The effects of the amino acid inhibitors glyphosate (GLY) and metsulfuron methyl (MSM) on pea callus growth was investigated. The enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthese, involved in the production of phenyalanine, tyrosine and tryptophan, is the target of GLY. Acetohydroxyacid synthase (AHAS) involved in valine, leucine and isoleucine production is the target of MSM.
GLY affected the fresh weight (FW) of pea callus in the range of 1 to 300 uM with an ISO level of 45 uM and an I90 level of 300 uM. Similar growth curves were obtained with MSM in the range 0.01 to 3 nm, the ISO levels was 0.83 nM MSM after 4 weeks and the I90 levels was 2 to 3 um MSM.
The shikimate pathway enzymes in GLY tolerant pea callus tissue were studied. Shikimate dehydrogenase activity was present in all samples, specific activity was 10 to 400 fold greater than EPSP synthase activity. It is concluded from these data that GLY tolerance is not due to overproduction of EPSP synthase.
The effects of the 3 herbicindes on membrane permeability were studied. All herbicides were seen to affect the membrane to some extent, MSM being the most toxic. Diflufenican (DFF) did not affect growth of either heterotrophic or photomiotrophic cell cultures of 1 uM although the tissues were visibly bleached. In terms of chlorophyll content the 50% inhibitian (I50) level was estimated to be 300 nM DFF. Reduction of carotenoids, chlorophylls and radioactive carbon dioxide uptake together with changes in the ultrastructure due to the inhibition of phytoene desaturase by the herbicide were demonstrated, the effects being enhanced in photonixotrophic callus. Differences in activity of DFF can be explained by the higher photosynthetic activity of photomixotrophine callus, causing the formation of highly destructive oxygen species. These would normally be quenched by carotenoids in uninhibited callus. Increased sucrose concentrations reduced photosynthesis and thus the activity of DFF. However, higher sucrose concentrations led to higher carotenoid contents, thus obscuring any herbicidal effect on carotenoid and chlorophyll content.
The ultrastructural study revealed the appearance of plastoglobuli, disappearance of thylakoids and the formation of lobes on nuclei, caused by DFF.
The effects of the amino acid inhibitors glycphosate (GLY) and metasulfuron methyl (MSM) on pea callus growth was investigated.
Isolation of viable protoplasts of 8 cultivars of field pea (Pisum sativum L) was achieved using 1% Driselase in an enzyme mixture. The yield of protoplasts increased using a buffer with 0.25 M sorbitol, 0.25 M mannitol and 1 mM L-arginine. Protoplast yield was genotype dependent and varied from 1.75E3 to 1.35E6 protein/g plant material. Protoplast viability varied from 80 to 90%. The culture of the protoplasts was not successful despite using 2 different basal media and 5 culture methods. The protoplasts showed budding characteristics within 24 h and died within 7 days.
Using the cultivar Belman, optimum somatic embryo production was achieved in vitro using nodal explants of shoots from a cotyledon-free embryo. Embryogenesis was genotype dependent since from the 16 cultivars screened, 3 cultivars (Alaska, Rondo and Ascona) did not show any embryo production. Replacement of 3% sucrose by 1.5% maltose and 1.5% sucrose enhanced the number of embryos produced per producing callus. Addition of proline (0 to 150 mM) improved the somatic embryo production for the cultivar Stehgolt, but using Belmanembryogenesis was not affected and root formation was observed.
Embryogenesis frequency in 5 cultivars (Solara, Belman, Progreta, Stehgolt and Maro) was not affected by the herbicides diflufenican (0 to 1 uM) and metsulfuron methyl (0 to 3 nM), but glyphosate (0 to 0.5 mM) reduced the yield of somatic embryos and differences in tolerance in vitro between the cultivars were observed.
In total 245 plants were regenerated. The plants were able to flower, form pods and produce viable seed. Whole plants from 8 cultivars have been regenerated from the 16 cultivars assessed. The plants showed growth characteristics caused by in vitro culture such as short height, short internode length and small leaf size, but these characteristics disappeared in the M1 generation.
Applications of glyphosate (0 to 750 g/ha) in vivo showed differences in tolerance between 5 cultivars (Stehgo lt, Maro, Belman, Progreta and Solara) used as assessed by reductions in shoot growth, fresh and dry weight. Metsulfuron methyl (0 to 50 g/ha) reduce the growth of the cultivars similarly. The cultivars however, were all equally tolerant following the application of diflufenican (0 to 4 kg/ha).
Protocols were developed for in vitro selection for resistance to foot rot fungus in the pea. Since cell suspension cultures were found to be unstable, plant material was grown in Petri dishes. Cultural conditions were optimized so that disease reactions of plantlets and callus tissue for partially resistant and susceptible genotypes of field pea (Pisum sativum) could be differentiated when inoculated with the foot rot pathogen Fusarium solani f sp pisi. Disease was assessed using both semiquantitative (disease score and fungal diameter) and quantitative methods (ergosterol assay). After optimization of inoculum concentration and incubation period it was possible to differentiate the disease reactions of plantlets using disease scores and amount of ergosterol in the tissues.
The infection potential of the pathogen was dependent on host plant resistance, inoculum concentration, incubation period, temperature and the auxin added to the media and were important in allowing differentiation of callus tissue cultures. These differentiation of callus tissue cultures. These differences were visible by measurement of the diameter of fungal growth on the callus and the amount of ergosterol in the tissues, but not with disease scores.
The plantlet test was used successfully with other foot rot pathogens Phoma medicaginis var pinodella and Aphanomyces euteiches allowing differentiation of the disease reactions of a partially resistant and susceptible genotype using disease scores. The ergosterol assay could be used to quantify infection caused by Phoma medicaginis var pinodella, but this sterol was detectable in mycelium of Aphanomyces euteiches. In this fungus the presence of cholesterol was detected which may be used to quantify infection.
Funding SchemeCSC - Cost-sharing contracts
6708 PB Wageningen
KA6 5HW Ayr