Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Summary

Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte.

Abstract

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 °C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 µmol m^-2s^-1 light intensity, at 22 °C to 24 °C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3–4 transgenic plants per 10,000 T1 seeds propagated.

Introduction

In this protocol, we describe the growth and establishment of stable transgenic lines for the extremophyte model Schrenkiella parvula. The availability of an efficient transformation system is a hallmark of any versatile genetic model. Plants that thrive in extreme environments, referred to as extremophytes, provide a critical resource for understanding plant adaptations to environmental stresses. Schrenkiella parvula (formerly Thellungiella parvula and Eutrema parvulum) is one such extremophyte model, with expanding genomic resources^1,2,3<....

Protocol

1. Plant Growth

1. Seed sterilization (optional)
   1. Prepare 50% bleach in double-distilled water (ddH₂O) with 1 or 2 drops of a non-ionic detergent (see Table of Materials) in a 50 mL tube. Invert the tube several times to mix the solution.
      NOTE: It is preferable to conduct seed sterilization in a laminar flow cabinet with a UV sterilized surface for 15 min.
   2. Add the bleach solution to ~100–200 S. parvula seeds in a 1.5 mL tube. Mix thoroughly and let the tube sit for 5 min.
   3. Remove the bleach from the tube and add 70% ethanol. Wash the seeds by p....

Results

We developed a transformation protocol that enables harvesting of T₀ seeds within 150 days, using a floral-dip method modified from that for A. thaliana. Figure 1 shows a summary of the timeline and S. parvula plants that represent the optimal stage for executing the transformation through floral-dip. We selected S. parvula plants with 70 –80 flowers in multiple inflorescences at 60–80 days after germination a.......

Discussion

The physiological state of the plant significantly influences the efficiency of transformation²⁵. The use of healthy and vigorous plants for transformation is a key requirement for successful transformation in S. parvula. Water or light stressed plants will have fewer flowers compared to the healthy plants ideal for transformation (Figure 1, center panel). S. parvula can grow at a light intensity less than 130 µmol m^-2 s^-1,.......

Materials

Name	Company	Catalog Number	Comments
Agar	VWR International, Radnor, PA	90000-762	Bacto Agar Soldifying Agent, BD Diagnostics
B5 vitamins	Sigma-Aldrich, St. Louis, MO	G1019	Gamborg’s Vitamin Solution
Desiccant	W A Hammond Drierite, Xenia, OH	22005	Indicating DRIERITE 6 mesh
Destination vector for plant transformation	TAIR	Vector:6531113857	pKGWFS7
Electroporation cuvette	USA Scientific	9104-5050	Electroporation cuvette, round cap, 0.2 cm gap
Electroporator	BIO-RAD Laboratories, Hercules, CA	1652100	MicroPulser Electroporator
Fertilizer beads	Osmocote Garden, Marysville, OH	N/A	Osmocote Smart-Release Plant Food Flower & Vegetable
Gel extraction kit	iNtRON Biotechnology, Boston, MA	17289	MEGAquick-spin Total fragment DNA purification kit
Gentamicin	Sigma-Aldrich, St. Louis, MO	G1914-5G	Gentamicin sulfate
Glufosinate-ammonium (11.3%) herbicide (BASTA)	Bayer environmental science, Montvale, NJ	N/A	FINALE herbicide
Kanamycin	VWR International, Radnor, PA	200004-444	Kanamycin monosulfate
MES	Bioworld, Dublin, OH	41320024-2	MES, Free Acid
MS salt	MP Biomedicals, Santa Anna, CA	092621822	Hoagland's modified basal salt mixture
N6-benzylaminopurine (BA)	Sigma-Aldrich, St. Louis, MO	B3274	6-Benzylaminopurine solution
NaCl	Sigma-Alrich	S7653	Sodium chloride
Non-ionic detergent	Sigma-Aldrich, St. Louis, MO	9005-64-5	TWEEN 20
Plasmid isolation kit	Zymo Research, Irvine, CA	D4036	Zyppy Plasmid Kits
Recombinase enzyme mix kit	Life Technology	11791-020	Gateway LR Clonase II Enzyme mix
Rifampicin	Sigma-Aldrich, St. Louis, MO	R3501-1G	Rifampicin, powder, >= 97% (HPLC)
Shaking incubator	ThermoFisher Scientific, Waltham, MA	SHKE4450	MaxQ 4450 Benchtop Orbital Shakers
Soil mix	Sun Gro	SUN239223328CFLP	Sun Gro Metro-Mix 360 Grower Mix
Spectinomycin	VWR International, Radnor, PA	IC15206705
Sterile 50ml conical tubes	USA Scientific, Ocala, FL	1500-1811	50 ml conical screw cap tubes, copolymer, racks, sterile
Sucrose	VWR International, Radnor, PA	57-50-1	Sucrose, ACS
Surfactant solution	Lehle seeds, Round Rock, TX	VIS-02	Silwet L-77
Topoisomerase-based cloning kit	Life Technologies, Carlsbad, CA	K240020	pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli
Tryptone	VWR International, Radnor, PA	90000-282	BD Bacto Tryptone, BD Biosciences
Yeast Extract	VWR International, Radnor, PA	90000-722	BD Bacto Yeast Extract, BD Biosciences