Improvement of embryogenic callus induction and shoot regeneration of buffalograss by silver nitrate

Addition of the ethylene antagonist, silver nitrate (AgNO₃), into callus induction medium significantly enhanced embryogenic callus production (both induction frequency and callus growth) of field-collected male immature inflorescence cultures of buffalograss NE84-45-3 and 'Texoka'. No stimulatory effect of AgNO₃ was observed on embryogenic callus induction for female immature inflorescence culture of a female genotype `609' and `Texoka'. Calli initiated on AgNO₃-containing media had more shoot-regenerating calli than those initiated on AgNO₃-free media, when they were transferred to the regeneration media. Benzyladenine at 2.2 μM gave the best response for regeneration, regardless of the callus source. Although average number of shoots regenerated per callus was lower for calli initiated on AgNO₃-containing media, total number of shoots regenerated was higher. The stimulatory effect, however, was environment and genotype dependent. While the addition of AgNO₃ significantly stimulated embryogenic callus induction of NE84-45-3 immature inflorescences collected in Fall 1995 and May 1997, it only slightly increased the embryogenic callus induction frequencies in May 1996 when rainy conditions occurred. For male inflorescences of `Texoka' collected in early May, AgNO₃ significantly enhanced embryogenic callus production consistently over the two-year period (1996, 1997). Published as Journal Series No. 1351, Agricultural Research Division, University of Nebraska. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation, characterization and evolutionary analysis of resistance gene analogs in annual ryegrass, perennial ryegrass and their hybrid

Recently, a number of disease-resistance genes related to a diverse range of pathogens were isolated from a wide variety of plant species. The majority of plant disease-resistance genes encoded a nucleotide-binding site (NBS) domain. According to the comparisons of the NBS domain of cloned R -genes, it has shown highly conserved amino acid motifs in this structure, which made it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate primers. We have designed three pairs of degenerate primers based on two conserved motifs in the NBS domain of resistance proteins encoded by R -genes to amplify genomic sequences from ryegrass ( Lolium sp.). Sixteen NBS-like RGAs were isolated from turf and forage type grasses. The sequence analysis of these RGAs revealed that there existed a high similarity (up to 85%) between RGA sequences among ryegrass species and other plants. The alignment of the predicted amino acid sequences of RGAs showed that ryegrass RGAs contained four conserved motifs (P-Loop, kinase-2, kinase-3a, GLPL) present in other known plant NBS-leucine rich repeat resistance genes. These ryegrass RGAs all belonged to non-toll and interleukin-1 receptor subclass. Phylogenetic analysis of ryegrass RGAs and other cloned R -genes indicated that gene mutation was the predominant source of gene variations, and the sequence polymorphism was due to purifying selection rather than diversifying selection. We further analyzed the source of gene variation in other monocots, rice, barley, wheat, and maize based on the data published before. Our analysis indicated that the source of RGA diversity in these monocots was the same as in ryegrass. Thus, monocots were probably the same as dicots in the source of RGA diversity. Ryegrass RGAs in the present paper represented a large group of resistance gene homologs in monocots. We discussed the origin and the evolution of R -genes in grass species.