Robust Genetic Transformation System to Obtain Non-chimeric Transgenic Chickpea

Abstract

Chickpea transformation is an important component for the genetic improvement of this crop, achieved through modern biotechnological approaches. However, recalcitrant tissue cultures and occasional chimerism, encountered during transformation, hinder the efficient generation of transgenic chickpeas. Two key parameters, namely micro-injury and light emitting diode (LED)-based lighting were used to increase transformation efficiency. Early PCR confirmation of positive in vitro transgenic shoots, together with efficient grafting and an extended acclimatization procedure contributed to the rapid generation of transgenic plants. High intensity LED light facilitate chickpea plants to complete their life cycle within 9 weeks thus enabling up to two generations of stable transgenic chickpea lines within 8 months. The method was validated with several genes from different sources, either as single or multi-gene cassettes. Stable transgenic chickpea lines containing GUS ( uidA ), stress tolerance ( AtBAG4 and TlBAG ), as well as Fe-biofortification ( OsNAS2 and CaNAS2 ) genes have successfully been produced.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants

“…Chimeric tissue formation (a single plant tissue containing a mixture of transformed and non‐transformed sections) during tissue culture transformation is a prevalent issue in legumes, including cowpea (Das Bhowmik et al ., 2019). Chimeric plants affect the segregation of the transgene to the subsequent generation and reduce the efficiency of recovering stable transgenic lines (Das Bhowmik et al ., 2019).…”

“…Several selection systems have been reported for cowpea transformation with different explant types (Manman et al, 2013), such as NPTII/kanamycin (Bett et al, 2019; (a) (b) (c) The Plant Journal, (2021), 106, 817-830 Chaudhury et al, 2007), NPTII/G418 (Solleti et al, 2008b), PMI/mannose (Bakshi et al, 2012), HPT/hygromycin (Kumar et al, 1996), BAR/glufosinate (Popelka et al, 2006) and ahas/imazapyr (Citadin et al, 2013;Ivo et al, 2008). It was reported that incomplete selection and tissue necrosis were associated with those selection systems (Manman et al, 2013) and resulted in lower transgenic plant recovery (Bakshi et al, 2011;Chaudhury et al, 2007;Solleti et al, 2008b) and a higher percentage of chimeric tissue formation in cowpea (Das Bhowmik et al, 2019). To identify more efficient selection agents for the de novo organogenesis described above, we tested and compared the selection efficiency of CTP-NPTII/kanamycin, CTP-NPTII/G418 and CTP-spcN/SPEC (Anada et al, 2017) after Agrobacterium-mediated transformation using Agrobacterium strain LBA4404 Thy-.…”

Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit

“…The cultivar, DCP 92-3 responds well in plant tissue culture and exhibit limited performance under drought conditions, and hence, the cultivar was used in the present study. Transformation efficiency obtained in the current study is 0.1%, however the efficiency can significantly be increased by modulating parameters like micro-injury to explants and LED-based light simulations, as reported [35]. Transgenic chickpea lines harbouring AtDREB1a gene driven by stress inducible promoter rd29a were developed and tested for various physiological parameters crucial to drought adaptation.…”

Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants

“…The infection medium also contained AS and DTT and had an acidic pH, which are all factors that have been shown to facilitate T-DNA transfer and to enhance transformation efficiency (25). After 5 days of co-cultivation the moderate survival of explants was observed but with higher transformation efficiencies (26). In this study, DTT was added to the CCM as an antioxidant, to reduce pruning and necrosis in explants which represents one of the primary factors affecting the conversion efficiency (19).…”