Segregation of genetic markers among plants regenerated from cultured anthers of broccoli (Brassica oleracea var. 'italica').

An experiment was conducted to determine critical factors in the recovery of embryos from cultured anthers of broccoli (Brassica oleracea var. 'italica') and to unambiguously distinguish whether embryos were of gametophytic origin. Among factors tested, genotype, genotype x anther developmental stage, and method of anther culture had a distinct impact on embryo recovery, whereas length of anther exposure to the culture medium did not. However, extreme heterogeneity of embryo emergence within and among replications precluded statistical contrasts. Among 762 plants derived from embryos of four independent cultivars, only one was determined to be of sporophytic origin by use of heterozygous codominant isozyme markers. Two of the cultivars tested were heterozygous at two or more loci. While segregation among loci was consistent with previously published linkage data, segregation of alleles was consistently non-random. In all of seven separate cases involving four cultivars, a significant over-representation of the fast-migrating class was observed. It appears, therefore, that populations of plants derived from microspores within cultured anthers of broccoli do not necessarily represent a random gametic array, and that care must be exercised in breeding and genetic applications.

Spontaneous electrophoretic and chromosomal variability in callus cultures and regenerated plants of celery.

Reciprocal sexual crosses were performed to produce plants heterozygous at four nuclear loci, two of which coded for phosphoglucomutase (PGM) and shikimic acid dehydrogenase (SDH) isozymes and could be visualized in petiole callus tissue. Clones of one reciprocal always exhibited the heterozygous phenotype at both isozyme loci, but 25.8% of clones of the other exhibited loss of the fast migrating electromorph at the PGM locus while remaining 100% heterozygous at the SDH locus. No reversion to heterozygosity was observed and the variant phenotype was transmitted to regenerated plantlets. All regenerates were karyologically and developmentally abnormal, and studies of the inheritance of this variability were therefore not possible. Epigenetic change, point mutation, and somatic recombination were ruled out as possible causes due to gross incongruencies with the phenomenon. No consistent differences between normal and variant clones were detected with respect to chromosome number, structure, and anomalous disjunction.

Chloroplast DNA evolution and the origin of amphidiploid Brassica species.

The origin and evolution of a hybrid species complex in the genus Brassica (cabbage, turnip, mustard, rapeseed oil) has been explored through mutational analysis of the maternally inherited chloroplast genome. A detailed chloroplast DNA phylogeny enables identification of the maternal parent for most of the amphidiploids examined and permits quantitative resolution of the relative time of hybridization as well as the relative divergence of the diploid parents. Contradictory chloroplast and nuclear phylogenies obtained for two accessions of the amphidiploid B. napus (rapeseed oil) lead to the hypothesis that introgressive hybridization has also figured in their recent evolution.

Cytogenetic analysis of plants regenerated from colchicine-treated callus cultures of an interspecific Hordeum hybrid.

Hybrids of Hordeum vulgare (HV) x H. jubatum (HJ) were synthesized for purposes of introgressive breeding, but were sterile and the recovery of pure diploid tillers by colchicine applications in vivo was difficult. Plant regeneration from colchicine-treated callus cultures of the hybrid (HV x HJ) was investigated as a means to produce high numbers of pure diploid, fertile intermediates. 10 of 50 plants regenerated in this manner exhibited variable chromosome numbers with means of approximately 37 (expected diploid number = 42). Cytological examinations of microsporogenesis in all such plants revealed a high incidence of bivalent formation at metaphase I (as compared to nearly complete asynapsis in the F1), but spindle and chromosome abnormalities in later meiotic stages led to complete sterility. Approximately 40% of HJ plants regenerated from colchicine-treated calli appeared to be pure tetraploids of high fertility. These techniques are hence useful for high frequency production of diploid or polyploid plants.

Chromosomal variability in tissue cultures and regenerated plants of Hordeum.

Spontaneous polyploidy, aneuploidy, and chromosomal rearrangements were observed in callus and suspension cultures of Hordeum vulgare, H. jubatum, and their interspecific hybrid. The extent to which each class of chromosomal variability was present in a culture depended upon differentiated state, age, and history. Cytological and isozymic analysis of subdivided callus cultures revealed spatial segregation of chromosomal variability. Cytogenetic analyses were performed to determine the expression of this in vitro chromosomal variability in corresponding regenerated plant tissues. A complete loss of polyploidy and a decrease in aneuploidy and chromosomal rearrangements were observed. Analyses of specific isozyme activities in regenerates suggested that a quantitative segregation of H. vulgare and H. jubatum genomes had occurred in tissue cultures of their interspecific hybrid. Possible uses of in vitro chromosomal variability for plant breeding and genetical studies are discussed.