In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance

Abstract

Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

Fig 1. Effect of basal media and pre-culturing.

Effect of different basal media on seed emergence (A). Seeds germinated on water, B5, ½ MS and MS. Effect of the stage of pre-cultured seeds on transformation frequency (B). Seeds were germinated and pre-cultured for 5, 7, 9 and 11 days. Graphs represent mean value ± SE followed by different letters are significantly different at P<0.05.

Fig 2. Effect of vacuum infiltration on transformation efficiency.

The graph represents mean value ± SE followed by different letters are significantly different at P<0.05.

Fig 3. In planta transformation of cumin seeds.

Seven days pre-cultured germinated seeds (A) were infected (co-cultivated) with Agrobacterium (B) followed by vacuum infiltration in a desiccator (C) and then transferred to the plastic pot for further growth (D). Putative transformants showed initiation of flower bud formation (E) and cross pollination by using ear bud (F). Randomly selected non transformed (control) seedling (G) and putatively transformed seedlings (H) showed histochemical GUS spots. Putative transgenic plants are grown further for seed setting and maturation (I-L).

Fig 4. Schematic representation of optimized in planta transformation protocol used for Agrobacterium-mediated genetic transformation of cumin seeds.

Fig 5. Molecular confirmation of in planta transformed transgenic lines.

PCR confirmation of putative transgenic lines using SbNHX1, gus gene and hptII genes (A); Southern blot analysis of randomly selected in planta transgenic plants (B) and overexpression analysis of the SbNHX1 gene in transgenic plants, analyzed by semi-quantitative RT-PCR (C). Lane M: Molecular weight marker ladder, lane PC: positive control, lane WT: wild type plant (negative control i.e. non-transformed plant) and lanes L: putative transgenic lines.

Fig 6. Bio-physiology of transgenic lines under salinity stress.

Estimation of electrolyte leakage (A), lipid peroxidation (B), proline (C), chlorophyll a (D), chlorophyll b (E), total chlorophyll (F) and carotenoid contents (G) of WT and transgenic plants. Graphs represent mean value ± SE followed by different letters are significantly different at P<0.05.