Assessment Of Somaclonal Variation Research Articles

Micropropagation and assessment of somaclonal variation in Galanthus transcaucasicus in vitro plantlets

Abstract In vitro culture of twin-scaling explants of Galanthus transcaucasicus with different concentrations of plant growth regulators (PGRs) including 0.5, 1, 2, 3, 4, 6, 8, and 10 mg L-1 naphthaleneacetic acid (NAA) and 0.5, 1, 2, 3, and 4 mg L-1 benzyladenine (BA) was studied. After 18 weeks, the number of regenerated bulblets and intensity of callus was measured. Subsequently, bulblets were transferred to a medium with 0.5, 1, 2, 3, and 4 mg L-1 NAA and 0.5, 1, 2, 3, and 4 mg L-1 BA and, after 15 weeks, the bulblets length and diameter were measured. The highest intensity of callus was obtained on 4 mg L-1 NAA or 8 mg L-1 NAA with 1 mg L-1 BA. The highest number of regenerated bulblets was detected with 6 mg L-1 NAA and 2 mg L-1 BA. The highest diameter of bulblets occurred on four mgL-1 NAA (9.4 mm), while the lowest was observed on 0.5 mg L-1 BA (1.83 mm). The analysis of genetic variation using ISSR revealed that there was no somaclonal variation among the regenerated plants from BA and low level of NAA, but there was a significant somaclonal variation at high concentrations of NAA.

Assessment of somaclonal variation in indirect morphogenesis-derived plants of Arracacia xanthorrhiza

Abstract: The objective of this work was to induce and detect somaclonal variation in arracacha (Arracacia xanthorrhiza) plants regenerated via indirect morphogenesis, in order to evaluate the potential of this technique to produce new genotypes for breeding purposes of this crop. Calli were induced from petiole segments on Murashige & Skoog (MS) medium supplied with 0.1 mg L-1 2,4-dichlorophenoxyacetic acid. The regeneration of plants via indirect morphogenesis was carried out on half-strength MS medium without plant growth regulators. Fifteen randomly chosen plants were subjected to flow cytometry and “inter-simple sequence repeat” (ISSR) analysis. Ploidy level remained stable in all tested regenerants (2n=4x=44), with no changes in the genome. Eighteen ISSR primers produced a total of 1,584 fragments in all samples. Two ISSR primers produced four polymorphic fragments in 26.7% of the tested samples. Somaclonal variation in arracacha is a result of plant regeneration via indirect morphogenesis and can be detected by ISSR markers.

ERRATA: Assessment of somaclonal variation in indirect morphogenesis-derived plants of Arracacia xanthorrhiza

ERRATA: Assessment of somaclonal variation in indirect morphogenesis-derived plants of Arracacia xanthorrhiza

Assessment of somaclonal variation during sugarcane micropropagation in temporary immersion bioreactors by intersimple sequence repeat (ISSR) markers

Somaclonal variation refers to the genetic and epigenetic changes in plants regenerated from plant tissue culture. In this study, using intersimple sequence repeat (ISSR) molecular markers, the somaclonal variation during micropropagation of sugarcane using temporary immersion bioreactors (TIBs) was evaluated. Apices of the cultivar Mex 69-290 were established and multiplied by ten subcultures in TIBs. After 30 d in each subculture, the number and length of shoots per explant were recorded. For the molecular analysis, ten plants were taken per subculture, and a total of 109 bands from ten ISSR primers were obtained. For each subculture, the polymorphism (%) was calculated. A dendrogram of genetic distances between subcultures and the donor plant was obtained using a matrix of Nei’s genetic distances and the unweighted pair group method with arithmetic mean (UPGMA). The results showed that the production of sugarcane shoots tends to increase until subculture 8, while shoot length decreases. ISSR markers showed the existence of somaclonal variation during micropropagation of sugarcane. The subcultures with the highest percentage of polymorphism (%) and genetic distances (GD) were the 1°, 9°, and 10° (with 10.1, 15.6, and 10.1% and 0.0222, 0.0181, and 0.0181 GD, respectively). The molecular and statistical analysis showed that in vitro establishment and the number of subcultures are both factors that affected the frequency of somaclonal variation during the micropropagation of sugarcane using TIBs. Thus, it is important to determine the optimal number of subcultures that can be made from an explant for each species to be micropropagated.

Somaclonal Variation During Picea abies and P. omorika Somatic Embryogenesis and Cryopreservation

AbstractEmbryogenic cultures of plants are exposed to various stress factors bothin vitroand during cryostorage. In order to safely include the plant material obtained by somatic embryogenesis in combination with cryopreservation for breeding programs, it is necessary to monitor its genetic stability. The aim of the present study was the assessment of somaclonal variation in plant material obtained from embryogenic cultures ofPicea abies(L.) Karst. andP. omorika(Pančić) Purk. maintainedin vitroor stored in liquid nitrogen by the pregrowth-dehydration method. The analysis of genetic conformity with using microsatellite markers was performed on cotyledonary somatic embryos (CSE), germinating somatic embryos (GSE) and somatic seedlings (SS), obtained from tissues maintainedin vitroor from recovered embryogenic tissues (ETc) and CSE obtained after cryopreservation. The analysis revealed changes in the DNA of somatic embryogenesis-derived plant material of bothPiceaspp. They were found in plant material from 8 out of 10 tested embryogenic lines ofP. abiesand in 10 out of 19 embryogenic lines ofP. omorikaafterin vitroculture. Changes were also detected in plant material obtained after cryopreservation. Somaclonal variation was observed in ETc and CSE ofP. omorikaand at ETv stage ofP. abies. However, most of the changes were induced at the stage of somatic embryogenesis initiation. These results confirm the need for monitoring the genetic stability of plants obtained by somatic embryogenesis and after cryopreservation for both spruce species.

Indirect organogenesis and assessment of somaclonal variation in plantlets of Vanilla planifolia Jacks

The low genetic variability of vanilla (Vanilla planifolia Jacks.) makes it susceptible to pests and diseases, which leads to a decrease in production. The genetic variability can be broadened by using in vitro plant tissue culture techniques. The use of indirect morphogenic routes allows increasing the percentage of somaclonal variation in regenerated plantlets. The objective of this research was to establish a protocol for indirect organogenesis in V. planifolia aimed at broadening the genetic base of the crop. A friable callus was induced from immature seeds grown in the dark, using MS medium supplemented with 2.27 μM thidiazuron (TDZ). Subsequently, 6.8 shoots per callus were regenerated in MS medium supplemented with 8.88 μM benzyladenine (BA). One hundred per cent rooting of regenerated shoots was achieved when MS medium was used with no plant growth regulators. Last, rooted plantlets were acclimatized in a greenhouse. A 91 % survival rate was observed. Molecular analyses on regenerated plantlets revealed the existence of a 71.66 % genetic polymorphism. Furthermore, morphological variation was confirmed by the presence of variegated individuals in regenerated plantlets. The proposed protocol can be useful in subsequent vanilla genetic improvement works.

Assessment of somaclonal variation in somatic embryo-derived plants of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson] using inter simple sequence repeat analysis and flow cytometry

article i nfo Background: Yacon (Smallanthus sonchifolius) is a root crop native to the Andean region. Low sexual reproductive capacity is a major constraint facing the genetic breeding of this crop. Biotechnological techniques offer alternative ways to widen genetic variability. We investigated somaclonal variation in regenerants of yacon derived from in vitro somatic embryogenesis using simple sequence repeat (ISSR) analysis and flow cytometry. Results: Twenty tested ISSR primers provided a total of 7848 bands in 60 in vitro regenerants and control plant. The number of bands for each primer varied from 3 to 10, and an average of 6.95 bands was obtained per ISSR primer. Eight primers were polymorphic and generated 10 polymorphic bands with 7.19% mean polymorphism. ISSR analysis revealed genetic variability in 6 plants under study. These regenerants had Jaccard's distances 0.104, 0.020, 0.040, 0.106, 0.163 and 0.040. Flow cytometric analysis did not reveal changes of relative nuclear DNA content in regenerants suggesting that the plants obtained via somatic embryogenesis had maintained stable octoploid levels. Conclusions: Our findings show that indirect somatic embryogenesis could be used in yacon improvement to widen genetic variability, especially when low sexual reproductive capacity hinders classical ways of breeding.

Assessment of somaclonal variation in sugarcane

A study was conducted from 2006 to 2008 to assess the variability arising from callus regeneration and its vegetative transmission in a subtropical variety of sugarcane, CoJ 64. Qualitative and quantitative characters were evaluated in the field at maturity in each of the 3 years at the same location. The frequencies of variants were compared to those found in the original variety. The tissue culture process resulted in increased morphological variability among the produced somaclones. Their variability was manifested according to the data for stalk diameter, length, internodes, millable canes, leaf length, width and sucrose characters. The variation among the somaclones was confirmed in the three consecutive years. Key words: Callus regeneration, vegetative transmission, sugarcane.

Assessment of somaclonal variation in Ducrosia anethifolia plants regenerated from long-term callus cultures using AFLP

Assessment of somaclonal variation in Ducrosia anethifolia plants regenerated from long-term callus cultures using AFLP

Assessment of somaclonal variation in purple coneflower (Echinacea purpurea (L.) Moench) by RAPD (Random Amplification of Polymorphic DNA) fingerprinting analyses

In order to induce somaclonal variation in purple coneflower (Echinacea purpurea (L.) Moench), the selected material from five elite plants was cultured in vitro. Optimum callus formation was observed on Murashige and Skoogs' (MS) medium supplemented with 10mg/l of 2,4-D. The best shoot regeneration was achieved upon transferring the callus to MS medium containing 2.5mg/l BAP and 0.5mg/l IAA. Complete plantlets were obtained upon transfer of the regenerated shoot to MS medium containing 1mg/l IBA. A number of 13 plants regenerated from callus were successfully transferred to the greenhouse, following previously standardized hardening procedures. DNA was extracted from the parental plants and from the callus regenerated plants. RAPD (Random Amplification of Polymorphic DNA) analyses were carried out to detect somaclonal variation. Two, out of 13 regenerated plants exhibited somaclonal variation. These four somaclones were different from the parental plants by at least one polymorphic amplification product. The two somaclones had common origin (the same elite plant) but they displayed non-maternal bands for two different primers, OPB 09 and OPX 03. The conclusion that can be drawn from here is that the somaclones are genotypically, and maybe even phenotypically different. The remaining regenerants were genetically stable as compared to the elite donor plants. RAPD markers were an efficient tool for the early detection of somaclonal variants in purple coneflower tissue culture.

An assessment of somaclonal variation in micropropagated plants of sugarcane by RAPD markers

Sugarcane variety Co 94012 is a new variety released in India through the use of somaclonal variation. Significant differences were found in quantitative and qualitative agronomic characters. Another somaclone VSI 434 is an extra early maturing variety with significant difference in morphological and qualitative characters over the parent CoC 671. The genetic variation of micropropagated plantlets was tested by RAPD analysis. The banding pattern of PCR amplified products from micropropagated plantlets showed that most of them were monomorphic in both the varieties. The amplicon pattern of VSI 434 and Co 94012 with primers OPA 17 and OPA 19 differed from parent CoC 671, respectively. But with other primers they were more or less monomorphic indicating that the amplicon from these two primers can be used as potential fingerprint for Co VSI 434 and Co 94012.

Assessment of somaclonal variation in Dieffenbachia plants regenerated through indirect shoot organogenesis

The occurrence of somaclonal variation among regenerants derived through indirect shoot organogenesis from leaf explants of three Dieffenbachia cultivars Camouflage, Camille and Star Bright was evaluated. Three types of somaclonal variants (SV1, SV2, and SV3) were identified from regenerated plants of cv. Camouflage, one type from cv. Camille, but none from cv. Star Bright. The three variants had novel and distinct foliar variegation patterns compared to cv. Camouflage parental plants. Additionally, SV1 was taller with a larger canopy and longer leaves than parental plants and SV2. SV2 and SV3 did not produce basal shoots (single stem) but basal shoot numbers between SV1 and parental plants were similar ranging from three to four. The variant type identified from regenerated cv. Camille had lanceolate leaves compared to the oblong leaves of the parent. This variant type also grew taller and had a larger canopy than parental plants. The rates of somaclonal variation were up to 40.4% among regenerated cv. Camouflage plants and 2.6% for regenerated cv. Camille. The duration of callus culture had no effect on somaclonal variation rates of cv. Camouflage as the rates between plants regenerated from 8 months to 16 months of callus culture were similar. The phenotypes of the identified variants were stable as verified by their progenies after cutting propagation. This study demonstrated the potential for new cultivar development by selecting callus-derived somaclonal variants of Dieffenbachia.

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.

Varietal Response of Wheat, Triticum aestivum L. To Tissue Culture and Assessment of Somaclonal Variation

Varietal Response of Wheat, Triticum aestivum L. To Tissue Culture and Assessment of Somaclonal Variation

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.

Assessment of somaclonal variation in apple. I. Resistance to the fire blight pathogen,Erwinia amylovora

SummaryAfter inoculation of glasshouse-grown somaclones regenerated from apple leaf discs (cv. Greensleeves) 33% of 270 showed an increase in resistance to the fire blight pathogen Erwinia amylovora. In contrast only 21% produced less severe symptoms than parental material after inoculation of plants held in culture in vitro. Sixteen somaclones which showed the highest levels of fire blight resistance were tested intensively as both glasshouse-grown plants and as micropropagated plants in culture. In tests conducted with glasshouse-grown plants up to 60% of plants of the most promising somaclones exhibited minimum symptoms after inoculation compared with 4% of ‘Greensleeves’ parental plants. The comparable figures for inoculations of in vitro cultured plants were 70% and 8%. The growth of E. amylovora was reduced in resistant somaclones compared with parental plants. Pretreating leaf discs with virulent E. amylovora cells prior to somaclone regeneration had no effect on the frequency of regenerated somaclones exhibiting increased resistance to the pathogen.

Assessment of somaclonal variation in apple. II. Rooting ability and shoot proliferationin vitro

Summary270 somaclones, regenerated from leaf discs of the apple cv. Greensleeves, varied in rooting ability, root number and shoot proliferation. The extent of variation in rooting ability was affected by treatments applied to the leaves before somaclone regneration, particularly infiltration with the antibiotic cefotaxime. Root number per rooted shoot increased with subsequent subculturing.

An assessment of somaclonal variation in linseed (Linum usitatissimum)

SummarySomaclonal lines of linseed from the parent cultivar Norlin were produced from a callus‐based in oitro regeneration system (the R0 generation). In field trials conducted over two seasons, 47 R1 (plants produced from the R0 generation) and 20 R2 somaclonal lines (plants produced from the R1 generation) were compared to the parent cultivar Norlin for quantitative characters. Irrespective of the genotype, traits in R1's and R2's were assessed on the basis of regression analysis as showing heritabilities of between 28% and 64%. Generally, the somaclonal variation assessed during these early generations revealed some detrimental traits, e.g. lower seed yield than the parent (control) cultivar and reduced 1000 seed weights, but a few lines were identified which had early or late flowering dates, improved seed yield and increased 1000 seed weights. It is concluded that somaclonal variation could be of value as an adjunct to classical breeding.

Somaclonal variation in high tannin sorghums

Genetic variants were found among over 6,000 primary plants (R1) regenerated from embryogenic tissue cultures of eight high tannin sorghums [Sorghum bicolor (L.) Moench]. Field assessment of somaclonal variation has progressed to the R2 population, with over 48,000 R2 seedlings (27,000 plants) in 1,126 rows from 1,055 R1 plants. A total of 43 variant phenotypes was recovered, including several types of chlorophyll deficiencies, dwarfism, short culm, sterility, narrow leaf, and several previously unreported variants, such as ragged leaf, multibranched heads, and Hydra, a developmental variant which produces large numbers of panicles. Variation production greatly depends on parent genotype and appears to increase with increasing time in cultures. The toal average somaclonal variation rate (based per 100 R1 plants) and somaclonal variant frequency (based per 100 R2 plants) estimated in the tested population were 11.3 and 1.6, respectively. Chimerism was found in regenerants. The estimated size of the mutated sector carried by mutant regenerants ranged from the whole plant to less than 3% of a single head. The average proportion of mutated R1 heads carrying large (80%-100%), medium (40%-80%), and small (<40%) mutated sectors was 38.7%, 26.0% and 35.3%, respectively. Some sector mutations do not appear until the R3 generation. In order to avoid losing variants, the population for selecting somaclonal variation should be as large as possible. Some of these variants found may be useful for further study or for use in breeding programs.

An assessment of somaclonal variation in Chrysanthemum morifolium: the generation of plants of potential commercial value

Chrysanthemum plants of the cultivar ‘Early Charm’ were regenerated from leaves and petals by direct explant regeneration or by an intermediate callus stage to obtain somaclonal variants. Explants provided quick and efficient systems for regeneration, with petals being a superior source of variation compared with leaves. Variants from a yellow genotype retained their normal colour, whereas in a white genotype plants were obtained with lilac-coloured ray florets. Two out of 6 variants from the white genotype were considered to be of commercial value. Chimerism, gene mutation and chromosome change are considered as possible causes for the somaclonal variations.