A Simple and Non-destructive Method of Direct-PCR for Plant Systems |
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Authors: | R. Sharma Vinod Kumar T. Mohapatra Vikas Khandelwal Govind K. Vyas |
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Affiliation: | (1) Plant Biotechnology Centre, S. K. Rajasthan Agricultural University, Bikaner, 334 006, India;(2) Seed Technology Research, Agricultural Research Station, S. K. Rajasthan Agricultural University, Durgapura, 302018 Jaipur, India;(3) NRC on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, 110 012, India;(4) Department of Genetics and Plant Breeding, N. M. College of Agriculture, Navsari Agricultural University, Navsari, 396450, Gujarat, India;(5) Institute of Applied Sciences and Biotechnology, Chemind Diagnosis and Biosolutions, Jaipur, 302019, India |
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Abstract: | To date, PCR is a fundamental tool for most of the research concerning plant diversity analysis, marker-assisted selection, genetic purity testing, disease diagnostics, and transgene analysis. In all of these analyses, good-quality DNA serves as a template for amplification of target sequences. Extraction of good-quality DNA requires many steps, making the whole process time consuming, tedious, labor intensive, and expensive due to costlier and toxic chemicals. To overcome these preparatory steps from PCR-based DNA amplification, we have developed a direct-PCR amplification method for plants without isolating DNA. The method is unique and beneficial over some previously described methods of direct-PCR which fail due to inefficient amplification of target DNA in the presence of PCR inhibitors and crop specificity. Moreover, such methods are non-specific and, being destructive, cannot be replicated; one cannot completely rely on them due to lack of reproducibility. This method was streamlined from our earlier observation that alcohol-desiccated tissues maintain intact DNA for a long time. This method is specific, rapid, and, being non-destructive, allows replication, giving advantages over existing methods. The method was tested over a wide range of plant species and found very effective and quick in generating data. The method was successfully used to test the genetic purity of pearl millet hybrid (RHB-127) and its restorer (RIB 3135-18) and CMS line (ICMA 93333A). Our method is especially important for developing inexpensive and high-throughput non-invasive genetic analyses. |
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