Somaclonal Variation in a Food Legume—Lathyrus sativus

Somaclones of Lathyrus sativus cv P-24 were obtained from leaf, internode and root derived callus cultures. These showed significantly decreased neurotoxin (ODAP) content up to 0.03% compared to that of parent cultivar P-24 (0.3%). The somaclones also showed higher seed yield than parent P24. In addition. somaclone Bio 1–22 showed significantly decreased time for flowering. Other heritable morphological variant features were leaf length, leaf breadth, internode length, flower colour, seed colour, 100 seed weight, neurotoxin content of leaves and red markings on the pods.     

RAPD Marker Linked to a Gene Conferring Resistance to Race IB-9 of Pyricularia grisea in a Somaclone of the Rice Cultivar Araguaia

The gene Pi-ar confers resistance to Pyricularia grisea in a somaclone of the upland rice cultivar Araguaia developed from callus culture of immature panicles. The somaclone SC09 exhibited resistant reaction to all of the 182 P. grisea test isolates belonging to 15 different races. The study on inheritance showed that the resistance to pathotype IB-9 of P. grisea is monogenic and dominant. In order to identify marker linked to this gene, the F₂ population from a cross between the highly susceptible cultivar Lijiangxintuanheigu (LTH) and the somaclone SC09 of rice cultivar Araguaia was screened using RAPD primers. Initially, the polymorphism between parents, the cultivar LTH and somaclone SC09 was analyzed using 577 random 10-bp primers. The susceptible and resistant bulks of the F₂ population, along with DNA of the two parents were tested with 176 primers that differentiated susceptible and resistant parents. Thirty-six primers differentiated the susceptible and resistant bulks, as well as the cultivar LTH of the somaclone SC09. However, one primer OPK17 was found to be closely linked (5.3 cM) to the resistance gene of somaclone and this can be used in the marker assisted selection.     

Variability for resistance to Fusarium solani culture filtrate and fusaric acid among somaclones in pea

Pea (Pisum sativum L.) somaclones of cultivars Adept, Komet and Bohatýr were obtained after selection in vitro with Fusarium solani filtrate and fusaric acid (FA). R2 regenerants were analysed by random amplification of polymorphic DNA (RAPD; OPAB4, P-14, UBC-556) and inter-retrotransposon amplification polymorphism (IRAP; Ogre) markers. Marker UBC-556 showed different banding patterns for each cultivar, but without specific bands for selected and control plants. Markers OPAB4, P14 and Ogre were useful for clear discrimination between selected and non-selected variants of all three cultivars. Flow cytometry analysis proved the same genome size of selected and non-selected pea lines. Therefore in vitro selection by pathogen derived agents could be the efficient method for obtaining of pea somaclones with increased resistance to F. solani.     

RAPD analysis of blast resistant somaclones from upland rice cultivar IAC 47 for genetic divergence

Seventeen somaclones of upland rice cultivar IAC 47 showing different plant types, and either resistance or susceptibility to leaf blast, were utilized for random amplified polymorphic DNA (RAPD) analysis. Somaclones exhibited differences in reaction to isolates of Pyricularia grisea. Two somaclones (SC02 and SC04) were resistant to all three field isolates of somaclones, while the cultivar IAC 47 was susceptible. The inheritance study of two distinct plant types, one with erect bright green leaves and the other with droopy yellow green leaves, showed that a single possibly different, dominant gene governs each plant type. Of 32 random decamer primers utilized, OPA02 and OPD02 detected polymorphisms between somaclones showing erect bright green leaves and droopy yellow green leaves. Reliable grouping exhibiting 80% similarity was achieved with 17 primers. Leaf blast resistance to race IC-2 of P. grisea was associated with the plant type of erect bright green leaves.     

RAPD Analysis of Maize Somaclones

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 17 decanucleotide primers used each allowed amplification of 2–17 fragments ranging 200–2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64–74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.     

RAPD-analysis of corn somaclones

The genetic difference between maize line A188 and A188-derived somaclones was assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total, 15 out of 17 decanucleotide primers used each allowed amplification of 2-17 fragments ranging 200-2000 bp. The RAPD patterns did not differ between individual plants of line A188, which demonstrated again its high genetic homogeneity. The difference between the initial line and the somaclones was high, ranging 64-74%. On evidence of the genetic divergence, the somaclones formed two clusters. The distribution of somaclones between these clusters was consistent with their origin.     

Molecular divergence of alfalfa somaclones

Summary Plantlets were regenerated from alfalfa callus following passage through a tissue culture medium which contained gibberellic acid. A proportion of these plantlets showed obvious morphological variations. Leaflet, stem and petiole tissue of these plants were extracted to yield a soluble protein homogenate which was characterized by polyacrylamide gel electrophoresis. Over 18 individual protein bands were resolved and visualized by staining with coomassie blue G250. Electrophoretic gels from regenerated plantlets and from the parent plant were scanned spectrophotometrically and analyzed. The relative quantity of each of the proteins resolved from plants was correlated with proteins of other plants via the Pearson's product-moment correlation. Cluster analysis was then performed using these correlation coefficients to judge relatedness among somaclones and the parent plant. Two of 22 somaclones (9.1%) differed significantly from the parent and from the other somaclones judged by quantitative protein pattern variations. Three distinctive lineages through tissue culture produced plantlets. Using a discriminant analysis strategy somaclones could be grouped according to lineage with 80.8% accuracy based upon distinctions between protein electrophoretic patterns. Two of the somaclone lineage groupings showed no overlap with the parental grouping which indicated significant molecular divergence of these plantlets as judged by quantitative protein differences.     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Development and characterization of salt tolerant somaclones in rice cultivar pokkali

Primary regenerants (1190) of a tall traditional salt tolerant rice cultivar pokkali were produced through in vitro culture from mature seed derived calli of fourth subculture. Out of 35000 SC2 regenerants, 26 promising lines with superior agronomic traits were chosen initially for evaluation. SC3 and SC4 generations were stringently evaluated under hydroponics with excess salt stress as well as under field conditions across two growing seasons in Bay Islands. A set of 10 promising somaclones was further evaluated at SC5 and SC6 of which BTS 2, BTS 13, BTS 18 and BTS 24 were found promising. In SC7 and SC8 yield trials in research farm, BTS 24 was found to produce a mean yield of 36.3 and 45.9 q ha-1 under saline and normal soil conditions, respectively. Somaclones varied significantly from the parent with respect to yield and yield attributes. Grain quality and biochemical parameters of all elite somaclones were different from the parent. However, somaclones did not deviate much from their parent in respect of disease and insect pest resistance pattern.     

Optimization of the choice of molecular markers for varietal identification in Vitis vinifera L.

 The aim of this study was to develop a cultivar identification tool based on molecular analysis and a statistical approach. From the PIC parameter we defined the D parameter, which evaluates the efficiency of a primer for the purpose of identification of varieties; i.e. the probability that two randomly chosen individuals have different patterns. D can be used to compare different types of markers even if only the allelic frequencies are known. We used this parameter to develop an algorithm for selecting the optimal combination of primers necessary to identify a set of varieties. The optimal combination of primers determined for a small elite group of varieties applied on a larger set induces a risk of confusion involving 1 of the elite varieties. We estimated the risk of confusion using the D value of each primer of the combination. We applied this methodology on a set of 224 varieties of Vitis vinifera screened with 21 RAPD primers and two microsatellite loci. The discriminating power of the primers did not only depend on the number of patterns it generates but also on the frequencies of the different patterns. A combination of 8 primers (6 RAPD and two microsatellite) was found to be optimum for the discrimination of these 224 varieties. A subset of 38 elite varieties was also investigated. The determined optimal combination of 4 primers (3 RAPD and one microsatellite) applied on the 224 varieties gave 9 risks of confusion involving 1 of the elite varieties. Confusion can happen between varieties with the same origin as well as between varieties of very diverse geographical origins. Received: 22 April 1998 / Accepted: 9 June 1998     

In vitro selection in resistance breeding of strawberry (Fragaria x ananassa duch.)

Genetic resistance to pathogenetic soil-borne fungus Verticillium dahliae Kleb. was examined in two strawberry somaclones. Strawberry somaclones were obtained in sterile culture from runner tips of cultivars 'Merton Dawn' and 'Selva'. In vitro selection was performed with the use of homogenate of liquid cultures of Verticillium dahliae. Microplants of both somaclones were inoculated at stage of 4. Leaves. Disease symptoms were observed at 15., 30., 45., 60. and 75. days post inoculation. Extent of leaf chlorosis was rated on a scale of 0-4. Under the controlled in vitro culture conditions a different response to infection by this pathogenic fungus was observed. After 75. days post inoculation the contribution of necrotic plants in somaclone of 'Merton Dawn' reached the value of 76%, whereas in somaclone of 'Selva' this value reached 86%. In control somaclones of 'Merton Dawn' and 'Selva' the contribution of necrotic plants after 75. days post mock-inoculation with sterile distilled water reached the considerably lower value of 13%. These results revealed that somaclone of 'Merton Dawn' was more genetically resistant to infection by V. dahliae than somaclone of 'Selva'. The observed response to in vitro infection caused by Verticillium dahliae in examined somaclones was similar in comparison with original cultivars. Furthermore, somaclonal variation induced in tissue cultured strawberry was sufficient to select variants that showed enhanced genetic resistance to Verticillium wilt caused by V. dahliae. In vitro selection can be efficiently used as an alternative program to conventional resistance breeding in strawberry.     

Analysis of specific RAPD- and ISSR-fragments in somaclonal maize (Zea mays L.) and development of SCAR markers based on them

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

Analysis of Specific RAPD and ISSR Fragments in Maize (Zea mays L.) Somaclones and Development of SCAR Markers on Their Basis

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

A 12-deoxywithastramonolide-rich somaclonal variant in Withania somnifera (L.) Dunal—molecular cytogenetic analysis and significance as a chemotypic resource

Withania somnifera, commonly known as ashwagandha or Indian ginseng, is a valuable medicinal plant, synthesizing a wide array of pharmacologically active secondary metabolites known as withanolides. In this study, we investigated variation among 54 regenerated plants attained through indirect organogenesis from leaf explants. Organogenic calli were induced on Murashige and Skoog medium containing 2?mg?l^?1 kinetin and 1?mg?l^?1 indole-3-butyric acid. High-performance liquid chromatography was used for quantitative determination of the major withanolides in the somaclones. One somaclone (WS-R-1) showed significantly higher accumulation of 12-deoxywithastramonolide (WS-12D; 0.516%) compared to the explant donor mother plant (0.002%). The incidence of somaclonal variation at the cytological level was investigated by studying mitosis and meiosis in relation to chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic chromosome count, or meiotic behavior. Fidelity at genomic level was evaluated by random amplification of polymorphic DNA (RAPD) analyses, which revealed multiple genetic polymorphisms between the WS-12D over-producing somaclone and the explant donor mother plant. This study demonstrates the capability of inducing chemotypic variability for the development of high-yielding clones due to molecular instability in W. somnifera using an in vitro approach.     

DNA Polymorphism among Rice Somaclones

Molecular markers were used to detect the influence of high concentrations of 2,4 dichlorophenoxyacetic acid (2,4-D) in the callusing media on DNA variations in regenerated rice plants. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) based RFLP analysis were carried out on 12 somaclones of Oryza sativa L. cv. B-370. In vitro culture induced DNA variations were detected in the regenerated plants but the effect of high auxin concentration in the medium could not be revealed. In a second study, fingerprinting of 15 semi-dwarf, high yielding somaclones of B-370 was carried out using RAPD technique. Amplification using 20 random primers produced a total of 167 DNA bands out of which 97 bands were polymorphic. A total of 32 unique DNA bands were detected across all the somaclones and they could be grouped based on their similarity to B-370. RAPD analysis helped to reveal similarity or differences among the somaclones while fingerprinting using additional RAPD markers was not successful.     

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B₅ medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R₁ generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R₂ generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.     

A New Somaclone of Prunus Avium Shows Diverse Growth Pattern under Different Spectral Quality of Radiation

The aim of this research was to set up a regeneration protocol from mature explants of Prunus avium L. cv. Hedelfinger and to develop an early screening method for selection of putative somaclones based on morphological and physiological traits regulated by the spectral quality of radiation. DNA analyses of a new somaclone named HS, conducted using Inter Simple Sequence Repeat (ISSR), revealed a polymorphism between the somaclone HS and wild type propagated by microcuttings. When grown under different spectral quality of radiation, somaclone HS showed a different pattern of growth and development compared to the wild type with the main modifications related to apical dominance and chlorophyll production. Somaclone HS showed reduced apical dominance compared to the wild type. Wild type shoots grown in darkness showed chlorophyll a and b contents at levels in both cases comparable to those recorded under red radiation while HS did not retain the same capability.     

Somacional variation and eyespot toxin tolerance in sugarcane

The analysis of sugarcane plants regenerated from culture for their reaction to eyespot (Helminthosporium sacchari) toxin is described. A total of 480 culture-derived plants (somaclones) from cultivar Q101 were characterized. Some of these plants derived from cultures which had been subjected to selection with the eyespot toxin and others were derived without overt selection. Leaves were assayed for their toxin reaction. A very high frequency of toxin-tolerant variants was found. The distribution was even further biased toward resistance in those plants regenerated from cultures exposed to toxin selection.A total of 85 somaclones was analysed for the stability of their increased toxin tolerance to the primary somaclone; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relate to the possibility of using consecutive vegetative segregation.Six tolerant variants were also passed through a second tissue culture cycle and 60 secondary somaclones were assayed. Twenty four (40%) of these plants had a similar tolerance to the primary somacione; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relative to the possibility of using consecutive cycles of culture to stack improved characters into a sugarcane cultivar.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.