Androgenetic Lines Research Articles

Mitochondrial and nuclear inheritance in an androgenetic line of rainbow trout, Oncorhynchus mykiss

Mitochondrial and nuclear inheritance of clonal rainbow trout produced by androgenesis were evaluated using DNA sequencing and amplified fragment length polymorphism (AFLP) analysis. All individuals were propagated using sperm from a single OSU clonal line sex inversed female crossed with multiple egg sources whose nuclear material was destroyed using Cobalt 60 gamma radiation. A heat shock was administered to developing haploid embryos to prevent the first cleavage and restore diploidy. Sequencing of 4713 continuous base pairs was performed on four rainbow trout and one cutthroat trout mitochondrial haplotypes spanning the ND 5 protein subunit of Complex I through the control region. Total sequence variation between haplotypes ranged from 0.34% to 6.14% with rainbow trout haplotype variation between 0.34% and 0.85%. No evidence was found to indicate point mutations associated with irradiation. DNA sequence for coded proteins was converted to amino acid sequence using the standard mammalian mitochondrial code to determine conservative and non-conservative amino acid substitutions. Total amino acid variation ranged between 0.60% and 3.10% with rainbow trout haplotype variation from 0.60% to 1.12% with non-conservative amino acid substitutions present between all mitochondrial haplotypes. AFLP analysis was performed on 119 clonal individuals to determine levels of nuclear genetic variation within the clonal line. Twelve primer sets revealed nine polymorphic bands among the 493 readable bands. Seventy individuals showed between one and five polymorphic loci with nuclear AFLP band variation ranging from 0.0% to 1.01% among all possible pairs. DNA from 12 individuals containing variable loci was re-extracted and AFLP amplifications repeated. Repeated primer sets showed little variation between extractions with polymorphic loci present in all repeated primer sets. AFLP primer sets having polymorphic loci were run using OSU by Arlee doubled haploids to determine map locations on the published linkage map of rainbow trout. Three of the nine polymorphic loci had differential polymorphism between the parents to allow mapping. Two loci mapped to linkage group II 46.2 and 32.6 centimorgans away from the putative centromere while the third locus was unassignable. The large distances between the two mapped loci and the putative centromere, along with the inability to assign the third locus, support the possibility that identified variable loci result from tetrasomic inheritance. The type of tetrasomic inheritance present is the result of distal crossover between homologous chromosomes during spermatogenesis, secondary tetrasomy. The low level of genetic variation found within the OSU clonal line is comparable to reported genetic variation within inbred lines of mice, zebrafish, and other species.

Acid phosphatase and leucine aminopeptidase isozymes as biochemical markers of homogeneity in oil seed rape androgenetic lines

Extracts of cotyledons of Brassica napus plants (seed progenies of doubled haploid plants) were separated by electrophoresis on polyacrylamide gels and stained for acid phosphatase (ACP-E.C. 3.1.3.2.) and leucine aminopeptidase (LAP-E.C. 3.4.11.1.) enzymes to investigate the possibility of utilising isozymes as markers of homogeneity (purity) of plant populations. One zone of activity for acid phosphatase and two zones of activity for leucine aminopeptidase were identified on gels, some variation in isozyme patterns occurred in several androgenetic lines. This method is appropriate and consistent for testing the homogeneity of breeding lines-progenies of double haploid (D.H.) plants.

Several nuclear genes control both male sterility and mitochondrial protein synthesis inNicotiana sylvestris protoclones

Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines of Nicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes, ms1, ms2 and ms3, while no changes can be seen in the mitochondrial genome. However, differences were found between the in organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.

Yield, Oil Content and Fatty Acid Composition Evaluated in Androgenetic Plants in Brassica juncea

AbstractHomozygous diploids were obtained through anther culture in Indian mustard, Brassier juncea, cultivar TM‐4, Seeds could be harvested from 97 such plants in the A; generation. The androgenetic lines were further selected and the yield and yield components were compared in the A4 and A5 generations, Androgenetic lines showed variations for most of the characters studied including seed oil content and fatty acid composition. However, it was possible to recover seven lines out of 25 which were equal in seed yield and three equal in oil content compared TO the anther donor parent TM‐4.

Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). II. Evaluation of first- and second-cycle doubled haploids

First- and second-cycle doubled haploids of burley tobacco (Nicotiana tabacum L.) were examined to ascertain the significance of source genotype on the performance of anther-derived material and to determine the magnitude of changes from the haploid-production and chromosome-doubling stages. The anther-derived lines evaluated in this study did not exhibit siginficant yield reductions. However, significant interactions between the cycle comparison and the source genotypes indicated that source genotype can affect the degree of vigor reduction observed. Significant variation was also observed among the second-cycle doubled haploids examined in this study. Partitioning of this variation indicates that most of it is due to alteration present at the haploid-production step instead of the chromosome-doubling step. In general, the variation present in the second-cycle doubled haploids was distributed around the values observed for the source first-cycle doubled haploids. Even though unexpected variation is often observed in anther-derived materials, these results suggest that reduced vigor is not intrinsically associated with androgenetic lines of burley tobacco. The most plausible explanations for the variation and vigor losses in anther-derived lines propose that natural phenomena, given the opportunity for expression via anther culture, are responsible. Spontaneous variants present in the gametic pool of inbreds or first-cycle doubled haploids offer one possible explanation for the performance observed in anther-derived lines of tobacco.

Diallel analysis of androgenetic plant production in hexaploid Triticale (X. triticosecale, Wittmack)

Studies were made on the genetic determination of androgenetic plant yield and its two components: embryo production and green plant regeneration. This involved the analysis of a complete 7×7 diallel cross of 4 androgenetic lines and 3 lines obtained by pedigree selection, one of them having the Triticum timopheevi cytoplasm. The three traits analysed are both heritable and environmentally influenced (by season and culture medium composition). The analysis of embryo production shows a mainly nuclear inheritance, with predominantly additive gene action, but also a favourable effect of Triticum timopheevi cytoplasm. Green plant regeneration has a more complex genetic determination, with additive as well as non-additive gene action and cytoplasmic influences. Hybrids appear superior to inbred lines for embryogenesis and green plant yield, but not for green plant regeneration. Androgenetic lines used as parents did not show superiority over other parents either in their own value or in the transmission of androgenetic abilities. Genetic improvement seems to be possible by recombination in progenies of hybrids between lines having complementary abilities.

Alterations of leaf characteristics and peroxidase activity in diploid androgenetic lines of Nicotiana sylvestris spegaz. et comes

Two series of diploid androgenetic lines of Nicotiana sylvestris and their original line were investigated for leaf characteristics and peroxidase activity. The first series included two androgenetic lines (‘flat’ and ‘crumpled’ phenotypes named after the leaf aspect) derived from the same doubled haploid regenerated plant. Compared to the original line, the leaves were less developed, although cell size and peroxidase activity was higher. The second series comprised androgenetic lines obtained by consecutive cycles of androgenesis. These plants had smaller leaves, larger cells, and increasing peroxidase activity through consecutive cycles of androgenesis. The ‘crumpled’ phenotype was also more and more marked with each cycle. Results are discussed in relation to other analyses performed on these plants (inheritance, DNA analysis, DNA content).

Genetic variability induced in Nicotiana sylvestris by protoplast culture

Plants were regenerated from axenic plantlets by mesophyll protoplast culture, without mutagenic treatment. Two different lines of Nicotiana sylvestris were used: an original line, and a diploid androgenetic line derived from it. The regenerated plants were either diploid and phenotypically similar to their respective protoplast source line, or tetraploid. Genetic studies carried out on several diploid regenerated plants revealed genetic variability. Eight of 13 selfed progenies of plants regenerated from the original line, and 1 of 8 selfed progenies of plants regenerated from the androgenetic line, produced new mutant phenotypes never observed in the protoplast source lines. Two plants regenerated from the same protoplast-derived callus produced different mutations. Selfed progenies without a recognizable mutant phenotype were also different from their respective protoplast source line for quantitative characters; protoplast culture induced a depressive effect on the size of plants derived from protoplasts at younger and older stages of development. The origin of this depression and of the mutations is discussed.

Genetic evidence for the hybrid nature of somatic hybrids from Datura innoxia Mill.

The hybrid nature of tetraploid somatic hybrids of two genetically different chlorophyll-deficient mutants from Datura innoxia Mill. was demonstrated with the aid of anther culture. Green and chlorophyll-deficient androgenetic lines could be regenerated from the pollen grains.