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American Journal of Plant Physiology
Year: 2007  |  Volume: 2  |  Issue: 6  |  Page No.: 333 - 343

Isolation of cDNA Clones of Genes Differentially Expressed During Somatic Embryogenesis of Pinus roxburghii

Ravindra B. Malabadi and K. Nataraja    

Abstract: Present research highlights for the first time the expression of cDNA clones of genes involved in programming the apical meristem cells towards somatic embryogenic pathway influenced by external environmental stimulus like cold-pretreatment. Differential display was used to isolate the genes which are expressed specifically in embryogenic tissue induced by cold-pretreatment of thin sections of vegetative shoot apices of mature trees of Pinus roxburghii. We have developed a rapid method which employs magnetic beads to capture mRNA from cell lysates. Of the 56 cold-enhanced embryogenic-associated cDNAs identified, 20 were cloned. Nine of the 20 fragments which generated single bands on re-amplification were selected for cloning and further analysis. During reverse northern hybridization, all the 20 clones selected generated a positive signal when probed with labeled cDNA from cold-enhanced embryogenic tissue, but no signal when probed with cDNA from the non-embryogenic tissue (control treatment). All the 20 clones thus contained inserts that were specific to cold-enhanced somatic embryogenesis. This approach allows us to perform differential display and construction of cDNA libraries from the small amount of embryogenic tissue and outline a PCR-based method for confirming differential expression of large number of cloned bands in cases where RNA quantities are limiting.

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