Factors affecting the establishment and maintenance of embryogenic callus and suspension cultures of wheat (Triticum aestivum L.).

Improved suspension cell culture systems are needed to facilitate the application of recombinant DNA technology for wheat germplasm enhancement. This study evaluated three wheat (Triticum aestivum L.) cultivars, and the effects of medium basal salts, 2,4-D, sucrose, and L-proline concentrations on the establishment of rapidly growing and highly embryogenic callus and suspension cultures. Percent embryogenic calli was visually estimated and verified with light and scanning electron microscopy. The most highly embryogenic callus was produced by cultivar Bobwhite on medium with MS basal salts, 5.6 µ M 2,4-D, 58 mM sucrose, and zero proline. The suspension cultures that produced the greatest number of regenerated plants utilized callus tissue produced on solid medium with MS basal salts, 87 mM sucrose, 9 µM 2,4-D, and no proline.

Cloning and expression of genes required for coronamic Acid (2-ethyl-1-aminocyclopropane 1-carboxylic Acid), an intermediate in the biosynthesis of the phytotoxin coronatine.

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.

Genetic analysis of a grass dwarf mutation induced by wheat callus culture.

Genetic variation induced by passage through tissue culture (somaclonal variation) has been characterized for many agronomic traits of wheat. The study presented here was conducted to genetically and phenotypically characterize a mutation influencing plant height that was induced by wheat callus culture. Dwarf plants were identified in the progeny of a tall plant regenerated from immature embryo-derived callus tissue of the hard red winter wheat 'TAM 105'. The dwarfs are significantly shorter, later in heading, and have a greater number of tillers, fewer seeds per spike, lower grain yield per plant, and lower floret fertility than 'TAM 105'. The dwarfs also exhibit branching at the aerial nodes when grown under cool temperatures (<20°C) and short daylengths (<12h). We hypothesize that a single, partially dominant gene which acts in a complementary manner with the grass-dwarf gene D1 is responsible for this phenotype. Based on phenotype and the dominance relationship between mutant and wild-type alleles, we hypothesize that the mutation is a new allele at either the D2 or D4 grass-dwarfism locus. The utilization of genotypes lacking any of the grass-dwarfism alleles would greatly reduce the chance of recovering these undesirable genotypes by mutations arising during tissue culture. It is also important to recognize the grass-dwarf phenotype. If transgenic plants, somatic hybrids, or regenerants from in vitro selection strategies have a grass-dwarf phenotype, they can be induced to enter reproductive development by long daylengths (>14 h) and high temperatures (>26°C).