequent rescue of the haploid embryos to regenerate green haploid plantlets. All the triticale and triticale x wheat crosses resulted in seed set in variable frequencies when hybridized with maize, I. cylindrica, pearl millet and sorghum. Seed set was also obtained with S. italica, F. arundinacea and P. minor in a few crosses in both groups. In general, all the triticale x wheat crosses, except for one in each case, resulted in embryo formation and green haploid plantlet regeneration when hybridizations were carried out with maize and I. cylindrica. However, the latter outperformed the former in embryo formation (25.48% vs. 20.0%) and regeneration (34.17% vs. 15.10%) frequencies, the differences being significant for regeneration frequencies. In the case of triticale hybrids, no significant differences between maize and I. cylindrica were observed for the three parameters of haploid induction. Embryo formation and regeneration were also observed in some of the triticale as well as triticale × wheat F₁ hybrids when hybridized with sorghum and pearl millet.     

QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of rice

Anther culturability of rice is a quantitative trait controlled by nuclear‐encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ‘Milyang 23’ and ‘Gihobyeo’. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F₂ populations, ‘MG RI036’/‘Milyang 23’, and ‘MG RI036’;/‘IR 36’ were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F₂ populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes.     

Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM

Isolated microspore cultures from two doubled haploid (DH) lines of wheat, Triticum aestivum L., were used to develop an in vitro chromosome-doubling protocol. During the initial 24 h or 48 h of culture the microspores were treated with either of the two antimicrotubule herbicides trifluralin or amiprophos-methyl (APM) in concentrations ranging from 0.1 μM to 10μM. Untreated control cultures yielded 209 embryos per 100000 microspores, which is the equivalent of one spike. Among the regenerated plantlets 67% were green, and 15% of the flowering plants were spontaneously chromosome doubled. Treatments with both the herbicides had a significant effect on chromosome doubling, measured as the percentage of fertile regenerants. With the best combination of treatment duration (48 h) and herbicide concentration (10/μM) the percentage of fertile plants among regenerants could be increased up to 74% with APM and up to 65% with trifluralin. The largest numbers of DH plants per spike could be obtained with herbicide concentrations at 1–3 μM. Treatments with either herbicide at these concentrations resulted in an estimated average between the two genotypes of 27 DH plants per 100 000 microspores. These results demonstrate the high potential of APM and trifluralin as chromosome-doubling agents in isolated microspore cultures. The in vitro treatment integrated into tissue culture procedures will constitute an efficient method for chromosome doubling in future wheat breeding     

Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry

A rapid and efficient microspore culture protocol was applied to produce homozygous progeny of crosses between low erucic canola and high erucic resynthesized rapeseed (Brassica napus L.). Microspores of Canadian cultivars ‘Excel’ and ‘Profit’ as well as three F₁ hybrids with the resynthetic line ‘RS239’ were treated with colchicine immediately after isolation. Flow cytometry was applied for early identification of doubled haploid (DH) regenerants. The diploidization rate was subsequently verified by scoring flower morphology. In vitro colchicine treatment had a positive effect on induced diploidization, and was associated with the frequency of preliminary spontaneous diploidization which was, however, determined by the genotype. In addition, the effects of colchicine treatment on embryoid formation and regeneration have been evaluated. The method presented is feasible for commercial large‐scale production of DHs in rapeseed as the genotype‐specific diploidization can be efficiently balanced by in vitro colchicine treatment. In addition, the use of flow cytometry immediately after in vitro culture allows efficient selection for DHs, thus saving labour and cost and in the laboratory and subsequent greenhouse phase.     

Crosses between cultivars and tissue culture-selected plants for salt resistance improvement in rice, Oryza sativa

Several R₂ somaclonal families were derived from plants regenerated from a salt‐resistant callus of the salt‐sensitive rice cultivar ‘I Kong Pao’ (IKP). The family R₂‐1‐23, in the presence of NaCl exhibited higher yield performances than the initial cultivar. This improvement in salinity resistance, however, was not transmitted to following generations; despite a higher number of spikelets per plant, family R₃‐1‐23 did not perform better than the initial cultivar because of a very low seed set. This somaclonal family, its initial being the cultivar IKP, the breeding line IR₃1785 (extremely salt‐sensitive) and the cultivar ‘Aiwu’ (moderately salt‐resistant), were used as parents for production ofhybrids. Four crosses, IKP×R3‐1‐23, IR31785 ×R3‐1‐23, IR31785× IKP and IKP בAiwu’, were performed. Most of the F₁ hybrids cultivated in the absence of salt exhibited increased performances compared with the mid‐parent, suggesting an heterosis effect for yield‐related parameters. F₂ populations were screened for salinity resistance and a clear improvement for yield in stress conditions was recorded for populations derived from IK×R3‐1‐23, IR31785×R3‐1‐23 and IR31785×IKP, although the mean level of increase over the mid‐parent (RIMP) varied depending on the population, the presence or absence of stress, and the quantified parameters. The results are discussed in relation to the usefulness of in vitro selection for obtaining interesting somaclonal variants useful to be integrated in classical breeding programmes for salinity resistance in rice.