The effect of the duration of callus culture on the accumulation of genetic alterations in regenerated pea Pisum sativum L

Two groups of regenerant plants were obtained from different pea genotypes (lines R-9 and W1 and cultivar Viola). The first group was derived after eight months of culture and the second, from calluses cultured for a prolonged (more than ten years) time. Using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) methods, the regenerants and the original lines were compared with regard to genetic differences. The regenerants from both groups were shown to differ in DNA polymorphism from the original lines and from one another. The divergence of the regenerants was also different, depending largely on the original genotype. Examination of genetic differences between the first and the second group showed that the variability increased with culturing time. This was particularly evident for regenerants of the Viola cultivar, in which variability ranged from 0-5% (first group of regenerants) to 10% (second group of regenerants).     

RAPD and ISSR analyses of regenerated pea Pisum sativum L. plants

Long-term pea callus cultures of different genotypes (mutants R-9 and W-1 and cultivar Viola) were used to regenerate plants (generation R0). The regenerants displayed changes both in qualitative and in quantitative traits. The most dramatic morphological alterations and complete sterility were observed in regenerants of the cultivar Viola. To estimate the genetic differences, regenerants were compared with the original lines with the use of RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) analyses. The extent of divergence varied among regenerants and depended mostly on the original genotype. The genetic difference from the original line was no more than 1% in W-1 regenerants, 0.7-5.3% in R-9 regenerants, and 10-15% in sterile regenerants of the cultivar Viola. The genetic variation of plants regenerated from a callus culture maintained for ten years did not exceed that of plants obtained from a culture maintained for two years.     

RAPD and ISSR analyses of regenerated pea <Emphasis Type="Italic">Pisum sativum</Emphasis> L. plants

Long-term pea callus cultures of different genotypes (mutants R-9 and W-1 and cultivar Viola) were used to regenerate plants (generation R₀). The regenerants displayed changes both in qualitative and in quantitative traits. The most dramatic morphological alterations and complete sterility were observed in regenerants of the cultivar Viola. To estimate the genetic differences, regenerants were compared with the original lines with the use of RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) analyses. The extent of divergence varied among regenerants and depended mostly on the original genotype. The genetic difference from the original line was no more than 1% in W-1 regenerants, 0.7–5.3% in R-9 regenerants, and 10–15% in sterile regenerants of the cultivar Viola. The genetic variation of plants regenerated from a callus culture maintained for ten years did not exceed that of plants obtained from a culture maintained for two years.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 71–77.Original Russian Text Copyright © 2005 by Kuznetsova, Ash, Hartina, Gostimskij.     

Quantitative trait variation in phenotypically normal regenerants of cotton

Summary Somaclonal variation for quantitative traits could affect the practical utilization of regenerants in cotton improvement. Three groups of experimental lines were derived to analyze variation, including one control group from the explant-source cultivar and two groups of R₃ somaclones from different R₀’s (R₀ = initial regenerant) free of observable chromosomal rearrangements. A three-environment field trial was conducted to evaluate group means, genetic variance, and line performance. Mean seedcotton yields of the somaclonal groups were reduced by 21 and 26% relative to the Coker 310 standard at two locations, but lint percentage and certain fiber properties were improved. Group-by-environment interactions were significant (P<0.05) for 10 of the 12 measured traits. Genetic variance tended to decrease in the somaclones, plant height being an exception. Line performance of the somaclones indicated that 50-boll weight, seed index, and fiber length did not reach the Coker 310 group means. These data suggest that genetic gain will be improved if regenerants of cotton are self-pollinated and the progenies evaluated for quantitative traits before crossing somaclones with the explantsource cultivar or other elite germplasm.     

Effect of media components on the gynogenic regeneration of onion (Allium cepa L.) cultivars and analysis of regenerants

Summary Two separate experiments were performed to analyze the effects of different media on gynogenic regeneration in four onion cultivars. In a two step flower/ovary culture procedure, 2,4-dichlorophenoxyacetic acid added to the induction medium was superior to phenylacetic acid in the highly regenerating cultivar, while the effect of thidiazuron in the regeneration medium was generally optimal in higher (2 mg/l) rather than in lower (0.2 mg/l) concentrations. Gellan-gum was compared to agar solidified media. A higher number of regenerants was achieved on the former, but an undesirable hyperhydricity of regenerants formed on gellan-gum solidified media greatly reduced the final survival of formed embryos. Analysis of the time interval needed for regeneration showed high variability (from 46 to 152 days after inoculation), which was particularly pronounced in genotypes with lower regeneration capacity. Simpler isozyme patterns of regenerants showed that all analysed regenerants of the cultivar with a high regeneration capacity were homozygous, while in the other three cultivars, a considerable percentage (11.1 to 36.4%) of heterozygous regenerants were also detected. Ploidy analysis of the regenerants with simpler isozyme patterns revealed that the majority of lines remain haploid. Identification of 2 homozygous triploid regenerants demonstrated that as in androgenesis, nuclear fusions can occur during gynogenic haploid regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - PAA phenylacetic acid - TDZ thidiazuron - B5 Gamborg et al. (1968) medium - BDS Dunstan and Short (1977) medium     

Utilizing the genetic diversity within rice cultivars

Plant breeding of rice emphasizes improvement in yield, disease resistance, and milling quality. Numerous other traits (e.g., bran carotenoids) that historically have not been selected for could provide added value in expanding niche markets, as well as be useful tools for understanding the genetic control of these traits. Residual heterozygosity is present in many rice cultivars; therefore, it is possible to select for different alleles within an existing cultivar. By identifying and using cultivars with high levels of variability for a trait, we were able to develop separate lines from single cultivars that showed high and low levels of that trait. The rice cultivar RU9101001 and the warm- and cold-sprouting lines that were derived from it were used to demonstrate that residual heterozygosity was present within a cultivar and that the original heterozygosity was separated in the derived lines. Rice simple sequence repeat markers were heterozygous in the parent RU9101001 cultivar, but the cold-sprouting lines were homozygous for one set of alleles and the warm-sprouting lines were homozygous for the other set. Through detailed phenotypic screening, we developed lines that exhibited low and high levels of the following traits in the specified cultivars: cold-sprouting from RU9101001 and Bonnet 73, postharvest yellowing from Tominishiki, early tillering from Hei Jaio and Tominishiki, and bran carotenoid levels from Spring. If variability exists in a cultivar, then utilization of residual heterozygosity may provide a quicker and more efficient means to develop lines with special characteristics using cultivars that are already agronomically valuable or to develop near isogenic lines for genetic and biochemical investigations.     

Development of a genomic library of near isogenic lines (NILs) in melon (Cucumis melo L.) from the exotic accession PI161375

A doubled haploid line (DHL) population of melon derived from a cross between the Korean cultivar “Songwhan Charmi” accession PI161375 (SC), included in the horticultural group conomon, and the Spanish cultivar “Piel de Sapo” (PS), included in the horticultural group inodorus, was used to develop a collection of near isogenic lines (NILs). These parental lines represent very different melon cultivar groups, with important differences at fruit, plant, disease response and molecular level. This cross is one of the most polymorphic ones within melon germplasm. Selected DHLs were backcrossed to PS and further backcrossing and selfing was performed, monitoring introgressions from SC using molecular markers covering the melon genetic map. A final collection of 57 NILs was obtained, containing a unique independent introgression from SC in the PS genetic background. The introgressions within the collection cover at least 85% of the SC genome with an average introgression size of 41 cM, corresponding to 3.4% of the SC genome. The average resolution for mapping genes or quantitative trait loci is 18.90 cM. This set of NILs is a potentially powerful tool for the study of quantitative trait locus involved in melon fruit quality and other important complex traits, and the introduction of new genetic variability in modern cultivars from exotic sources. The NILs can also be used as pre-competitive breeding lines in melon breeding projects.     

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.     

Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating different alfalfa cultivars in Italian soils

We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.     

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.     

Suitability of RAPD for analyzing spined and spineless variant regenerants of pineapple (Ananas comosus L., Merr.)

A random amplified polymorphic DNA (RAPD) analysis of spineless (variant phenotype) plants obtained from micropropagated dormant pineapple (Ananas comosus L., Merr.) axillary buds was performed using arbitrary 10-mer oligonucleotide primers. This was done to investigate the genetic fidelity of the regenerants and to distinguish these variants from regenerants bearing the normal spined phenotype. Of the 58 arbitrary primers used, 29 produced bands unique to the spineless phenotype, and 30 produced bands unique to the spined phenotype. A total of 914 bands were scored, 55 of which were polymorphic to the spineless phenotype and 51 of which were polymorphic to the spined phenotype. On the basis of RAPD amplification products, genetic similarity was estimated in both types of regenerants using similarity coefficients (Nei and Li, 1979). The characteristic finger-prints generated by each probe emphasize genetic variability of regenerants. This technique is suitable for analyzing variant regenerants induced in vitro.     

Genetic Diversity and Dynamics of Sinorhizobium meliloti Populations Nodulating Different Alfalfa Cultivars in Italian Soils

We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.     

Stable transformation via particle bombardment in two different soybean regeneration systems

The Biolistics^® particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for -glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.     

Genetic stability analysis of chrysanthemum (Chrysanthemum x morifolium Ramat) after different stages of an encapsulation-dehydration cryopreservation protocol

Genetic stability in chrysanthemum (cultivar ‘Pasodoble’) apices was studied at each step of an encapsulation-dehydration cryopreservation protocol: control shoots (A), nodal segments after cold treatment (N), apices after osmotic stress (0.3 M sucrose) and cold treatment (P), encapsulation and culture in 0.8 M sucrose (S), dehydration (D), and cryopreservation (Cr). Two different markers were employed: RAPDs and AFLPs. Throughout the process, the origin of the apices (in vitro shoot from which they were excised) was recorded. Eight complete lines (from which DNA could be amplified after all the steps considered) were studied. Two out of twelve arbitrary primers showed polymorphisms. Three RAPD markers were replaced by three new ones in the Cr sample in one line. Using a different primer, a 700 bp fragment was absent from all samples from the 0.3 M sucrose-culture step (‘P’) onwards, in all the lines studied. The sequences of these fragments were studied to find similarities with known sequences. Polymorphic AFLP fragments were also observed, and most of the differences appeared from step ‘P’ onwards, pointing out the possible effect of this process (preculture on 0.3 M sucrose) in the DNA variation. These results show that genetic variation can appear throughout the cryopreservation process, and the low temperature itself is not the only stress risk of the technique. Therefore, genetic stability of the regenerants obtained after cryopreservation should be monitored.     

Analysis of genetic distance by SSR in waxy maize

We examined the genetic diversity of 80 inbred waxy maize lines using 22 SSR molecular markers that could be used to achieve heterosis in waxy maize. Eighty inbred waxy maize lines with different phenotypes, 40 yellow, 25 white, 13 black, and two red lines were analyzed by SSR molecular marker fingerprint and cluster analysis. Using a standard genetic distance of 0.55, the 80 waxy maize inbred lines were clustered into nine groups. Among them, group II, group V, groups VII and VIII, and group IX were divided into three subgroups at a genetic distance of 0.46, into two subgroups at 0.49, into two subgroups at 0.46, and into four subgroups at 0.493, respectively. All but one of the yellow waxy maize inbred lines were clustered in groups VI, VII, VIII, and IX. Group IX (30 lines) contained 28 yellow lines; the other 11 yellow lines were distributed among groups VI, VII and VIII. Among the 25 white lines, 21 were clustered in groups III, V, VI and the third subgroup of group II. The black line N72 was in a group of its own. The black lines N75, N76 and N78 were distributed in groups VII, VIII and IX, respectively. The other nine black lines were clustered in group II. The red lines were distributed in the second subgroup of group II and there was no difference in genetic distance between them. In conclusion, there were considerable genetic differences among waxy maize inbred lines of different colors. The mean genetic distance of inbred lines of the same color was significantly less than that of lines of different colors. Therefore, we concluded that it was more accurate to determine the difference between the populations using the highly stable DNA genetic markers.     

Genetic diversity changes in Indian lentils over the times

The genetic diversity of 96 genotypes of lentil comprising 34 cultivars, 46 advanced breeding lines, and 16 germplasm lines were studied using 260 SSR markers. These markers generated a total of 749 alleles. The alleles/locus ranged from 2 to 16 with an average value of 2.87. Polymorphic information content varied from 0.02 to 0.91 with a mean of 0.30. Major allelic frequency ranged from 0.14 to 0.99 with a mean of 0.77. Studied genotypes were clustered into two groups according to their breeding history. Advanced breeding lines derived from exotic lines were clustered in one group, while another group accommodated most of the cultivars and advanced breeding lines with common cultivars in parentage. The germplasm lines were sub-clustered within first group. Cumulatively, first three principal components contributed 21.2% to the total variability. Advanced breeding lines showed higher number of alleles/locus and gene diversity (He) than other sets of genetic materials. In present study, no significant differences were observed between cultivars developed in different decadal groups for both NA and He. Moreover, genetic diversity changes between small and large seeded lentil cultivars were also found non-significant in this study. These findings showed that the use of alien genes can help to diversify active gene pool for developing improved new cultivars in lentil.     

Molecular marker analysis of hypoploid regenerants from cultures of barley x Canada wild rye.

Canada wild rye (CWR, Elymus canadensis L., 2n = 4x = 28) is a potential source of genes for disease resistance and environmental tolerance in barley (Hordeum vulgare L., 2n = 2x = 14). Tissue cultures were initiated from immature inflorescences of CWR x 'Betzes' barley hybrids to promote CWR introgression into barley through possible tissue culture induced chromosome breakage and exchange. Among the plants regenerated, some were missing one (2n = 20) or part of one (2n = 20 + telo) chromosome. The objective of this study was to identify the missing chromosome or chromosome arm in these regenerants through the analysis of molecular (RFLP) markers that previously had been mapped in barley. Forty-six hypoploid regenerants that traced to 30 separate explants obtained from 10 interspecific hybrid plants were evaluated. DNA was digested with the restriction enzyme HindIII, Southern blotted, and probed with 39 genomic and cDNA barley clones that identified sequences polymorphic between barley and CWR. Eight of these probes identified band loss patterns that separated the regenerants into two groups. One group, all with barley cytoplasm, were missing a CWR chromosome homoeologous to barley chromosome 3; a second group, all with CWR cytoplasm, were missing a CWR chromosome homoelogous to barley chromosome 7. These results indicated that chromosome elimination in culture was not random. The two cytoplasm groups were further differentiated by probes that identified band shifts. These band shifts were caused by differences in DNA methylation. Key words : Hordeum vulgare, aneuploidy, Elymus canadensis, tissue culture.     

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD)

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts.     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Differential sensitivities of transpiration to evaporative demand and soil water deficit among wheat elite cultivars indicate different strategies for drought tolerance

In order to satisfy increasing wheat demands, scaling up wheat production will require boosting yield in suboptimal, drought-prone areas. Under rain-fed environments, one promising option is the identification of traits allowing for soil water conservation until the next rain episode. This can be achieved either by limiting transpiration rate (TR) of the crop to a maximum level when atmospheric drought (or VPD for vapor pressure deficit) is too high or by decreasing stomata conductance earlier in the soil drying cycle. Although promising, those strategies were never explored in wheat. A first objective of this study was to investigate the extent of the genetic variability of TR sensitivity to both VPD and soil water deficit among a group of eight elite wheat lines, which are cultivated under south Australian conditions. Those consisted of seven differentially drought-adapted lines and one check cultivar. TR responses to VPD were highly variable among genotypes, with six lines displaying a breakpoint in their TR response to VPD that ranged from 2.4 to 3.9 kPa, while two others had their TR increasing linearly as VPD increased. Transpiration response to a progressively decreasing fraction of transpirable soil water (FTSW) was investigated in those lines. A significant genetic variability in the responses among genotypes was observed. They revealed different FTSW thresholds at which transpiration started to decrease at levels ranging from 0.43 to 0.52, and different slopes for the decrease. A second objective was to investigate the existence of phenotypic correlations between the parameters characterizing transpiration sensitivities to both sources of water deficit (i.e., VPD and FTSW). Significant correlations were observed revealing that genotypes with conservative water use in their response to high VPD were also conservative in response to decreasing FTSW and that the drought tolerance of other lines might stem from an apposite strategy, invoking decreased sensitivity of TR to both sources of drought. Those findings provide new options for breeding drought tolerant lines based on this germplasm.