William R. Scowcroft

Heritable somaclonal variation in wheat

SummaryEfficient tissue culture and regeneration methods were established using immature wheat embryos as expiants. Genotype differences in culturability were evident, and from the ten accessions most amenable to culture, a total of 2,846 plants were regenerated. Extensive somaclonal variation for morphological and biochemical traits was observed among 142 regenerants of a Mexican breeding line, ‘Yaqui 50E’, and their progeny. Variant characters included height, awns, tiller number, grain colour, heading date, waxiness, glume colour, gliadin proteins and α-amylase regulation. The variant characters were heritable through two seed generations and included traits under both simple and quantitative genetic control. Segregation data suggested that mutations both from dominance to recessiveness, and from recessiveness to dominance, had occurred. Most mutations in the primary regenerants were in the heterozygous state but some were true-breeding and presumed to be homozygous. Chromosome loss or addition did not account for the variation and none of the variant phenotypes was observed in over 400 plants from the parental seed source. The distinctive parental gliadin pattern was maintained in the somaclones thus excluding seed contamination or cross-pollination as a source of the variation.

Somaclonal Variation and Crop Improvement

There has been a remarkable escalation of interest in tissue-culture derived plant variation (somaclonal variation) in the last few years. Earlier authors were aware to some extent that abnormalities could result from a tissue culture cycle (42,65,67). However it is only more recently that the thought has been seriously entertained that some of this variation may be useful for varietal improvement (95,100). It is our contention that the lateness of this realization was a consequence of the fact that so few tissue culturists were engaged in careful analysis of the regenerated plants and also failed to see cell culture manipulation in a genetic context. We were committed to the idea of variation but envisaged it would only happen after specific manipulations (somatic hybridization or DNA-mediated transformation). Underlying the development of these important means of modifying the plant genome was the presupposition that tissue culture was cloning.

Molecular analysis of a somaclonal mutant of maize alcohol dehydrogenase

SummaryPlants regenerated from tissue cultures of maize were screened for variants of ADH1 and ADH2. Root extracts of 645 primary regenerant plants were tested, and one stable mutant of Adh1 was detected. The mutant gene (Adh1-Usv) produces a functional enzyme with a slower electrophoretic mobility than that of the progenitor Adh1-S allele, and is stably transmitted to progeny. The mutant was not present among four other plants derived from the same immature embryo, and therefore arose as a consequence of the culture procedure. The gene of Adh1-Usv was cloned and sequenced. A single base change in exon 6 was the only alteration found in the gene sequence. This would translate in the polypeptide sequence to a valine residue substituting for a glutamic acid residue, resulting in the loss of a negative charge and the production of a protein with slower electrophoretic mobility.

Somaclonal variation in wheat: genetic and cytogenetic characterisation of alcohol dehydrogenase 1 mutants.

SummaryThe progeny of 551 regenerants of the hexaploid wheat cultivar ‘Millewa’ were analysed for somaclonal mutants at the threeAdh-1 loci in hexaploid wheat. Seventeen regenerants gave rise to progeny having altered ADH1 zymograms. Progeny with altered zymograms in 13 of these regenerants were aneuploid. The remaining 4 regenerants gave rise to euploid progeny with altered ADH1 zymograms. The genetics of three of these somaclonal mutants is described in detail. These regenerants were interpreted to possess a 4Aα isochromosome, a 3BS/4Aα translocation and a 7BS/4Aα translocation, respectively.

Chloroplast DNA variation in isonuclear male-sterile lines of Nicotiana

SummaryChloroplast DNAs of six isonuclear malesterile tobacco lines and their respective parental species were analysed with the restriction endonuclease, EcoR1. Four of the lines had the same fragmentation pattern as their respective maternal species. Two lines had a pattern which was different to either parental species. The results show that nucleotide changes can occur in chloroplast DNA of isonuclear male-sterile lines, and are discussed in relation to the possible involvement of chloroplast DNA in cytoplasmic male-sterility in tobacco.

The induction of nitrogenase activity in Rhizobium by non-legume plant cells

SummaryNitrogen fixation was induced in a strain of “cowpea” rhizobia, 32Hl, when it was grown in association with cell cultures of the non-legume, tobacco (Nicotiana tabacum). Rhizobia grown alone on the various media examined did not show nitrogenase activity, indicating the involvement of particular plant metabolites in nitrogenase induction. Nitrogenase activity, as measured by C2H2 reduction, was maximized at an O2 concentration of 20% and at an assay temperature of 30°C, the conditions under which the plant cell-rhizobia associations developed. Glutamine, as a nitrogen source, could be replaced by other organic nitrogen sources, but NH4+ and NO3- repressed nitrogenase activity. Nitrogenase activity induced in rhizobia when cultured adjacent to, but not in contact with, the plant cells could be stimulated by providing succinate in the medium. At least 12 other strains of rhizobia also reduced C2H2 in association with tobacco cells; the highest levels of activity were found among cowpea strains.

Nitrogenase activity in cultured Rhizobium sp. strain 32H1

Nutritional and physical conditions affecting nitrogenase activity in the strain of “cowpea” rhizobia, 32H1, were examined using cultures grown on agar medium. Arabinose in the basic medium (CS7) could be replaced by ribose, xylose, or glycerol, but mannitol, glucose, sucrose, or galactose only supported low nitrogenase (C2H2 reduction) activity. Succinate could be replaced by pyruvate, fumarate, malate, or 2-oxoglutarate, but without any carboxylic acid, nitrogenase activity was low or undetectable unless a high level of arabinose was provided. Inositol was not essential. Several nitrogen sources could replace glutamine including glutamate, urea, (NH4)2SO4 and asparagine.The maximum nitrogenase activity of cultures grown in air at 30°C was observed under assay conditions of pO2=0.20–0.25 atm and 30°C incubation. Greatest activity occurred after a period of rapid bacterial growth, when viable cell count was relatively constant.Compared with results obtained on the CS7 medium, nitrogenase activity could be substantially increased and/or sustained for longer periods of time by using 12.5 mM succinate and 100 mM arabinose, by increasing phosphate concentration from 2 to 30–50 mM, or by culturing the bacteria at 25°C.

Nitrogen fixation in cultured cowpea rhizobia: Inhibition and regulation of nitrogenase activity

Abstract Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by NH 4 + but this was relieved by increased O2 tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by NH 4 + , glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or NH 4 + inhibited cultures. These results indicate that NH 4 + inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis.

Protoplasts and Variation from Culture

There is now ample evidence of the occurrence of genetic variation in plants following a tissue or cell culture cycle. Acknowledgement of the existence of this phenomenon has been increasing over a number of years as illustrated in the reviews of Morel (1971), Murashige (1974), Green (1977), Skirvin (1978), Oono (1978), Thomas et al. (1979), Shepard et al. (1980), Larkin and Scowcroft (1981), and Larkin and Scowcroft (1983a). Many questions arise concerning this so-called somaclonal variation — when does it occur, what are the molecular mechanisms, what is the spectrum of genetic changes, are there mutational hot spots, etc?