Rice Genetics IV - IRRI books - International Rice Research Institute

These three plasmids were used to transform rice suspension cells. Transgenicrice plants were regenerated. Transgene copy numbers were estimated by comparingthe intensity of hybridization bands of BamHI-digested genomic DNA from transgenicplants to bands of known quantities of plasmid DNA. The copy numbers of thetransgene, including rearranged copies, varied between 1 and 9 in different plants(Cheng et al 2000). The p5cs expression was first analyzed at the mRNA level byRNA blots. Two-month-old R 2 plants were subjected to water-stress treatment bywithholding water for 6 d, whereas nonstressed plants were supplied with water continuouslyand used for basal mRNA-level analysis. To detect the salt-stress-inducedmRNA level, 200 mM NaCl solution was used to water p5cs-transgenic and uidAtransgenicplants (as control) for 48 h. Our results indicated that the p5cs mRNAproduction driven by the inducible promoter was low, but it did increase from 3- to 7-fold with different transgenic lines. Plants with constitutive p5cs expression gavehigh p5cs mRNA levels with or without stress, as expected.Next, we measured the free proline level in transgenic rice plants. One R 2 line ofAct1-p5cs plants (L1) and two R 2 lines of AIPC-p5cs plants (L5 and L7) were chosen.Before water-stress treatments (0-d sample), the proline level produced by the constitutiveexpression of the p5cs transgene (L1) reached 319%, the value of the L3 controlplant. When a stress-inducible promoter (AIPC) was used, the proline level reachedonly 138% (L5) and 121% (L7) of that of L3. As the stress proceeded, however, theproline levels of lines L5 and L7 increased at relatively higher rates and reached finallevels approaching that of line L1 after 8 d of water stress. For salt stress, the resultsare generally similar to those of the water-stress treatment (Cheng et al 2000).Growth performance of transgenic plants under water-stress conditions. Chenget al (2000) tested the growth performance of R 2 plants in soil. Under nonstress conditionsin soil, no significant differences were observed between p5cs-containingtransgenic plants and AIPC-uidA control plants in their growth performance duringthe entire period of the experiment. Upon withholding water from the trays, the absolutewater content in the soil decreased from 35% to 12% after 7-d water stress.The plants were watered for 2 d and then stressed again by withholding water foranother 7 d. Following two cycles of water stress, the leaves of AIPC-uidA controlplants started to turn yellow and the Act1-p5cs plants showed a low growth rate,whereas the two AIPC-p5cs plants with a stress-inducible promoter showed healthygrowth. After four cycles of water stress, more severe symptoms, such as leaf chlorosis(in both the L3 control and Act1-p5cs plants) or death of leaf tips (in control plantsonly), were found. The AIPC-p5cs plants still showed a relatively high rate of growthand less severe leaf chlorosis. Data in Table 2 show the average fresh shoot and rootweights of the plants after four cycles of 7-d water stress. The results indicated that,under water stress, the AIPC-p5cs plants (L5 and L7), which contained a stress-induciblepromoter to drive the p5cs expression, grew much faster than the Act1-p5csplants (L1), which contained a constitutive promoter for driving the p5cs expression.The difference between using the two types of promoters was highly significant(P