Somaclonal variation in Lotus corniculatus L. in relation to plant breeding purposes

Summary Seventy-two plants regenerated from leaf-derived calli of a single plant of Lotus corniculatus have been evaluated for several morphological and agronomical traits. The analysis of selfed and polycross progenies of the regenerants indicates that the variation among regenerants was, at least in part, of genetic origin. Most of the mutations induced by tissue culture were recessive and were detected only after sexual propagation. Although in vitro culture had a depressive effect for most of the traits, the selfed progenies of 2 regenerants displayed higher values for leaflet width and seed yield than the selfed progeny of the initial plant. However the somaclonal variation did not increase the variation for any trait with respect to the variation of the donor cultivar of the initial plant.     

Genetic stability at nuclear and plastid DNA level in regenerated plants of <Emphasis Type="Italic">Solanum</Emphasis> species and hybrids

In this work we detected the extent of variability at nuclear and cytoplasmic DNA level of regenerated plants belonging to Solanum genotypes with a different genetic background and somatic chromosome number. As for the nuclear characterization, a total of 66 (18.5%) polymorphic bands were scored using 13 ISSR primers on 45 randomly selected regenerants. Our results show that the regenerants obtained from clone cmm 1T and, at lower level, those from cph 1C are unstable under in vitro conditions or rather more prone to in vitro-induced stress leading to somaclonal variation than the other genotypes used. Two types of changes were observed: disappearance of parental ISSR fragments, termed “loss”; appearance of novel ISSR fragments, termed “gain”. The most frequent event occurring in the regenerants was the loss of fragments (41 bands). Regenerated plants were analyzed with seven plastid universal primers to determine the cytoplasmic composition at chloroplast level. All cpDNA primer pairs tested produced amplicons of the same size in all genotypes analyzed and no polymorphic fragments were observed with any universal primers used. Our results show that under in vitro culture conditions genotype affects the integrity of the genome. In addition, the absence of polymorphism at plastid level confirms the greater genetic stability of cytoplasmic DNA.     

Suitability of RAPD analysis for the detection of somaclonal variants in oil palm (Elaeis guineensis Jacq)

Random amplified polymorphic DNA (RAPD) analysis using arbitrary 10-mer oligonucleotide primers was employed in order to investigate the genetic fidelity of somatic embryogenesis-derived regenerants of oil palm (Elaeis guineensis Jacq). Clonal palms bearing the ‘mantled’ phenotype were identified in the field and the ability of RAPD markers to distinguish these variants from palms of the normal type was assessed. Of the 387 arbitrary primers used, 259 (67%) were successfully used to amplify oil palm DNA genomic fragments with consistently reproducible banding. Of these 387 primers, 73 (19%) primers enabled the identification of polymorphism between clones. No intraclonal variability and no differences between mother palms and regenerants could be identified using the total number of markers scored (8900). Twenty-four of these 73 primers were chosen for use in a larger experiment aimed at comparing, first, the mother palm genome with that of its clonal offspring and, second, true-to-type and variant regenerants. Thus, the regeneration protocol based on somatic embryogenesis set up for oil palm clonal propagation does not induce any gross genetic changes. The results obtained revealed however, that the RAPD approach is not suitable for the detection of the ‘mantled’ variant phenotype. The use of RAPD markers for the detection of somaclonal variation in oil palm is discussed.     

RAPD-based detection of genomic instability in soybean plants derived from somatic embryogenesis

Random amplified polymorphic DNA (RAPD) markers were used to evaluate genetic stability of regenerants of soybean plants obtained through somatic embryogenesis using 180 μM 2,4‐dichloro‐phenoxy acetic acid. Twenty primers were used to screen 44 regenerants from the cultivar ‘Spring’ and 28 from the cultivar ‘CAC‐1’. Three primers were polymorphic for two of the ‘Spring’‐derived regenerants, with a somaclonal frequency of 4.5%. Four primers were polymorphic for the ‘CAC’‐l‐derived regenerant, with a somaclonal frequency of 3.57%. The results indicate the usefulness of RAPD markers to detect genetic instability in soybean primary regenerant plants derived from somatic embryogenesis, and as a certification tool for monitoring genetic stability during the regeneration process.     

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.     

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.     

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     

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     

Inter and intra-species Inter Simple Sequence Repeat (ISSR) variations in the genus Cicer

Intra and inter-species ISSR variation and use of ISSR markers in determination of genetic relationship were investigated in an accession collection representing twoperennial and six annual Cicerspecies. Screening of Ciceraccessions with SSR primers revealed highly reproducible amplicon profiles with relatively high multiplex ratios. Many of the primers generated amplicon profiles with which not only the differences among species can readily be identified, but also polymorphisms within species could be detected more efficiently. PCR products at 150 gel positions detected using six SSR primers in Cicer accessions were treated as dominant DNA markers and utilized to compute the distances among accessions and species. Cluster analysis of accessions and species revealed groupings that corroborate our previous studies of relationships based on allozyme and AFLP analysis. Consistent with the AFLP analysis carried out in the same accession collection, ISSR-based groupings indicated that perennial C. incisumis genetically close to the annuals of the second crossability group (C. pinnatifidum,C. bijugum, C. judaicum) while C. reticulatum is the closest wild species to the cultivated chickpea. ISSR-based variation estimates were relatively higher when compared to previous estimates computed from RAPD and AFLP data. Technically, ISSR analysis combines the PCR-based targeting of microsatellite-associated polymorphisms with no prior sequence requirement and stringent PCR conditions. Similarly, when compared to AFLP analysis, it is less technically demanding allowing to survey polymorphic loci in the genome. Thus, ISSR-PCR technology is a reliable, fast, and cost-effective marker system that can be used to study genetic variation and genetic relationships in the genusCicer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somaclonal Variation in Tissue Culture of Triticum aestivum×Agropyron desertorum F1 Hybrid

Callus induced from immature inflorescences of the partially self-fertile hybrids (2 n = 35; ABDPP) between Triticum aestivum (2n = 42; AABBDD) and Agropyron desertorum (2n = 28; PPPP) led to the regeneration of 88 plants on MS medium supplemented with 2 mg/l of 2,4-D. These regenerants were used to investigate somaclonal variation and to obtain more selfed derivatives. Immature inflorescences at the stage of developing floral primordia gave the best response in terms of callus induction and plant regeneration. The regenerants exhibited great variability for most morphological traits. Although the regenerants did not exhibit variation in chromosome number, they did show a higher degree of meiotic instability than the initial hybrid. In particular, the regenerants gave much higher selfed seed-set (5.49 %) than the donor hybrid (0.46 %), so that a total of 484 selfed seeds were obtained.     

Development of a new initial breeding material in sunflower (Helianthus annuus L.) using direct organogenesis and somatic embryogenesis

Summary Immature zygotic embryos from three inbred lines of sunflower (Helianthus annuus L.) were used as donor material for the induction of direct organogenesis and somatic embryogenesis. The scope of the spontaneously appearing somaclonal variation has been studied among the regenerants produced according to the method of Freyssinet & Freyssinet (1988), and was compared to the results obtained after gamma ray mutagenesis (7 and 10 Gy). Freshly excised immature zygotic embryos were used for irradiation treatment. Genetic changes occurring spontaneously during the regeneration procedure were observed for date of flowering, vegetation period, plant height, head diameter, 1000 seed weight, kernel oil content, and fatty acid composition. The mutagenic treatment had a marked stimulatory effect on the frequency of the regenerants produced and the degree of the observed changes. The 7 Gy treatment was most efficient for the majority of the characters studied. The type of changes was correlated with the genotype. The degree and type of somaclonal variation, with or without mutagenic treatment, should be sufficient for an application in the production of new breeding material.     

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.     

Assessment of natural and induced genetic variation in Alstroemeria using random amplified polymorphic DNA (RAPD) markers

Summary We have used random amplified polymorphic DNA (RAPD) markers to study genetic variation in Alstroemeria. The first objective was to examine the discriminatory power of RAPD markers in different genotypes of Alstroemeria obtained by traditional breeding. All genotypes examined, including commercial Alstroemeria varieties, could be distinguished on the basis of their RAPD profiles. Progeny plants could be distinguished from their parents. A second objective of this study was to investigate whether RAPD markers can be used as a routine tool to detect mutant plants, as an alternative to glasshouse testing. To address this objective, we analysed Alstroemeria plants that carried phenotypically visible mutations that either were induced by irradiation using X-rays or were the result of somaclonal variation. In eight out of a total of 13 mutant Alstroemeria plants obtained after irradiation or tissue culture we detected no polymorphisms when compared to control plants that were considered to be non-mutated. Only in five of the mutant plants analysed we detected one to two polymorphisms. These results suggest that frequent genome rearrangements had not occurred in the mutant plants analysed. These results also demonstrate that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It that the RAPD technique is an inappropriate tool for the rapid screening of Alstroemeria for induced variation. It seems probable that this conclusion would be equally applicable in other plant genera in which induced variation has occurred. However, the RAPD technique is a simple and effective tool for genetic fingerprinting of Alstroemeria varieties, provided their differences are due to sexual propagation.     

Variation in Phenotype and Chromosome Number of Plants Regenerated from Protoplasts of Dihaploid and Tetraploid Potato

An investigation was made on the origin and basis of somaclonal variation in potato. Plants were regenerated from protoplasts of dihaploid and tetraploid potato, and the degree of variation in phenotype and chromosome number was analysed. All the regenerants from dihaploid potato showed variation in chromosome number, while only part of these varied in plant morphology. In tetraploid potato, 60 % of the regenerants showed variation in chromosome number, all of which exhibited phenotypic changes. The data suggest that the explant source and the initial ploidy level of the genotype play an important role in influencing the degree of somaclonal variation.     

Evaluation of Somaclonal and Mutagen Induced Variation in Fingermillet

The effects of gamma rays and EMS on plant regeneration and somaclonal variation in fingermillet were studied. While EMS had an inhibitory effect on plant regeneration, gamma irradiation in low doses (5 Gy) was stimulatory. A wide range of variation was observed for almost all the traits and it was similar in both mutagen treated and untreated regenerants and seeds. This indicates that tissue culture itself induces variation and it can be exploited to advantage for crop improvement.     

刺槐体细胞无性系变异的RAPD检测

植物离体培养过程中都有可能产生体细胞无性系变异,为了从质量上保证二乔刺槐与四倍体刺槐组培苗,检测其是否变异是很有必要的。本试验中,以扦插苗为对照,用10个随机引物进行RAPD扩增。共扩增出33条带,多态性谱带比例为72.7%。聚类分析结果表明,二乔刺槐扦插苗与组培苗、四倍体刺槐扦插苗与组培苗的遗传距离分别为0.18、0.17。二乔刺槐与四倍体刺槐两者的遗传距离为0.25。可见,尽管组培苗与扦插苗有一定程度的变异,但并未影响到其基因型。     

In vitro screening and field evaluation of tissue-culture-regenerated sorghum (Sorghum bicolor (L.) Moench) for soil stress tolerance

Summary Sorghum bicolor (L.) Moench is generally quite sensitive to salt and acid (high aluminium) soil stresses, but quite tolerant of drought stress. As with any stress phenomenon, intra-specific variability exists within the genus. In vitro cell selection and somaclonal variation offer an alternative to traditional breeding methodology for generating improved breeding lines for hybrid development. A field selection protocol was developed for the three soil stresses and inter-stress evaluations were conducted in an effort to find multiple, stress-tolerant genotypes. The acid soil-drought stress, super-tolerant selections were located by the R₇ generation when exposed to a combined aluminium-drought stress field environment and when the regeneration population (number of regenerated lines from one callus source) was maintained at 15,000 plants or higher. A variant frequency of 0.1 to 0.2% for stress tolerance and acceptable agronomic traits among the surviving somaclones, provided an adequate number of phenotypes with desirable agronomic characteristics and a high level of soil stress tolerance. Subsequent research verified that the stress-tolerant regenerants had superior acid soil and drought stress tolerance to that of the donor parents, that their yield capabilities under stress were superior to their parents, and that their stress tolerance attributes were transferred in hybrid combinations. In vitro selection was not effective in increasing the number of field stress survivors. In fact, superior germplasms were developed from non-stressed callus or salt-stressed callus. In vitro selection reduced regeneration frequency and subsequent survival of plants under field stress. In vitro-stressed regenerants should be subjected only to non-stressed environments to maintain population numbers for field selection and thereafter should be subjected to stress environments during later (R₅₊) generations. The optimal strategy for the exploitation of somaclonal variation may be through short-term cell culture (< 12 months) with no attempt at in vitro selection.     

RAPD variability in rice (Oryza sativa L.) plants derived from desiccation-tolerant calli

Genetic modification from selfed progenies of 18 rice (Oryza sativa L.) plants regenerated from callus tissues which survived desiccation, were investigated at the DNA level using the random amplified polymorphic DNA (RAPD) method. Twelve 10-mer random primers were used to amplify DNA of progenies from the regenerated plants, and a total of 228 PCR products and 1780 DNA fragments were obtained by primers, generating between four to thirteen major bands. The size of the amplified fragments ranged from 0.2 to 2.55 kb. The results showed that 10 out of 12 primers produced polymorphic bands, two primers (RA31 and RA185) showed no polymorphism among plants tested. A dendrogram of the genetic distance was constructed based on their polymorphism, demonstrating that somaclonal variation exists in rice plants regenerated from callus which survived the desiccation treatment. Part of this variation can be useful in rice breeding. This revised version was published online in August 2006 with corrections to the Cover Date.     

Application of cell and tissue culture and in vitro selection for disease resistance breeding — a review

Summary Somaclonal variation, i.e. the variation induced by cell and tissue culture, offers an opportunity to broaden the genetic variation of crops. As a result of somaclonal variation a wide range of plant characteristics can be altered. However, the selection of agronomically important traits, e.g. disease resistance, has many limitations. The efficiency of selection can be increased by the application of in vitro selection procedures. Selection strategies that may be applied to obtain disease resistant somaclonal variants are described. Their merits and limitations, in relation to the efficiency of the procedures, the frequency of disease resistant variants and the genetics of the resistance obtained, are discussed.     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.