In vitro regeneration and Agrobacterium-mediated genetic transformation of Dragon's Head plant (Lallemantia iberica)

Abstract

Dragon's head plant (Lallemantia iberica), is a flowering species belongs to the mint family (Lamiaceae). The species contains valuable essential oils, mucilage and oil which are used in pharmaceutical and food industries. Tissue culture is a feasible strategy to attain large-scale production of plantlets with a huge potential to produce plants with superior quality. The objective of this study was to develop a simple and efficient method for regeneration and transformation of L. iberica. To reach this goal, the regeneration ability of various explants including leaf, cotyledonary node, hypocotyl and cotyledon segments was investigated in MS medium supplemented with diverse concentrations of NAA (Naphthalene acetic acid) and BAP (6-Benzyl Amino Purine). According to the results, cotyledonary nodes showed the best regeneration response. The maximum rate of regeneration (and number of induced shoots was achieved in 1 mg l^-1 BAP in combination with 0.05 mg l^-1 NAA from the cotyledonary nodes. Additionally, through the optimized regeneration technique Agrobacterium-mediated transformation of L. iberica was successfully accomplished. Gene transfer was assessed on leaf samples from regenerated plantlets under a fluorescent microscope to detect the GFP signals. Moreover, transgene integration and its expression were confirmed by PCR and RT-PCR analysis, respectively. The establishment of these efficient regeneration and genetic transformation methods paved the way for further application such as plant improvement, functional analysis and gene editing.

Figure 1

Genetic map of pXK2FS7 binary vector containing two reporter genes of GFP and GUS driven by the CaMV 35S promoter.

Figure 2

Response of explants on plant regeneration medium containing 1 mg l⁻¹ BAP plus 0.05 mg l⁻¹ NAA. (a,b) direct shoot formation after two weeks on cotyledonary node; (c) direct shoot regeneration from hypocotyl; (d) roots induced after two weeks on medium containing 0.1 mg l⁻¹ NAA.

Figure 3

The effect of different concentrations of NAA on root induction. Means with only the same letters are not significantly different at the 5% level based on Duncan’s multiple range test (p ≤ 0.05). Data shown represent means of induced roots ± SD (n = 3).

Figure 4

Acclimatization of regenerated plantlets. (a) The early stages of adaptation: transfer the plantlets to the pots containing 1:1 ratio of coco peat and perlite mix and covering them with transparent plastic shield; (b) making 2–3 holes in the shields to easy adapt plants; (c) removing of the plastic cover after 3–4 weeks; (d) transfer of plantlets to larger pots containing mixture of farm soil, compost, and sand at the late stage of adaptation; (e) a plant at flourishing stage.

Figure 5

Explants on regeneration medium containing 30 mg l⁻¹ and 60 mg l⁻¹ kanamycin. (a) Explants on a medium containing 30 mg l⁻¹ kanamycin after 3 weeks; (b) explants on a medium containing 60 mg l⁻¹ kanamycin after 3 weeks.

Figure 6

Regeneration of explants in selective medium containing 60 mg l⁻¹ Kanamycin plus 400 mg l⁻¹ cefotaxime after inoculation of explants with Agrobacterium cells. (a) Regeneration of putative transgenic plantlets (green plantlets) and non-transformed plantlets (Red circles) after two weeks of culture; (b) Sub-culturing of putative transgenic plantlets; (c) further development of plantlets in a new medium; (d,e,f) root initiation of regenerated shoot in MS medium containing 0.1 mg l⁻¹ NAA and 60 mg l⁻¹ kanamycin; (g) acclimatized plantlets; (h) mature plant producing seeds; (i) T₁ transgenic progeny plants.

Figure 7

The effect of (a) different infection time and (b) concentrations of acetosyringone on gene transfer percentage. Means with only the same letters are not significantly different at the 5% level based on Duncan’s multiple range test (p ≤ 0.05). Data shown represent means of transformation frequency ± SD (n = 3).

Figure 8

Green Fluorescence emission of green leaves from greenhouse-grown putative transgenic plants, (a) leaf sample from putative transgenic plant, (b) leaf segment of a wild-type plant (non-transgenic).

Figure 9

Gel electrophoresis of PCR on genomic DNA from the putative transgenic T₀ plants using the (a) GFP and (b) GFP-GUS primers; (c) gel electrophoresis of PCR on cDNA from the putative transgenic T₀ plants using the GFP primers (d) PCR on DNA from four T₁ plants using the GFP-GUS primers. M) DNA Ladder 100 bp; NC) Negative Control; PC) Positive Control containing linear plasmid of binary vector; W) non-transgenic plants; lanes 1, 2, 3 and 4 are transgenic lines screened by GFP visualisation.