Expanding the genetic variability of flatpea using tissue culture, mutagenesis, and intercrossing techniques

Flatpea (Lathyrgs sylvestris L.) is a potentially valuable forage legume but contains high levels of 2,4-diaminobutyric acid (DABA), a compound that can have adverse effects on some animals, including rats and poultry. To increase genetic variability in foliar DABA content and other traits of interest, three approaches were utilized: (1) regeneration of flatpea plants from tissue culture to produce potential somaclonal variants, (2) seed irradiation and screening of potentially mutated progeny, and (3) intercrossing among flatpea accessions. Low-frequency whole plant regeneration of flatpea was obtained from hypocotyl-derived callus cultures. Auxin concentrations above 5.0 µM resulted primarily in root formation without shoots. Conditions for optimum whole plant regeneration were as follows: callus was initiated on a modified Schenk-Hildebrandt low-ammonium medium containing 5.0 µM IAA (indole acetic acid) and 2.5 µM zeatin for callus initiation; after two subcultures, calli were transferred to a regeneration medium containing 0.1 µM IAA and 1.0 µM zeatin. Calli and regenerated root tissue contained 47 and 381 as much DABA as leaf tissue from 'Lathco' flatpea plants, respectively.

Initial tests established that the effective range of gamma irradiation for seed treatment was between 10.0 and 17.5 kR. Within this range, reduction in percentage of both seedling height and plant survival was a linear function of dose. Individual M₂ plants that contained reduced levels of DABA were identified. No significant trend in DABA concentration with increasing gamma irradiation was apparent.

Flatpea pollination methods were evaluated prior to utilization of intercrossing for inducing genetic variability. Appropriate flower stages for emasculation were determined by in vitro germination of pollen. Lines that produced high numbers of seeds per pollination were identified by crossing in all possible combinations among seven flatpea accessions. Cross-pollinations resulted in significantly higher seed set than selfing. Pollination using caged honeybees was the most efficient method of hybridization. Greater range and coefficient of variation in DABA concentration were observed for flatpea accessions than for intercrossed progeny produced by honeybee pollination.