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Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive.

Transgenics for the expression of beta-carotene biosynthetic pathway in the endosperm were developed in indica rice background by introducing phytoene synthase (psy) and phytoene desaturase (crtI) genes through Agrobacterium-mediated transformation, employing non-antibiotic positive selectable marker phosphomannose isomerase (pmi). Twenty-seven transgenic lines were characterized for the structural organization of T-DNA inserts and the expression of transgenes in terms of total carotenoid and beta-carotene accumulation in the endosperm. Ten lines were also studied for the inheritance of transgenic loci to the T(1) progenies. Copy number and sites of integration of the transgenes ranged from one to four. Almost 50% of the transgenic lines showed rearrangement of T-DNA inserts. However, most of the rearrangements occurred in the crtI expression cassette which is adjacent to the right T-DNA border. Differences in copy numbers of psy and crtI were also observed indicating partial T-DNA integration. Beyond T-DNA border transfer was also detected in 25% of the lines. Fifty percent of the lines studied showed single Mendelian locus inheritance, while two lines showed bi-locus inheritance in the T(1) progenies. Some of the lines segregating in 3:1 ratio showed two sites of integration on restriction digestion analysis indicating that the T-DNA insertion sites were tightly linked. Three transgenic lines showed nonparental types in the segregating progenies, indicating unstable transgenic locus. Evidences from the HPLC analysis showed that multiple copies of transgenes had a cumulative effect on the accumulation of carotenoid in the endosperm. T(1) progenies, in general, accumulated more carotenoids than their respective parents, the highest being 6.77 mug/g of polished seeds. High variation in the carotenoid accumulation was observed within the T(1) progenies which could be attributed to the variation in the structural organization and expression of transgenes, minor variations in the genetic background within the progeny plants, or differences in the plant microenvironments. The study identified lines worthy of further multiplication and breeding based on transgene structural integrity in the segregating progeny and high expression levels in terms of the beta-carotene accumulation.

Calli of two genotypes of barley, 'Dissa' and W 193, were used for selection of resistance against fusaric acid, a pathotoxin of Fusanitm. Callus was initiated from 7-to 10 days old immature embryos. 1000 calli of the 'Dissa' and 500 of the W 193 genotypes were grown for 4 selection cycles on medium with 0.8 mM fusaric acid. In the first selection cycle, about 80 % of the calli were killed; after the 4 selection cycles, 8 to 11 % resistant calli were obtained and plants were regenerated. Resistant calli maintained on non-toxic medium showed retention of resistance ability after 3 months of sub~culttaring. Plants could be regenerated from the surviving calli and testing by leaf bioassay revealed that many were resistant to the same toxin concentration employed for callus selection (100 %), while some were only resistant up to a concentration of 75 %.

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