Jhy-Jhu Lin

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.

Regeneration of both plant tissues and transgenic plant tissues using a new plant hormone, 5-bromoindole-3-acetic acid

The present invention describes the use of 5-bromoindole-3-acetic acid (5-B-IAA) as an auxin affecting plant cell growth. The invention relates to the use of 5-B-IAA compositions to affect growth in monocotyledonous as well as in dicotyledonous plants. The invention also describes the use of 5-B-IAA in plant growth affecting compositions for the regeneration of both plant tissues and transgenic plant tissues. Further, the invention provides plant growth affecting compositions comprising 5-B-IAA alone or in a mixture comprising one or more additional plant growth regulators, such as cytokinin, etc.

Optimization of the transformation efficiency of Agrobacterium tumefaciens cells using electroporation

Abstract The procedure for electroporating the recombinant plasmid, pBI121, into Agrobacterium tumefaciens LBA4404 cells has been optimized. A 2-fold increase in the transformation efficiency of A. tumefaciens LBA4404 was observed using a 3-h expression period after electroporation compared to a 1-h expression period. The total number of viable cells remained similar using either a 1-h or a 3-h expression period. No significant difference in the transformation efficiency was shown using either one pulse or multiple pulses at field strengths of 8.9 kv/cm or 16.7 kv/cm. The transformation efficiency obtained by using AB broth, a well characterized medium for growing Agrobacterium cells, as the expression broth was similar to the efficiency using YM broth. Furthermore, using LB broth with 1% mannitol as the expression medium resulted in a transformation efficiency that was only slightly lower than the efficiency using YM broth. These results indicate that mannitol may have contributed to improving the transformation efficiency of A. tumefaciens cells after electroporation.

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).

Reporter Genes and Transient Assays for Plants

The introduction of DNA into plant cells, protoplasts, or intact tissue s has been accomplished by a variety of mechanisms, including electropo-ration, electrofusion, particle bombardment, liposome transfer, the us e of bacterial vectors, polyethylene glycol treatment, and other procedures. As new techniques are developed or modified, it is necessary to use a reliable gene-expression system to monitor DNA uptake, transcription , and translation. A series of DNA plasmids containing reporter genes encoding readily assayed enzymes are available for this purpose. Several reporter gene systems have been used in experiments to transform plant s and to perform transient assays with plant material. In_ general, thes e reporter genes encode enzymes whose activities can be detected through assays and stains, thus facilitating the identification of transformed cell s and quantification of the transformation process. Reporter genes also provide a method for analyzing regulatory characteristics of promoters , such as promoter strength and tissue specificity, when the promoter fro m a gene of interest is coupled to the reporter gene. Reporter gene constructs are comprised of a reporter gene togethe r with active promoter and terminator regions cloned into a plasmid vector. Sometimes these constructs lack promoter regions, so different pro