Genetic Variability in the Progeny of Androgenic Dihaploid Plants and Selection of High Agronomic Performing Lines in Brassica Juncea

Androgenic lines of Brassica juncea cv. PR-45 raised by anther culture, were screen for genetic variation. 393 androgenic plants were transferred to pots to study the R₀ generation. These plants showed substantial variation for different characters. Seed progenies of 27 lines of R₀ plants were sown in the field to study R₁ generation. Androgenic plants within lines were significantly homogeneous for the various agronomic characters studied. Two lines were shorter (18 – 20 %) than the control plants, with a remarkable feature of early maturation. Three lines showed 27 – 31 % higher yield than the parent cultivar.     

Variation ofHelminthosporium resistance and biochemical and cytological characteristics in somaclonal generations of barley

SC₂ and SC₃ progenies of nineteenin vitro regenerated barley plants (SC₁) from resistant calli selected against purified culture filtrate ofHelminthosporium sativum and one parent ‘Dissa’ genotype were studied for stability of resistance and protein, soluble protein, maltose and saccharose contents. Cytological studies were also carried out on the SC₃ generation. Stability of resistance toHelminthosporium sativum was found in 50% of the somaclonal lines. Significant variation among different somaclonal lines and among different callus lines from which the plants were regenerated were found for yield, disease score and biochemical characters assessed except saccharose content in the somaclonal lines. Significant increase and decrease over the donor parent for most of the characters were obtained. Cytological abnormalities such as multilobed nuclei, multinucleate cells, abnormal anaphase and mixoploidy were also observed.     

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.     

Agronomic evaluation of tissue-culture-derived soybean plants

Summary Genetic alterations of regenerated plants based on the tissue culture process (somaclonal variation) have become common for many plant species including soybean [Glycine max (L.) Merr.]. The objective of this study was to test for the presence of tissue-culture-derived genetic variation in eight agronomic traits in homozygous progeny regenerated by organogenesis using the commercially important cultivar Asgrow A3127. A total of 86 lines derived by repeated self-pollination of nine regenerated plants was grown in two locations for 2 years. When compared to the unregenerated parent, statistically significant variation (P<0.05) was found for maturity, lodging, height, seed protein and oil, but not for seed quality, seed weight, or seed yield. All of the variation noted was beneficial and did not involve decreased yield. Since the differences were not large, the results indicate that the tissue culture process is not necessarily detrimental to plant performance, which is an important consideration since tissue culture techniques are used in many genetic engineering methods.     

Integration,expression and inheritance of two linked T-DNA marker genes in transgenic lettuce

T-DNA integration and stability were assessed in Agrobacterium-derived transgenic lettuce lines carrying a chimaeric CaMV 35S promoter-driven gus-intron gene and a chimaeric nos.nptII.nos gene. T-DNA integration was predominantly complex in transgenic plants derived from an A. tumefaciens strain carrying the supervirulent plasmid ToK47. Truncation of the right side of the T-DNA was observed in first seed generation R₁ plants from one line. Complex T-DNA integration patterns did not always correlate with low transgene expression. Despite a high T-DNA copy number, ca. 30% of the lines analysed showed high transgene expression in the R₁ generation. High transgene expression was stable at least to the R₄ seed generation in selected high-expressing lines. Transgene expression was lost in the R₂ generation in a low expressing line, while complete, heritable transgene silencing from the R₀ to R₂ generations was also observed in another line. A 50-fold variation in -glucuronidase (GUS) activity and a 16-fold variation in NPTII protein content were observed between R₁ plants derived from different R₀ parents. Reactivation of transgene expression with 5-azacytidine in partially silenced lines indicated that low expression was associated with DNA methylation.     

Somaclonal Variation in Rice after Two Successive Cycles of Mature Embryo Derived Callus Culture in the Presence of NaCl

Two successive cycles of mature embryo-derived callus culture separated by one cycle of sexual reproduction of R₀ regenerated plants were performed using two rice (Oryza sativa L.) cultivars in order to gain information upon the nature of somaclonal variation in this species. Plants regenerated after one cycle of tissue culture exhibited higher variability and lower performances than those of initial cultivar. A second cycle performed using R₁ embryos as explants showed that the cellular component of salt resistance in terms of growth and regenerating abilities selected during the first cycle could be transmitted to the progenies. The extent and the nature of somaclonal variation depended on the identity of R₀ mother plant and culture conditions, somaclonal variation being strongly reduced in some families obtained from salt-treated calli.     

Somaclonal variation in high tannin sorghums

Summary 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.     

Agronomic evaluation of inbred lines derived from tissue cultures of maize

Summary Tissue culture-induced variation has been proposed as a novel source of variation for crop improvement. In maize (Zea mays L.), chromosome aberrations and qualitative genetic variants have been induced during in vitro culture. The proportion of regenerated plants carrying such variants has been shown to increase with culture age. The objective of this research was to evaluate the relationship between culture age and somaclonal variation for several agronomic traits. Six sib-pollinated ears of S₀ (F₂) plants in four OH43 ms/A188 populations each provided control seed and embryos for culture initiation. S₂ lines derived from control seed and from plants regenerated 4 and 8 months after culture initiation were grouped according to their source ear and grown in 6 separate trials. A total of 305 tissue culture-derived and 48 control lines were evaluated as lines per se and in a testcross at each of three locations. Tissue culturederived lines and their testcrosses generally had lower grain yield and moisture. Since grain yield and moisture were not positively correlated in any trial, the highest yielding lines could be selected without increasing grain moisture. Grain yield and plant height tended to decrease with culture age. Although tissue culture-derived lines were, on average, inferior, the highest yielding line per se in three of six trials and the top-ranked line in five of six trials for yield and moisture were derived from tissue culture. The results indicate that tissue culture may generate variation for agronomic traits. Some of the variation, particularly the trend towards earlier maturity, could be useful. However, this method may require screening large populations because of the tendency to generate a large proportion of inferior lines.Contribution from Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108. Minnesota Agric. Exp. Stn. Scientific Journal Series Paper No. 15,172     

A Comparative Evaluation of Somaclonal,Gamma Ray and EMS Induced Variation in Brassica juncea

Tissue culture induced somaclonal (SC), chemical mutagen EMS (E) and gamma ray (I) induced variation among 269 third generation families in each category generated from a popular variety of Brassica juncea were evaluated for important agronomic traits, namely flowering time, plant height, number of primary and secondary branches, seed yield per plant and per plot. The frequency of somaclonal-lines significantly superior to control was higher than such EMS or irradiated lines, in general. Somaclones also showed higher heritability values. One somaclone, SC-122, is significantly superior to control for upto five traits, simultaneously, demonstrating the practical utility of somaclonal variation in improving productivity.     

Screening for grain polyphenol variants from high-tannin sorghum somaclones

Several hundred somaclones established from plants regenerated from embryogenic callus cultures of six high tannin sorghum lines were screened for variants with altered levels of polyphenols in the grain. Grain from over 6000 plants including the R ₁ (primary), R₂, and R₃ generations were analyzed for total phenols, flavan-4-ols, and proanthocyanidins (condensed tannins). Although many variants which had lost the ability to synthesize chlorophyll were found, none of the somaclones tested had lost or greatly reduced the ability to synthesize any of the polyphenols assayed. However, we did observe statistically significant differences in polyphenol concentration between tissue culture-derived R₁ plants and the parental controls. In the R₂ generation the proportion of somaclones which differed significantly from the parents varied from 47% to 68% depending upon genotype. The average somaclonal variation rate and somaclonal variant frequency estimated in the tested population for the three polyphenol characteristics ranged from 37.3% to 40.7% and 5.3% to 7.8%, respectively. Variants with decreased levels of polyphenols were usually epigenetic and reverted back to normal levels in subsequent generations, but those with increased levels usually persisted after two meiotic cycles, indicating they are heritable. Variants with polyphenol levels increased up to 80% or decreased by 30% were selected for in the R₃ generation.     

Exploitation of wild Cicer species for yield improvement in chickpea

 Chickpea (Cicer arietinum L.) ranks third in the world, and first in the Mediterranean basin, for production among pulses. Despite its importance as a crop and considerable research effort, traditional breeding methods have so far been unable to produce cultivars with a large impact on chickpea production. Interspecific hybridization is known to improve yield in many crops. Therefore, an attempt was made to increase the seed yield in chickpea through the introgression of genes from wild relatives at the International Center for Agricultural Research in the Dry Areas (ICARDA), Syria, from 1987 to 1995. Four crosses, ILC 482 (C. arietinum)×ILWC 179 (C. echinospermum) and ILC 482×ILWC 124 (C. reticulatum) and their reciprocals, were made. Pedigree selection was used to advance the material. Heterosis was recorded visually in F₁s, and single plant measurements for seed yield were recorded in F₂ populations. Promising and uniform progenies were bulked in the F₅ generation. Out of 96 F₆ lines, 22 were selected on the basis of seed yield and other agronomic characters, and evaluated in a replicated trial for seed yield and 14 agronomical, morphological and quality characters. A high level of heterosis was observed in F₁s. Several F₂ plants produced two to three times more seed yield than the best plant from the cultigen. Nine F₇ lines out-yielded the cultigen parent by up to 39%. Over 2 years, 12 lines had a higher yield than the cultigen parent. These lines were not only high yielding but also free of any known undesirable traits from the wild species, such as spreading growth habit, pod dehiscence, and non-uniform maturity. Quality traits, such as seed shape, type, colour, weight, and testa texture, protein content, cooking time and an organoleptic test of a Middle East dish, Homos Bi-Tehineh, were also similar to the cultigen parent. Both C. echinospermum and C. reticulatum contributed towards the increased yield. Received: 11 July 1996 / Accepted: 15 November 1996     

Somaclonal variation in some agronomic and quality characters in wheat

Summary A total of 256 selected lines derived from tissue culture of three hexaploid wheat cultivars were grown in a replicated hill plot experiment to examine somaclonal variation in agronomic characters. The lines were derived by single plant selection for various characters from a total of 100 regenerants, and were either SC₃ or SC₄ generation in the test. Significant variation was found in all the characters measured: height, grain number per spike, kernel weight, yield, total dry weight and harvest index. In most cases, variation could be identified which was both less than and greater than the parental controls. However, there was also an apparent effect of the parent cultivar on the total amount and direction of the variation. For two cultivars, lines could be traced back through the culture phase to individual explant embryos. Many of the original embryos contributed significant variation, and most characters showed significant variation arising from more than one embryo. In the following year, 32 lines selected from the hill plot experiment were grown in larger replicated plots and yield, harvest index and a number of grain and baking quality characters were measured. No lines selected for high yield or harvest index maintained significant improvements over their parental controls. However, significant variation was displayed for many of the quality characters examined. Significant increases in kernel weight, hardness and protein content, and a significant reduction in yellow pigmentation represented potentially useful improvements. Only unfavourable variation was seen in flour yield and in mixograph height, time and breakdown.     

The expression and perpetuation of inherent somatic variation in regenerants from embryogenic cultures of Pennisetum glaucum (L.) R. Br. (pearl millet)

Summary Genetic analysis was conducted on the qualitative and quantitative traits of sexual progeny derived from embryogenic cultures of two inbred lines of Pennisetum glaucum (L.) R. Br. (pearl millet). These lines included a genetically stable inbred of Tift 23 BE and a genetic marker line, derived from Tift 23BE, which bore qualitative genetic markers for a dominant purple plant trait (P) and two recessive traits, early flowering (e₁) and yellow stripe (ys). Tissue culture regenerant populations (R₀) and progeny populations (R₁) produced from these plants by selfing showed no qualitative genetic variation when derived from the genetically stable inbred Tift 23BE. In contrast, stably inherited qualitative variation for a number of genetic markers was observed in R₀, R₁, and R₂ progeny of the genetic marker line. In a population of 1,911 plants regenerated over a 12-month period, 0.02% of the population lost or showed reduced expression of the purple plant trait and 92% of plants were chlorophyll deficient. Plants showing reduction or loss of anthocyanin synthesis also flowered later. None of the purple plants showed any significant variation in flowering time. The incidence of chlorophyll deficiency increased with time in culture, 51 % of the progeny regenerated after 1 month were chlorophyll deficient, while 100% of the plants regnerated after 12 months were chlorophyll deficient. Qualitative variation was also observed in control populations of the genetic marker line where 1 plant in a total of 1,010 lacked purple pigmentation and a total of 6% showed chlorophyll variation in the first generation (S₀). The presence of qualitative variation in controls suggests that the inherent variation present in the original explant was expressed and perpetuated in vitro. Quantitative variation was observed for a number of traits in the first sexual cycle (R₁) of the marker line but did not occur in a subsequent generation, suggesting that this variation was epigenetic.     

In vitro culture provides additional variation for pigeonpea

Summary The present study is an attempt to exploit somaclonal variation for the varietal improvement of pigeonpea [Cajanus cajan (L) Millsp.]. The pigeonpea plants were regenerated from cotyledon explants, and their progeny was screened for variability. The regenerated R1 plants exhibited a spectrum of alterations in floral morphology and architecture that were absent in the control population. The field-sown R2 plants segregated for traits such as flower color, leaf shape, seed size, color and strophiolation, flowering habit, and fertility. Tissue culture produced different mutational events resulting in both dominant and recessive alleles. Significant variation was observed for plant height, seed mass, and damage due to the insect pest Helicoverpa armigera. The R3 plants, obtained from seed of R2 generation selected for traits such as white seed coat, strophiolation, reduced plant height, seed mass and low damage due to Helicoverpa, maintained the traits when compared with the seed-derived control populations. The results indicate a definite gene for white seed coat and the possibility of additional genes for pest tolerance and high seed mass in an adapted background. Submitted as Journal Article No. 1906 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Somaclonal variation in plants regenerated from cultures of soybean

Plants were regenerated from embryogenic and organogenic cultures derived from immature embryos of nine soybean (Glycine max L. Merr.) genotypes and extensive qualitative variation was noted in different regenerated families. Three lethal sectoral albinos were seen in the regenerated plants (R₀). Variants observed in later selfed generations included twin seeds, multiple shoots, dwarfs, abnormal leaf morphology, abnormal leaflet number, wrinkled leaves, chlorophyll deficiency, partial sterility and complete sterility. The frequency of possible mutations ranged from 0 to 4% in R plants as determined by studies of corresponding R₁, R₂, R₃ and R₄ families. No significant differences were seen in the frequencies of possible mutations for embryogenic as compared to organogenic culture derived plants. Chlorophyll deficiency, sterility and wrinkled leaf traits were followed in two or more generations and showed that these traits were inherited stably. The known traits of this nature are controlled by single recessive nuclear genes. Other traits occurred more randomly and not in all generations. The genetic basis of the random variation is not known at the present time. This study indicates that heritable somaclonal variation does occur in tissue culture derived plants of soybean.Abbreviations R₀ Original regenerated plant - R₁ Selfed seeds of R₀ plants - R₂ Selfed seeds of R₁ plants - R₃ Selfed seeds of R₂ plants This research was supported by funds from the Illinois Agricultural Experiment Station and Agrigenetics Inc.     

Selection of somaclonal variants with low-temperature germinability in melon (Cucumis melo L.)

Summary Plants were regenerated from adventitious buds and somatic embryos (R₀) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R₁) of R₀ plants. Among 5,618 R₁ seeds harvested from 23 R₀ plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R₂ seeds harvested from 2 R₁ plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R₁ seeds harvested from 50 R₀ plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R₂ seeds harvested from 17 R₁ plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R₃ seeds were collected from these R₂ plants following self-pollination. Forty-five of the 47 lines (R₃) originated from 10 R₀ plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.     

Selection for high seed oil content in soybean families derived from plants regenerated from protoplasts and tissue cultures

Recurrent selection for high seed oil content was carried out with 2,008 progeny of 28 plants regenerated via embryogenesis, 95 via organogenesis and 25 from protoplasts via organogenesis from five different soybean cultivars. Two lines derived from plants regenerated from the cultivar Jack with small increases in seed oil content emerged after three selection cycles in the field but in both cases the protein content was decreased and the seed yield of one of the lines was also decreased. Apparently somaclonal variation for seed oil content can arise, but on the basis of the decreases in protein and yield found in this study, this small change is not useful for soybean improvement. Received: 22 July 2000 / Accepted: 28 July 2000     

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.     

Identification of minor DNA variations in rice somaclonal variants

Some somaclonal variants derived from a landrace rice variety, Indrayani, were shown to be high yielding and resistant to multiple diseases in previous analysis carried out in our laboratory. An attempt was made to assess the effect of culturing and regeneration of rice plants on DNA variation at microsatellite loci in R₂ progeny of callus-derived rice plants. Different somaclones of the rice line Indrayani differing in yield and disease response (high, low and no change in yield, as compared to the original genotype) were used as genetic material for these analyses. Analysis of microsatellite loci was accomplished by digesting DNA from regenerated rice somaclones and assaying for polymorphisms at microsatellite loci by in-gel hybridization with synthetic oligonucleotide probes such as (GATA)₄, (CAC)₅ and (TG)₁₀. Specific variation at a PCR-amplified locus containing three internal microsatellite repeats (1E₆) using restriction site fingerprinting was also investigated. The locus-specific amplification of a sequence-tagged microsatellite marker followed by digestion with HinfI and Sau3AI restriction endonucleases showed differences in some somaclonal variants. The technique used in this study enables monitoring of DNA changes in successive generations of somaclonal variants as a measure of DNA variability and possibly to identify the regions which are responsible for specific traits. Received: 7 November 1997 / Revision received: 22 April 1997 / Accepted: 5 June 1998     

Somaclonal variability as a source for creation of new varieties of finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

Data on selection and characteristics of somaclonal variants of finger millet (Eleusine coracana (L.) Gaertn.), obtained as a result of regeneration of plants from callus tissue, are presented. Among the genetically stable lines tested, the somaclonal variant SE-7 was the most interesting owing to its acquired important agricultural traits, such as a higher seed and biomass yield, rapid seed germination at lower temperatures, and shortening of the main plant development stages. Data analysis showed that somaclonal variability can serve as a source of initial material for further selection of new finger millet varieties.