A grobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC

Abstract

A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg L^-1 and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg L^-1. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03251-x.

Keywords: Agrobacterium tumefaciens; Artemisia pallens; Candidate genes; GUS; Stable transformation; Suspension cells.

Fig. 1

Different explants of Artemisia pallens on MS medium supplemented with 2,4-D (2. 0 mg L⁻¹). a Attached cotyledon explant, b individual cotyledon explant, and c compound leaf explant

Fig. 2

Callus induction from different explants of Artemisia pallens on MS medium supplemented with 2,4-D (2.0 mg L⁻¹). a 2 months old calli onto medium obtained from cotyledon explant b friable cream-yellow callus, c soft watery non-embryonic callus, and d hard nodular callus

Fig. 3

Initiation and proliferation of suspension cultures from callus derived from cotyledon explants of Artemisia pallens on MS medium supplemented with 2,4-D (2.0 mg L⁻¹) and ZEA (0.25 mg L⁻¹). a Fully developed suspension cultures, b proliferated fine cells after two to three months of cultures initiation, c filtered suspension cells or clumps, and d packed cell volume used for transformation with Agrobacterium tumefaciens

Fig. 4

Evaluation of various factors using GUS histochemical assay a Non-transformed suspension cells and calli (control) after GUS histochemical assay, b transformed suspension cells and calli showing blue color after GUS histochemical assay, c effect of temperature on GUS histochemical assay d effect of cold shock on GUS histochemical assay e effect of pluronic acid F-68 on GUS histochemical assay

Fig. 5

Effect of various factors on GUS fluorometric activity nmol (4MU/mg/min) of suspension cells transformed with Agrobacterium tumefaciens

Fig. 6

Proliferation of transformed suspension cultures and development of calli of Artemisia pallens. a Transformed cells onto filter paper observed under a stereomicroscope, b yellowish and creamish calli after 3 months of cultures observed under the stereomicroscope, c and d transformed calli viable and untransformed calli dead after two to three subcultures on selection medium, e and f transformed calli showing blue color in GUS histochemical assay

Fig. 7

Molecular confirmation of transgenic callus lines Artemisia pallens analysis for gusA gene presence in hygromycin-resistant callus lines using gusA gene-specific primers. L1: Marker DNA ladder (Lamda DNA/EcoRI+HindIII marker, Thermo Scientific); L2: No template (Negative control); L3: Positive control (plasmid DNA); L4: Wild type (control plant DNA); L5-L10: Different transformed hygromycin-resistant callus lines using PCR

Fig. 8

Confirmation of the presence of a single peak in melt curve analysis by RT-PCR. Melt curves of 3 candidate reference genes showing single peaks a ApAct ( β-actin), b ApArf (ADP-ribosylation factor), c ApUbi (ubiquitin) d L1: 100 bp DNA ladder, L2: No template (Negative control), L3: ApAct, L4: ApArf, L5: ApUbi, and L6: Appds e the relative expression of uidA gene in transgenic callus lines of Artemisia pallens