Abstract
Groundnut (Arachis hypogaea L.) is an important crop in terms of income and nutrition. Despite its importance, groundnut yield is limited by environmental factors such as drought. This work reports the genetic transformability of Kenyan groundnut (Arachis hypogaea L.) genotypes with isopentenyl transferase (IPT) gene towards drought tolerance. The cotyledonary nodes of six Kenyan adapted groundnuts genotypes (ICGV 12991, CG 7, Red Valencia, ICGV 90704, Chalimbana, and JL 24) were transformed using Agrobacterium tumefaciens strain EHA 101 carrying PNOV-IPT binary vector containing an IPT gene, which was driven by SARK promoter and terminated by TNOS terminator. The vector also contained the phosphomannose isomerase (PMI) gene for the selection of transformed tissues. Putative transformants were tested for the presence of the transgene by PCR designed to amplify the IPT gene sequence. Gene expression was confirmed by RT-PCR. Transformation frequency was calculated as a percentage of the number of putative transformants divided by the total number of infected cotyledonary nodes. This ranged from 9.87% for ICGV 90704 to 19.77% for JL 24. Transformation efficiency was calculated as a percentage of the number of PCR positive plants divided by the total number of cotyledonary nodes infected. This ranged from 0% for ICGV 12991 and Chalimbana to 1.74% for JL 24. The data suggest the possibility of transforming groundnuts with the IPT gene and regenerating normal transgenic plants. This information will be useful during the transformation of groundnut towards different factors that affect production.
Highlights
Groundnuts (Arachis hypogaea L.) are among the oilseed crops that are cultivated around the world. e production of this crop encounters abiotic stresses such as waterlogging, salinity, and drought, which lead to very low productivity
Gene Construct. e PNOV-isopentenyl transferase (IPT) binary vector containing an IPT gene driven by SARK promoter and terminated by a TNOS terminator was used for the transformation of groundnuts in this study (Figure 1). e vector contained the phosphomannose isomerase (PMI) gene as a selectable marker gene. e IPT gene was donated by Dr Edwardo Blumwald from the University of California, Davis
In order to overcome other challenges associated with leaf senescence, expression of the IPT gene should be during plant maturation, but before the onset of senescence [3]. erefore, the IPT gene should be driven by the drought inducible SARK promoter so that it can be useful in enhancing tolerance to drought stress by delaying drought-induced leaf senescence in locally adapted groundnut genotypes
Summary
Groundnuts (Arachis hypogaea L.) are among the oilseed crops that are cultivated around the world. e production of this crop encounters abiotic stresses such as waterlogging, salinity, and drought, which lead to very low productivity. E production of this crop encounters abiotic stresses such as waterlogging, salinity, and drought, which lead to very low productivity. To solve these challenges, different transformation methods are currently being used to complement the conventional breeding of groundnuts. Some of the successful transformation methods in legumes involve the use of cotyledonary nodes and embryonic axes as explants [1]. Six Kenyan adapted groundnut genotypes were taken through Agrobacterium-mediated transformation with IPT gene for drought tolerance. Putative transformants were regenerated using regeneration media supplemented with 20 g/L mannose concentration for selection. After the regeneration of putative transformants and successful hardening, the presence of the transgene was confirmed by the use of PCR and RT-PCR molecular techniques
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