The freezing tolerance of 38 independent transgenic potato lines derived from the cultivar Desiree was tested in vitro using plantlets. The lines were transgenic for the DREB1A gene under control of the rd29A promoter, both of which were derived from Arabidopsis thaliana. The level of damage caused by freezing varied significantly among the transgenic clones and a non-transgenic control (cv. Desiree). Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation, but freezing tolerance was not dependent on the number of insertions. Northern blot analysis using a DREB1A cDNA probe revealed high levels of DREB1A expression among the transgenic clones during the initial cold exposure at 4 degrees C (after 2 h) and in the early stages of freezing (-20 degrees C, 1-10 min). Furthermore, a linear correlation was detected between the level of expression and the phenotypic response for all lines except D138. Thus, in the case of potato, a significant increase in freezing tolerance was observed in vitro on a small scale following the introduction of rd29A::DREB1A. Additional testing will show whether this strategy can be used for tolerance breeding in potato and to increase the freezing tolerance of other agriculturally important crops.
Plants respond to dehydration stress and tolerate water-deficit status through complex physiological and cellular processes. Many genes are induced by water deficit. Abscisic acid (ABA) plays important roles in tolerance to dehydration stress by inducing many stress genes. ABA is synthesized de novo in response to dehydration. Most of the genes involved in ABA biosynthesis have been identified, and they are expressed mainly in leaf vascular tissues. Of the products of such genes, 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis. One of the five NCED genes in Arabidopsis, AtNCED3, is significantly induced by dehydration. To understand the regulatory mechanism of the early stages of the dehydration stress response, it is important to analyse the transcriptional regulatory systems of AtNCED3. In the present study, we found that an overlapping G-box recognition sequence (5′-CACGTG-3′) at −2248 bp from the transcriptional start site of AtNCED3 is an important cis-acting element in the induction of the dehydration response. We discuss the possible transcriptional regulatory system of dehydration-responsive AtNCED3 expression, and how this may control the level of ABA under water-deficit conditions.
We developed and modified a precise, rapid, and reproducible protocol isolating high‐quality RNA from tissues of multiple varieties of cassava plants (Manihot esculenta Crantz). The resulting method is suitable for use in mini, midi, and maxi preparations and rapidly achieves high total RNA yields (170–600 μg·g−1) using low‐cost chemicals and consumables and with minimal contamination from polysaccharides, polyphenols, proteins, and other secondary metabolites. In particular, A260 : A280 ratios were > 2.0 for RNA from various tissues, and all of the present RNA samples yielded ribosomal integrity number values of greater than six. The resulting high purity and quality of isolated RNA will facilitate downstream applications (quantitative reverse transcriptase‐polymerase chain reaction or RNA sequencing) in cassava molecular breeding.
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