Noboru Inagaki

Early salt-stress effects on expression of genes for aquaporin homologues in the halophyte sea aster (Aster tripolium L.)

Six partial PCR fragments that encode aquaporin homologues in the halophyte sea asterAster tripolium were cloned using genomic DNA and cDNA as templates. The deduced amino acid sequences were highly similar to those of other major intrinsic proteins (MIPs) in plants. The expression patterns of sea astermip genes in leaves and suspension-cultured cells during a 24-hr NaCL stress period were studied with PCR samip (sea aster major intrinsic protein) fragments. Three samip (A-C) fragments were obtained by PCR with genomic DNA as templates, and 3 samip (D-F) fragments were obtained by RT-PCR. Among the 6 genes, expression ofSamip A, B, andC was significantly inducible by salt stress. The level ofSamip A, B andC mRNAs was dependent on NaCL concentration in both whole plants and cultured cells. However, no significant accumulation ofSamip transcripts was observed in salt-adapted cells. These observations suggest thatSamip A, B andC genes play an important role in the early salt-adaptable stage in sea asters.

In vitro evaluation of osmotic stress tolerance using a novel root recovery assay

We established a novel in vitro method, termed the root recovery assay, to evaluate the survival under osmotic stress of lettuce (Lactuca sativa L.) seedlings. Under salinity and drought stress, combination of the root-bending assay and root recovery assay showed the same trends in dry weight and survival rate as a hydroponic culture. Both in vitro assays and hydroponics ranked the three lettuce cultivars in the same order of drought tolerance. The root-bending assay evaluated the plant’s growth and the root recovery assay indicated the plant’s survival. In addition, the combined assay required less space and approximately half the time period compared with the hydroponic culture. These results suggested that application of the root-bending and root recovery assay should be a rapid and space-saving method with which to evaluate the osmotic stress tolerance of lettuce from both growth and survival standpoints.

Growth and protein profile responses in the halophyte sea aster (Aster tripolium L.) suspension-cultured cells to salinity

We established salt tolerant cell lines, which survived and grew under high salinity conditions with 150 mM (S-150) and 300 mM (S-300) NaCl, to study the effects of salt stress on the proliferation and protein profile of these cells in the halophyte sea aster,Aster tripolium L. These salt-adapted cell lines were produced from leaves and selected by repeated suspension subculture in media containing NaCl every 25 days for five cycles. S-150 cells displayed no inhibition in their growth compared to control cells maintained under non-stressed conditions. S-150 cells exhibited approximately a 15-fold increase in both fresh and dry weight during the 25 days under saline conditions. S-300 cells showed positive growth under severe salt stress, but their dry matter gain was significantly less than that of the S-150 cells, with only a 2.5-fold increase in dry weight. We also detected changes in the protein profile of salt-adapted cells with two specifically induced polypeptides (basic 58.4 and acidic 24.8 kDa) and one enhanced polypeptide (basic 15.1 kDa) in the soluble fraction, and one specifically induced polypeptide (42.0 kDa) in the insoluble fraction.

Callus formation, plant regeneration, and transient expression in the halophyte sea aster (Aster tripolium L.)

An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500xa0mgxa0l−1 casamino acids, and supplemented with 5.4xa0μM a-naphthaleneacetic acid (NAA) and 4.7xa0μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9xa0μM NAA. A high yield of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells, and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was obtained with 1xa0×xa0105 protoplasts ml−1, 25xa0μg batch−1 of plasmid vector, and 30% polyethylene glycol 4,000.

In vitro evaluation of tipburn resistance in lettuce ( Lactuca sativa . L)

Lettuce tipburn is an irreversible physiological disorder caused by calcium deficiency that decreases the crop value. Breeding a tipburn-resistant cultivar is the only causal therapy in many cases. In this study, we investigated an efficient method to evaluate lettuce resistance to tipburn in vitro. Seedlings of 19 lettuce cultivars representing three head types were cultured on agar medium containing EGTA, which chelates Ca2+. The percentage of tipburned leaves decreased proportionally with EGTA concentration. Susceptible cultivars were distinguished at 0.01xa0mM EGTA, whereas resistant cultivars were classified at 1.0xa0mM EGTA. Based on mean values of tipburn measurements, tipburn susceptibility was highest for ‘Leaf Lettuce’, followed by ‘Butterhead Lettuce’, and then ‘Crisphead Lettuce’. Two cultivars were selected for further tests using hydroponic and pot culture. The rank order of susceptibility to tipburn in these experiments was consistent with that of the in vitro assay. The in vitro evaluation of lettuce susceptibility to calcium deficiency is useful for initial screening of lettuce cultivars against tipburn incidence. Resistant cultivars identified in this study are practical candidates for cultivation in controlled environments, such as a plant factory, while sensitive cultivars are also useful as indicator plants to monitor environmental conditions.