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.

Keywords: Agrobacterium; LED light; chimeric chickpea; legume transformation; micro-injury of in vitro explants; transgenic chickpea.

FIGURE 1

Schematic representation of steps involved in developing the improved protocol for Agrobacterium-mediated genetic transformation and regeneration of chickpea using half embryo explants.

FIGURE 2

Schematic representation of the constructs made for chickpea plant transformation. (A) Reporter gene construct with constitutive promoter CaMV 35s driving GUS and NPTII gene; (B,C) Abiotic stress tolerant gene constructs with AtBAG4 gene from Arabidopsis and TlBAG gene from Tripogon loliiformis, respectively, driven by CaMV 35s promoter and NPTII driven by S1 promoter for both genes; (D,E) Multigene construct for iron biofortification with chickpea Nas2 (CaNas2) and rice Nas2 (OsNas2) genes driven by Nos promoter, soybean Ferritin (GmFerritin) driven by CaMV 35s promoter and NPTII driven by S1 promoter.

FIGURE 3

The experimental process of Agrobacterium-mediated transformation using half embryo explants. (A) Seed sterilization and imbibition in sterile water; (B) Dissection of embryos into half along its axis; (C) Needle micro-injury; (D) 5 day old explants co-cultivated in B5 medium turns purple and start germinating; (E) Shoot multiplication in regeneration and selection (RS2) medium; (F) Putative shoots selected for GUS expression and PCR analysis; (G) Grafting of PCR positive in vitro shoots; (H) Acclimatized grafted shoot in the soil.; (I) Fully acclimatized T₀ plants.

FIGURE 4

Transient histochemical GUS staining of 5-day old half embryo explants co-cultivated in B5 medium and stable GUS staining of in vitro shoots derived after third round of regeneration and selection. (A,B) Non-transgenic control without transformation; (C) 5-day old half embryo explant with micro-injury under LED light; (D) In vitro shoots derived after third round of regeneration and selection with micro-injury under LED light; (E) 5-day old half embryo explant without micro-injury under LED light; (F) In vitro shoots derived after third round of regeneration and selection without micro-injury under LED light; (G) 5-day old half embryo explants with micro-injury under fluorescent light; (H) In vitro shoots derived after third round of regeneration and selection with micro-injury under fluorescent light; (I) 5-day old half embryo explants without micro-injury under fluorescent light; (J) In vitro shoots derived after third round of regeneration and selection without micro-injury under fluorescent light.

FIGURE 5

Molecular characterization of fully transformed and chimeric in vitro shoots through PCR analysis. (A) Amplification of GUS (uidA) gene from leaves of in vitro shoots obtained with micro-injury under LED light (lane 1–6), without injury under LED light (7–12), with micro-injury under fluorescent light (17–22) and without injury under fluorescent light (23–25); (B) Amplification of NPTII gene from leaves of in vitro shoots obtained with micro-injury under LED light (lane 1–6), without injury under LED light (7–12), with micro-injury under fluorescent light (17–22) and without injury under fluorescent light (23–25). Lane 13,26 - Blank; Lane 14,27 - Negative (water) control; Lane 15,28 –Plasmid (uidA) control; 16,29 - Non-transgenic control; (C) Non amplification of virC gene from in vitro leaves tissues confirms absence of Agrobacterium traces on leaves. Lane – 1–15, 19, 20, 21, 22, 26, 27 samples; 16, 23 – Negative (water) control; 17, 24 - Non-transgenic control; 18, 25 – blank; “+” – virC control from Agrobacterium.

FIGURE 6

Stable GUS expression in vegetative and reproductive parts of 2nd generation progeny. (A) Two week old non transgenic seedlings without any GUS expression; (B) Two week old transgenic seedlings with GUS expression in shoots roots, leaves and cotyledons; (C) Flowers of non-transgenic seedlings without any GUS expression; (D) Flowers of transgenic seedlings with GUS expression in sepals, petals stamens and stigma; (E) Stamens of non-transgenic seedlings without any GUS expression; (F) Stamens of transgenic seedlings with GUS expression.

FIGURE 7

Southern blot hybridization of the transgenic plants with (A) a probe for the gene AtBAG4 to detect the presence of integrated transgene; (B) a probe for the gene TlBAG to detect the presence of integrated transgene; (C) a probe for the selection marker, NPTII, which was linked to and assumed to act as a surrogate for the presence of the OsNas2 and CaNas2 transgenes.