Shigeo Nishimura

Metabolic Alterations in Organic Acids and γ–Aminobutyric Acid in Developing Tomato (Solanum lycopersicum L.) Fruits

Salt stress improves the quality of tomato fruits. To clarify the mechanism(s) underlying this phenomenon, we investigated metabolic alterations in tomato fruits exposed to 160 mM salt, focusing on metabolism of organic acids related to the tricarboxylic acid (TCA) cycle and gamma-aminobutyric acid (GABA). Quantitative analyses revealed that most amino acids increased in response to salt stress throughout fruit development, and the effect of the stress was greater in the pericarp than in the columella, whereas organic acids did not show a remarkable tendency to salt stress. The transcript levels of 20 genes encoding enzymes of the TCA cycle and peripheral pathways were also analyzed in salt-stressed fruit. Genes responsive to salt stress could be categorized into two types, which were expressed during early development or ripening stages. During fruit development, phosphoenolpyruvate carboxylase 2 and phosphoenolpyruvate carboxykinase displayed contrasting expression patterns between early development and ripening, suggesting a switch of carbohydrate metabolism after the turning stage. Our results revealed a new metabolic pathway for GABA during the development of tomato fruits. At the start of ripening, GABA is first converted to malate via succinate semialdehyde, and it passes into a shunt through pyruvate. Then, it flows back to the TCA cycle and is stored as citrate, which contributes as a substrate for respiration during fruit maturation.

Efficient shoot regeneration from hairy roots of Antirrhinum majus L. transformed by the rol type MAT vector system

Abstractu2002Eleven independent GUS-positive hairy roots were induced by co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing the rol type MAT vector pNPI702. The MAT vector pNPI702 possesses a GUS gene under the 35u2009S promoter and a removal element in which the 7.6-kb DNA fragments containing the rolA, B, C and D genes and recombinase gene with a 35u2009S promoter are located between two directly oriented recombination site sequences. A total of 326 adventitious shoots regenerated from 11 independent hairy root lines cultured on 1/2MS medium without plant growth regulators at 25u200au200a°C under a 16/8u2009h (day/night) photoperiod after 8u2009weeks of stock-culture of hairy roots and 4u2009weeks of culture of the green segments of hairy roots. Regenerated plants showed either a normal or dwarf morphology. GUS activity was observed in the hairy roots and regenerated shoots. The presence of the GUS gene in the regenerated, morphologically normal plants was confirmed by PCR analysis.

The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.

Improved culture conditions for somatic embryogenesis using an aseptic ventilative filter in eggplant (Solanum melongena L.)

Abstract Embryogenic calli were induced from a cotyledon cutting of Solanum melongena L. on Murashige and Skoog (MS) solid medium supplemented with 50 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Calli were proliferated in MS liquid medium supplemented with 50 μM 2,4-D. Non-vitrified somatic embryos were formed and efficiently developed into cotyledonary embryos on transfer to MS hormone-free medium with high concentration (1%) of gelrite in a culture vessel capped with an aseptic ventilative filter. Somatic embryos developed into normal plants when transferred to half-strength MS solid medium without hormones. In contrast, vitrified and swollen embryos were formed on the same medium with 0.2% gelrite in culture vessels capped with aluminum foilm could not germinate normally and developed in part into callus tissue. The established system here was applied to 10 cultivars, and eight of them could form non-vitrified embryos that developed into normal plants. The effects of an aseptic ventilative filter on somatic embryogenesis are discussed.

Transformation of Antirrhinum majus L. by a rol-type multi-auto-transformation (MAT) vector system

A total of 11 independent beta-glucuronidase (GUS) positive hairy roots were induced following co-cultivation of leaf explants of Antirrhinum majus L. with Agrobacterium tumefaciens strain GV2260 containing rol-type multi-auto-transformation (MAT) vector pNPI702. A total of 326 adventitious shoots were regenerated from the hairy root lines on 1/2 MS medium without plant growth regulators at 25 degrees C under a 16 h/day photoperiod condition 4 months after infection of the A. tumefaciens GV2260. The absence of the rol genes in five plants was verified by polymerase chain reaction (PCR) and Southern blot analysis. Acclimatized transformants exhibited normal phenotypes in height and in the morphology of leaves and flowers. Furthermore, the GUS gene was strongly expressed in the leaves, inflorescence of the transformed plant, and the progeny. This result demonstrates that the rol-type MAT vector can be used to study gene functions controlling the morphogenesis of Antirrhinum majus plants.