Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L

Abstract

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters-such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD_600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride-were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

Keywords: Agrobacterium tumefaciens; Medicago sativa L. (alfalfa); green fluorescent protein (GFP); transient gene; β-glucuronidase (GUS).

Figure 1

A schematic diagram of the plasmid vector pCAMBIA1304, represented within Agrobacterium tumefaciens, was used for the transformation of various alfalfa tissues. This vector includes several key elements: a kanamycin resistance gene (KanR) for bacterial selection, a hygromycin resistance gene (HygR) for plant cell selection, and an mgfp5-GUS fusion gene expression cassette, driven by the CaMV 35S promoter and terminated by the NOS poly-A sequence, serving as a reporter gene. The pCAMBIA1304 vector is relatively small, measuring 12,362 base pairs, and features a high copy number in E. coli, ensuring efficient DNA yields.

Figure 2

Different developmental stages of callus induction: (A) approximately 2 mm excised leaf segments cultured on callus induction medium; (B) 2-week-old callus derived from segmented leaf; (C) 3-week-old leaf-derived callus. Scale bars: 200 pixels (A, C); 50 pixels (B).

Figure 3

Histochemical localization of transient GUS gene expression in alfalfa different types of explants: (A) GUS gene expression in segmented leaf explants (0.2 cm), indicated by dark blue spots; (B) prominent GUS gene expression observed in the midrib region of a larger leaf explant (0.4 cm); (C) the circle shows a magnified view (6×) of a single blue spot with GUS expression in multiple cells within the midrib area; (D) dark-to-light blue pattern within single or multiple cells, indicating GUS-positive cells’ distribution across the leaflet; (E) active GUS expression in the axillary bud and nodal regions of the stem, marked by dark spots with enhanced expression; (F) subtle GUS gene expression observed in the petioles; (G) high levels of GUS gene expression observed in the lateral root area; (H) scattered GUS expression observed in 3-week-old leaf-derived callus. Arrows indicate specific regions of GUS gene expression in different explants and leaf-derived callus. Scale bars = 200 pixels.

Figure 4

Optimization of transient GUS gene expression in alfalfa: Percent transient GUS gene expression response relative to the control alfalfa under varying rates of different parameters. (A) Effects of different explants on transient GUS gene expression. (B) Effect of age of leaf on transient GUS gene expression. (C) Effects of different sizes of leaf explants on transient GUS gene expression. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 5

Effect of wounding types on transient GUS gene expression in alfalfa leaf explants: Percentage expression response in comparison to control under varying leaf tissue wounding conditions. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 6

Effect of Agrobacterium concentrations on transient GUS gene expression in alfalfa leaf explants: Percentage transient response across varied effect of Agrobacterium concentrations compared to controls. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 7

Impact of preculture of leaf tissues on transient GUS gene expression in alfalfa: Percentage expression relative to the control (in days). All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 8

Influence of infection periods on transient GUS gene expression in alfalfa leaf explants: Percentage response rate compared to the control. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 9

Evaluating transient GUS gene expression in alfalfa leaf explants: Percentage response under varying effect of co-cultivation period relative to the control. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 10

Effect of acetosyringone on transient GUS gene expression in alfalfa leaf explants: Percent expression compared to control plants. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 11

Effect of calcium chloride on transient GUS gene expression in alfalfa leaf explants: Percentage transient response under different calcium chloride concentration treatment relative to control. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 12

Assessment of the effects of silver nitrate on transient GUS gene expression in alfalfa leaf explants: Percent expression response compared to the control plants. All experiments were replicated four times (n = 64). Results were pooled over experimental runs. Vertical bars are represented as means ± standard errors.

Figure 13

GFP fluorescence in alfalfa leaf explants (A–F) and embryonic calli (G–L): After co-cultivation with Agrobacterium at OD_600nm = 0.6 for 3 days, followed by two washes with liquid MS medium to eliminate Agrobacterium. (A) (chloroplast autofluorescence), (B) (GFP fluorescence): Fluorescence image of clustered multiple cells in 3-week-old wounded leaves (scale bar = 500 µm); (C) (chloroplast autofluorescence), (D) (GFP fluorescence): Isolated GFP spots in excised leaf explants (scale bar = 1 mm). (E) (chloroplast autofluorescence), (F) (GFP fluorescence): GFP expression in the wounded areas of leaf explants (scale bar = 1 mm). (G) (chloroplast autofluorescence), (H) (GFP fluorescence): GFP transient expression in 1-week-old pro-embryonic callus derived from leaves (scale bar = 500 µm). (I) (chloroplast autofluorescence), (J) (GFP fluorescence): GFP transient expression in 2-week-old pro-embryonic callus derived from leaves (scale bar = 500 µm). (K) (chloroplast autofluorescence), (L) (GFP fluorescence): GFP transient expression in 3-week-old pro-embryonic callus derived from leaves (scale bar = 1 mm). GFP expression was especially prominent in globular embryos from 3-week-old leaf-derived calli grown under non-selection conditions. Images were taken using an Olympus SZX12 Stereo Fluorescence Microscope with a GFP filter (Olympus America Inc., Melville, NY, USA). Arrows indicate GFP expression.

Figure 14

Molecular confirmation of alfalfa transient gene expression and analysis for the presence of the GUS (β-glucuronidase) gene in putative transgenic alfalfa explants using GUS-specific primers. Lane 1: M (marker DNA ladder, 100–4000 bp, FlashGEl^® DNA marker, Lonza Rockland, Inc., Rockland, ME, USA); Lanes 2–6: putative transgenic alfalfa explants, leaf (L), stem (S), petiole (P), root (R), and callus (C); Lanes 7–11: non-transgenic alfalfa explants, leaf (NTL), stem (NTS), petiole (NTP), root (NTR), and callus (NTC).

Figure 15

Step-by-step protocol for transient expression in Medicago sativa L. (alfalfa).