Gametoclonal Variation Research Articles

Homozygote Depression in Gamete-Derived Dragon-Fruit (Hylocereus) Lines.

Putative gamete-derived progenies from two Hylocereus species, the diploid H. monacanthus and the tetraploid H. megalanthus, were studied with the dual aims to confirm their gamete origin and to evaluate their potential use as genetic resources. An additional goal was to determine the origin (allotetraploid vs. autotetraploid) of H. megalanthus by exploring morphological variations in the di-haploid (2x) H. megalanthus progeny. Gamete origin was proved in all five H. monacanthus lines obtained and in 49 of the 70 H. megalanthus lines by using flow cytometry and simple sequence repeat (SSR) markers. The five double-haploid (2x) H. monacanthus lines showed low vigor and abnormal flower development, with malformed ovules and aborted pollen grains. Only one flower set fruit, giving several viable seeds. For H. megalanthus, both abnormal ovules and defective anthers were observed in the di-haploid (2x) and double di-haploid (4x) lines. Among the 46 di-haploid lines, only 14 set fruit. Another 13 di-haploid lines formed flower buds that abscised before anthesis or soon after pollination. The severe sterility of the double-haploid H. monacanthus and the reduced fertility of all the di-haploid and double di-haploid H. megalanthus lines can be linked to their reduced heterozygosity, which drastically affected the development of normal female and male organs. We thus concluded that chromosome doubling, as occurred spontaneously in the double-haploid H. monacanthus and the double di-haploid H. megalanthus, is not sufficient to restore fertility in Hylocereus. We also observed very low gametoclonal variation among the di-haploid (2x) H. megalanthus lines, a finding that supported an autotetraploid, rather than an allotetraploid, origin of this species. Nonetheless, despite the above-described challenging limitations, these gamete-derived lines are currently being bred as the seed parent, offering unique possibilities for genetic research and additional breeding.

Characterization of Leaf Essential Oil Composition of Homozygous and Heterozygous Citrus clementina Hort. Extan. and its Ancestors

Clementine is a natural tangor, resulting from an interspecific cross between mandarin and sweet orange. Gametic embryogenesis, allowing the single-step development of complete homozygous line from the heterozygous parents, increases the efficiency of perennial crop breeding programs. Tri-haploids have been regenerated through pollen embryogenesis (specifically, by anther culture) of Citrus clementina Hort. ex Tan., cv. Nules. Two of them (HOMO1 and HOMO2) have been acclimatized and grafted in vivo in 2000. Research regarding the chemical characteristics of plant regenerated by pollen embryogenesis or gynogenesis are useful to study the “gametoclonal variation”, as well as for further application of haploidy technology in Citrus breeding. For these reasons and with the aim to characterize these homozygous clementine genotypes, in this study leaf essential oils were extracted by Clevenger apparatus and their chemical composition was investigated by capillary GC-MS, comparing them with oils obtained from the heterozygous plant and from its ancestors (clementine is a hybrid between sweet orange and mandarin). The obtained oil compositions, submitted to a statistical analysis, are partitioned into groups using principal components analysis (PCA) and, compared to those of both parents, distinguished in three major chemotypes sabinene/linalool, γ-terpinene and methyl N-methylanthranilate.

Evidence for gametoclonal variation in potato (Solanum tuberosum L.)

Gametoclonal variation that occurs in gametic cells in culture and is recovered in their regenerated derivatives has not been reported in potato (Solanum tuberosum L.). Based on a set of 24 differentiating phenotypic traits, canonical variates analysis genetically distinguished the androgenic (di)haploid (2n = 2x = 24) D4 from its tetraploid (2n = 4x = 48) anther-derived sibs and anther donor JTH/C-107. Nuclear microsatellite analysis over six polymorphic loci indicated that meiotic rearrangements and mutant alleles were primarily associated with the release of gametoclonal variation. The incidence of null alleles in D4 at the loci STACCAS3 and STM0031 was also indicative of mutations occurring within the priming sequence. Microsatellite results were supported by random amplified polymorphic DNA (RAPD) assays that characterized a total of 567 loci (bins) representing 4,258 amplified fragments. Sixty-five new RAPDs that were absent in the anther donor and in either of its parents, viz., S. phureja Juz. & Buk. IVP-35 and S. tuberosum cv. Kufri Jyoti were present in D4, indicating the occurrence of extensive recombinational events. The results have been discussed in the context of microsatellite null alleles providing the most conclusive evidence for gametoclonal variation.

Induced androgenesis in tomato (Lycopersicon esculentum Mill.). III. Characterization of the regenerants.

We present data on the morphological, cytological, biochemical and genetic characteristics of tomato regenerants obtained through anther culture. As a result of induced androgenesis, more than 6,000 rooted regenerants were developed that differed both from the donor plants and among each other with respect to habitus and leaf, flower and inflorescence morphology. Cytological analysis revealed a great variability in chromosome number in the cells of the regenerated plants. While most of the regenerants were mixoploid, the majority of the cells had a haploid chromosome number. R(1) and R(2) progenies were tested for their resistance to Clavibacter michiganense subsp. michiganense ( Cmm 7). Some of the regenerants were resistant to the pathogen. A biochemical analysis of fruit from R(3) and R(4) plants showed a higher content of dry matter, sugars and vitamin C in the regenerant plants obtained from the hybrids than in those from the cultivars and control plants. The values of the parameters of hybrid regenerants grown in the greenhouse were about 1.5-fold higher than those of the hybrid regenerants grown in the field, and this trend is clearly expressed in all of the hybrid regenerants. The results obtained suggest that induced androgenesis and gametoclonal variation may be used as an additional tool to create a large range of new forms. The application of the latter in breeding programs would accelerate the development of tomato lines and varieties that would be more productive, disease-resistant, highly nutritive and flavour-acceptable.

Comparative Molecular Analysis of Winter Wheat Cultivars and Their Doubled Haploid Derivatives

Genetic stability of doubled haploid (DH) lines of androgenetic origin is the prerequisite of their breeding value. In our investigations GK Gobe: a traditional cultivar, GK Delibab: a cultivar of doubled haploid origin, various DH lines of GK Gobe (first cycle DH lines) and DH lines of GK Delibab (second cycle DH lines) were compared with PCR-based molecular techniques. High molecular weight DNA was isolated from randomly selected individuals of these groups and analysed by RAPD, SSR and AFLP methods. The objective of these analyses was to determine the existence or magnitude of difference between cultivars produced by classical and haploid methods (populations are represented by the individuals of each group), and to find which marker system would be most suitable to investigate the homogeneity of DH populations. From the 30 RAPD primers tested, only 6 differentiated the two cultivars (GK Gobe and Delibab). Individual polymorphism could not be observed. There were 7 fragments generated on 12 loci with 9 SSR primers, which were suitable to differentiate the two cultivars. Individual polymorphisms could not be detected between the cultivars, or within the group of first and second cycle DH lines. In the AFLP analyses, 7 of 8 primer combinations were suitable to show differences, resulting in an average of 100–150 fragments. Eighty-one polymorphic fragments were obtained with these 7 primer combinations. Twenty-three of the 81 polymorphic (bands) markers could detect individual differences. Nine of them were suitable to separate cultivar GK Gobe and its DH group. Based on AFLP fragments, the fewest individual polymorphisms were obtained within the DH group of GK Delibab (second cycle DH lines). This result is probably due to repeated application of haploidization techniques. Presumably, the repeated DH production increased the level of homogeneity, as suggested by the significant decrease of the number of polymorphic bands in the second cycle DH lines. Each group consists of individuals, which can be genetically considered as progeny mixture of homozygous recombinants used for the establishment of the given population. Differences between the DH lines of one cultivar may be due to the consequences of the gametoclonal variation, which is fixed at individual level in the homozygous (DH) genome.

RESISTANCE RESPONSES OF PLANTS REGENERATED FROM TOMATO ANTHER AND SOMATIC TISSUE CULTURES TO CLAVIBACTER MICHIGANENSE SUBSP. MICHIGANENSE

The production of gametoclonal and somaclonal variants in tomato is of great importance for the genetic improvement of tomato hybrids and cultivars. The possibility to obtain tomato plants resistant to Clavibacter michiganense subsp. michiganense (Cmm) through anther and tissue culture was investigated in the present study. Regenerants from anther and tissue cultures and their progenies (R1-R3) in the cultivars Roma ms and Bella, lines L. 24–13, and L. 6944, as well as the hybrids Roma ms × UC 82A, Roma ms × L. 31, Roma ms × Bella, and Cristy, were tested for resistance to Cmm. The regenerants differed in their resistance to Cmm. All regenerants from the anther culture of the genotype Roma ms and Roma ms × UC 82A are susceptible to the disease, while those from Bella and Roma ms × L. 31 vary from susceptible to resistant. The highest number of regenerants obtained from somatic tissue culture lacking disease symptoms until the end of the vegetation was observed in the genotypes Cristy and L. 24–13. Variation in regenerant resistance is found in R1, Rb, and R3. The results suggest that the gametoclonal and somaclonal variation may be efficiently applied to obtain tomato plants resistant to Cmm.

Second cycle gynogenesis in onion, Allium cepa L., and genetic analysis of the plants

AbstractHaploid induction in onion via gynogenesis provides inbred lines that can be used in production of hybrid cultivars. Despite several successful reports, the gynogenic induction rate is still relatively low, ranging mainly from 0.5 to six embryos per 100 cultured flowers. In the present study, gynogenesis was induced in selfed progeny of three genetically homozygous and stable fertile doubled haploid lines previously obtained through in vitro gynogenesis. The aims were to acquire information on whether the gynogenic responsiveness of a particular heterozygous cultivar is correlated with higher or lower gynogenic capacity in the second cycle of gynogenesis, to evaluate the non‐genetic component of the variability, and to monitor by random amplified polymorphic DNA (RAPD) analysis the onset of eventual gametoclonal variation induced during the gynogenic processes. The results from the gynogenic induction of 9708 flowers showed significant differences among three lines, one of them being extremely responsive, showing a mean yield of 118.3 haploid embryos per 100 cultured flowers (118.3%). The high embryo yield of this line was determined by the line's particular genotype. However, the induction rate of gynogenic embryos obtained within 24 genetically uniform donor plants of this responsive line varied from 67 to 196% and this variation can be attributed to uncontrolled environmental conditions. A total of 157 plants sampled from two lines and obtained through a first (RI) and a second (SCG) cycle of gynogenesis, was analysed by RAPDs. The single band profiles were compared either between or within the RI and SCG plants of each line. A total number of 4600 RAPD fragments were scored and no variation was found in the highly responsive line, while in the less responsive one, minor changes were detected for the presence/absence of two RAPD bands. These results indicate high genetic stability of onion homozygous lines passed through two cycles of gynogenesis.

Non-random gametoclonal variation in rice regenerants from callus subcultured for a prolonged period under high osmotic stress

Gametoclonal variations were investigated in rice plants regenerated from anther-derived callus after subculture for two different periods (4–6 and 20 weeks) in medium with two levels (292 and 438 mM) of mannitol. A high osmotic stress by mannitol in subculture medium contributed to the maintenance of high regeneration ability. The frequency of diploid regenerants (R0 plants) increased and that of haploids decreased from callus subcultured for the longer period in medium with the higher concentration of mannitol. R1 regenerant lines displayed variations in all six agronomical traits studied at the frequencies of 63% and 88% after subculture for 4–6 weeks and 20 weeks, respectively. High homozygosity was suggested in most of the R1 lines for these traits by the similar levels of variance to those in the control variety. A principal component analysis showed a skewed distribution of variants towards dwarfness and less panicle weight in the regenerants from callus subcultured for 20 weeks with 438 mM mannitol. Two short culmn variants were shown to be due to single or double recessive mutations. The results suggested that prolonged subculture under high osmotic stress causes non-random selection of homozygous gametoclonal variations in rice anther culture.

In vitro production of haploid plants

Although several methods have been developed for producing haploid plants, the in vitro techniques are much more efficient than inter-specific hybridization or treatment with plant-growth regulators, temperature or irradiation. Androgenesis is the most universal of these techniques but ovule culture and the bulbosum method could complement or replace anther culture in those species or genotypes with less responsive male gametes. Genotype, environment, physiological status of the donor plant, and culture conditions and components all need to be taken into account when developing procedures for producing haploid and dihaploid plants. Suitable methods are already well established for a number of important crops. However, many problems, related to regeneration frequency, gametoclonal variation and albinism, are still unsolved. It is now clear that haploids and dihaploids form the ideal system for genetic manipulation in plants. Their key role in producing new theoretical and applied knowledge in plant science is an important aspect of our review.

In VitroCulture of Wheat and Genetic Transformation — Retrospect and Prospect

Abstract This review focuses on the improvement of wheat (Triticum aestivum L.) via tissue and cell culture and its use in gene transfer techniques. Success of the latter critically depends on the ability to regenerate plants from cells or tissues cultured in vitro. Hence, we have devoted attention to the attempts made so far in obtaining regenerants from diverse explants. Although it is known that immature embryos are the best source for initiating morphogenic cultures, basic information related to the process of differentiation can also be gained by studying less responding tissues. The opportunity provided by anther and microspore culture in wheat improvement and the progress made is also presented. To enhance tissue culture responses, identification of chromosomes, gene loci, and genes is of cardinal importance. We have also surveyed the progress made in this regard by conventional but incisive plant-breeding techniques. Gene rearrangements in tissue culture leading to the appearance of somaclonal or gametoclonal variation are of interest in selection of useful cell lines. The last part of the review is devoted to work done on transient gene expression and transformation with emphasis on recent developments.

Studies of gynogenesis in onion ( Allium cepa L.): induction procedures and genetic analysis of regenerants

Gynogenesis of four onion cultivars was induced, starting from ovules and ovaries, using a 2-step culture procedure. The induction frequency was much higher from ovaries and was strongly affected by genotype. Thidiazuron (2 mg/l) had a positive effect on regeneration and subsequent in vitro culture of regenerants. Esterase isozyme analysis showed that 59% of regenerants were homozygous. Chromosome counting of root tip cells and flow cytometry of leaf tissue showed haploid, doubled haploid and mixoploid structures. Within cloned plants, some showed segregation of the ploidy level with a tendency towards endoreduplication. Analysis of random amplified polymorphic DNA (RAPD) profiles of homozygous regenerants revealed several missing bands compared with donor plants, but micropropagated clones of different ploidy level were identical among each other. Gametoclonal variation was detected by the appearance of a novel RAPD band, present in two out of 12 regenerants and not in the donor plants.

Variation in Doubled Haploid Plants of Wheat Obtained through Wheat (Triticum aestivum) × Maize (Zea mays) Crosses

AbstractVariation was investigated in 110 doubled haploid (DH) lines of wheat derived from wheat × maize crosses. Field observation revealed visible variations in 15 DH2 lines such as extreme dwarfism, low seed fertility, alteration of spike type and stripes. Six agronomic traits, i.e., heading date, spike number/ plant, culm length, spike length, seed fertility and grain weight were statistically analyzed in the DH2 and DH3 generations. Out of the 88 DH2 lines/DH3 groups, 26 %/64 % showed significant differences from the parental variety in the means of one or more traits. Ranges of the DH3 lines were larger than those of the DH2 lines, except for spike number/plant. Furthermore, analyses of variance within and between DH lines showed the presence of heterogeneity/heterozygosity in the DH2 lines/plants. These results indicated the occurrence of gametoclonal variation in the DH lines. It is considered that most of the variations detected were due to the colchicine treatment rather than to the 2,4‐D treatment or in vitro culture.

Molecular and morphological evaluation of doubled haploid lines in maize. 1. Homogeneity within DH lines

The homogeneity of anther culture-derived lines of maize has been evaluated by means of field observations and molecular markers. The homogeneity of the doubled haploid (DH) lines was shown by the absence of segregation for morphological oligogenic traits. The intravariance for polygenic traits for 42 DH and two conventionally derived lines was similar, which confirmed the homozygosity of the DH lines. More than 100 RFLP markers were tested on 189 DH lines derived from two crosses, DH5 x DH7 and A188 x DH7, and 60 single-seed descent (SSD, F6) lines derived from A188 x DH7. The overall rate of heterozygosity for all of the DH lines was approximatively 1% and pertained to 6 lines out of 189, while it was 8.5% for the SSD lines after four selfings. A precise description of the material used suggested that the events which led to this unexpected heterozygozity in DH lines were more likely to have occurred after rather than during the androgenetic process. Nine duplicated pairs of genotypes were found within the DH lines, indicating that a single microspore-derived structure can fragment to give two identical plantlets. Despite the extensive screening with more than 100 markers, only 2 lines showed unexpected banding profiles, and these were probably gametoclonal variants. The use of a direct regeneration system that avoids any callus phase might explain this low frequency of gametoclonal variation.

Prolamin Analysis of Progenies from Androgenetic Plants of Triticale

AbstractThe present work reports results of the analysis of variability of prolamins in five breeding lines of triticale and 61 androgenetic progenies obtained from them by in vitro culture of the anthers. The phenotypic analysis of the prolamins showed heterogeneity even between seeds harvested from the same plant regenerated by in vitro culture. The variability of prolamins in this material could be, perhaps, explained under different hypotheses: unreduced microspores, embryo fusion. The percentage of androgenetic plants which generated uniform progenies for the prolamins was 14.7. The plants obtained by in vitro culture showed a significant increase of the phenotypic variability for both specific bands and patterns of prolamins as compared with the parental materials. Some plants showed missing groups of prolamins in their electrophoretic profiles, which completely affected specific regions of the proteinogram. This could be explained by gametoclonal variation based on either structural rearrangements, or aneuploidy affecting chromosomes carrying genes involved in the expression of prolamins.

Some Aspects of Development and Application of Plant Biotechnology in Bulgaria

ABSTRACTBiotechnological methods which supplemented the conventional breeding methods are extremely valuable today because of the high contamination level of the environment with insecticides, herbicides and artificial fertilizers. The review describes current investigations of tissue and cell cultures in Bulgaria which began at the end of the sixty's. The main directions of our investigations are: clonal micropropagation combined with virus elimination; overcoming the incompatibility in remote hybridization; haploid production; somaclonal and gametoclonal variation; in vitro mutagenesis and cell selection; protoplasts culture, somatic hybridization and transformation in economically important plant species.

Application of Tissue Cultures for Obtaining Disease Resistant and Male Sterile Forms of Tobacco

ABSTRACTThe aim of the investigation is the obtaining of disease-resistant forms of tobacco. Three in vitro methods have been used: somaclonal variation, gametoclonal variation and genes transfer of resistance form wild tobacco species. As source material we used the following wild species: Nicotiana goodspeedii, N. alata, N. sanderac, N. africana, N. paniculala and cultivars Burley 21, Harmanliiska basma 163, Vrania 96 and Krumovgrad 58. More than 30 000 regenerants were obtained from anthers and stem explants tissue cultures. Some of them were resistant to economically important diseases as Lycopersicum virus 3, Peronospora labacina, Thielavia basicola, TMV, Erysiphe cichoraccarum. The disease resistance was inherited in R1 and R2 progenies. Cytoplasmic male sterile tobacco forms have also been obtained.

Characterization of Potato Virus Y Resistance from Gametoclonal Variation in Flue-Cured Tobacco

A previously identified gametoclonal tobacco variant, NC 602, exhibits resistance to certain potato virus Y (PVY) strains. NC 602 was subjected to a second cycle of anther culture and colchicine treatment. The resulting doubled haploid lines were evaluated for back mutation to susceptibility and any additional mutation that might provide resistance to tobacco etch virus (TEV). The PVY resistance was stable in anther culture, and although no TEV resistance was found, three lines were identified with delayed symptom onset (...)

FOOD QUALITY GENES AND GENETIC MODIFICATION OF FRUIT AND VEGETABLE PRODUCE

For many branded food products, the raw material is purchased as a commodity. Food companies provide added-value based on processing technology or marketing. The tools of plant biotechnology, which shorten the time for crop improvement and permit development of novel germplasm, offer the food industry the opportunity to modify raw materials and develop proprietary branded products. Such modifications will permit development of plant cultivars specifically selected for traits with added-value for the processor or the consumer. Biotechnology-developed cultivars offer the opportunity to develop produce that can be branded. The cell genetics tools of clonal propagation, somaclonal variation, gametoclonal variation, and protoplast fusion permit new cultivar development in an intermediate time scale, making them attractive for market introduction of proprietary products. When integrated with conventional breeding, these intermediate-term technologies will permit modification of fruits and vegetables over the course of the next several years. The longer-term technologies of plant genetic engineering will continue to have an impact on manipulation of specific traits, resulting in second-generation products specifically designed for consumers.

The value of gametoclonal variation in breeding for quantitative traits in flue-cured tobacco (Nicotiana tabacum L.)

In tobacco (Nicotiana tabacum L.), anther-derived doubled haploid populations have been shown to exhibit large amounts of unexpected genetic variation and a severe depression in cured leaf yield when compared to conventionally inbred genotypes from comparable sources. A previous study had predicted that the yield depression observed in a doubled haploid population-derived from a near homozygous cultivar, NC95, might be overcome through a recurrent selection program. In the current study, progress from three cycles of full-sib family selection for improved yield in an anther-culture derived population of NC95 was measured, as well as the remaining genetic variation within the population. A design II experiment was conducted in the population following three cycles of selection. Results indicate that the NC95 yield level has been recovered in the third selection cycle population. Although most of the genetic variation in the population appears to be exhausted, the additive genetic variance among maternal half-sib families for yield is significant, and it appears that continued yield improvement can be made through recurrent selection. Significant additive-genetic variance for yield was found among maternal half-sib families but was essentially zero among the paternal half-sib families, suggesting that remaining genetic variation is not being transmitted through pollen. One possible explanation results from the phenomenon of DNA amplification that can occur during the anther culture process, and that may enable extraordinary recombinational events and reduce the viability of male gametes.

Agronomic Performance of Wheat Doubled‐Haploid Lines Derived from Cultivars by Anther Culture

AbstractTo determine the level of gametoclonal variation among doubled‐haploid lines (DHLs) of Triticum aestivum L. developed using anther culture techniques and its effect on agronomic performance, 70 anther culture‐derived DHLs of ‘Kitt’ were compared for 7 agronomic traits to 50 single‐seed descent‐derived lines (SSDLs) of ‘Kitt’ and to the cultivar ‘Kitt’. In a second experiment, 26 DHLs of ‘Chris’ were compared for 7 agronomic traits to 29 SSDLs of ‘Chris’ and to the cultivar ‘Chris’. Each experiment was grown as a randomized complete block design with three replications in three environments. For ‘Kitt’, the DHLs averaged significantly lower grain yields than the comparable SSDLs. For ‘Chris’, the DHLs averaged lower, but not significantly lower, grain yield than the SSDLs. In both ‘Kitt’ and ‘Chris’, the genetic component of variance for yield of the DHLs was significantly larger than that of the SSDLs indicating the presence of gametoclonal variation. The lower average grain yield of the DHLs was explained by a larger group of low‐yielding DHLs than was present in the SSDLs. Six ‘Kitt’ DHLs and 3 ‘Chris’ DHLs were lower yielding than the lowest yielding SSDL, respectively. Elite DHLs and SSDLs were similar for mean grain yield performance. Though the DHLs and SSDLs were significantly different for some yield components, the affected yield component changed with the cultivar and there was no consistent effect. Significant genotype × environment interactions were detected for some traits which were caused by changes in the magnitude of differences, rather than reversals in ranking, indicating that low yielding DHLs could be culled on the basis of visual selection or single‐environment testing. Hence, gametoclonal variation was induced by the anther culture techniques used in this study, tended to be deleterious for grain yield, and was sensitive to the growing environment. However, as the DHLs and SSDLs had similar expected population means based upon expected gains from selection, this gametoclonal variation should not be a major hindrance to wheat breeding.