Jonathan Kuo

Identification of molecular markers in soybean comparing RFLP, RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.

Plant hormone effect of antibiotics on the transformation efficiency of plant tissues by Agrobacterium tumefaciens cells

The inhibition of bacterial growth by carbenicillin and cefotaxime was demonstrated using three different Agrobacterium tumefaciens strains, LBA4404, C58 and EHA101. LBA4404 was the most sensitive strain to carbenicillin and cefotaxime. Significantly toxic effects were observed when tobacco leaf explants were grown on MS medium containing 250 μg/ml carbenicillin and 1 μg/ml 2,4-dichlorophenoxyacetic acid (2,4-D). The regeneration of tobacco shoots from tobacco leaf explants was decreased as the addition of carbenicillin increased from 250 μg/ml to 2000 μg/ml in MS medium containing 0.5 μg/ml of 6-benzylaminopurine (BA). Interestingly, tobacco leaf explants grown on the MS medium containing carbenicillin alone up to 1000 μg/ml did not show any toxic effects. Moreover, chemical structure comparisons revealed that carbenicillin contains auxin related structures like 2,4-D or α-naphthalenacetic acid (NAA), suggesting that the toxic effects of combinations of carbenicillin and 2,4-D may be due to high auxin activity levels. Further studies demonstrated that by adjusting the levels of carbenicillin and 2,4-D, the transformation efficiency of Arabidopsis thaliana ecotype Columbia and Landsberg was significantly improved from 20% to >90% using Agrobacterium-mediated transformation.

Development of a rat parathyroid hormone 2 receptor antagonist.

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptors endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine(4), tyrosine(5), tryptophan(6), histidine(7)-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.

Amplified fragment length polymorphism analysis of vandaceous orchids

Abstract We investigated the application of the PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), in orchids. The optimal AFLP patterns have been determined using primer combinations of EcoRI +4 and MseI +3 selective nucleotides. The same reproducible AFLP patterns were demonstrated in genomic DNAs isolated both from: (1) different orchid tissues, e.g. leaves and flowers; and (2) orchid flowers collected at different times. Genomic variations among different cultivars of vandaceous orchid hybrids were successfully determined by AFLP analysis. More than 10% of the AFLP bands were polymorphic DNA when siblings, derived from the same original crosses (two cultivars of Aranda Christine, five cultivars of Mokara Willie How), were used. Only 0.3–0.7% of the AFLP patterns were shown to be polymorphic when different cultivars, originating from somatic mutations during meristem culture for massive propagation, were used (two cultivars of Ar. Christine, four cultivars of M. Chark Kuan).