Name: plant growth and agrobacterium-mediated floral-dip transformation of the extremophyte schrenkiella parvula
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula at JoVE.com

This work was supported by a National Science Foundation award MCB 1616827.

1. Plant Growth
<ol>
	<li>Seed sterilization (optional)
	<ol>
		<li>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.</li>
		<li>Add the bleach solution to ~100&#8211;200 S. parvula seed...

<ol>
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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 &#181;mol m-2 s-1,...

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 resources1,2,3,4,5. However, transformation protocols have not yet been reported for S. parvula in published studies.
The genome of S. parvula is the first published extremophyte genome in Brassicaceae (mustard-cabbage family) and shows an extensive overall genome synteny with the non-extremophyte model, Arabidopsis thaliana1. Thus, comparative studies between A. thaliana and S. parvula could benefit from the wealth of genetic studies performed on A. thaliana to make informative hypotheses on how the S. parvula genome has evolved and regulated differently to cope with extreme environmental stresses5,6,7. S. parvula is one of the most salt-tolerant species (based on soil NaCl LD50) among known wild relatives of A. thaliana8. In addition to the NaCl tolerance, S. parvula survives and completes its life cycle in the presence of multiple salt ions at high concentrations toxic to most plants7. In response to the abiotic stresses prevalent in its natural habitat, it has evolved various traits, among which several have been studied at the biochemical or physiological level 8,9,10,11.
Since 2010, there have been over 400 peer-reveiwed publications that used S. parvula as a target species or used it in a comparison with other plant genomes. However, a clear bottleneck could be identified with a closer look of what type of studies have been conducted. The majority of these reports discuss the potential use of S. parvula in future studies or use it in comparative genomic or phylogenomic studies. Due to the lack of a proof-of-concept transformation protocol established for S. parvula, it has not been used in functional genomic studies, despite having one of the highest quality plant genomes available to date (&#62;5 Mb contig N50) assembled and annotated into chromosome-level pseudomolecules1.
The Agrobacterium-mediated floral-dip transformation method has become the most broadly used method to create trasngenic lines in A. thaliana, and the development of a reproducible system of transformation was a critical factor in its success as a genetic model12,13. However, not all Brassicaceae species have been shown to be successfully transformed using the floral-dip method developed for A. thaliana. Specially, the Brassicaceae Lineage II species that include S. parvula has been recalcitrant to floral-dip based transformation methods14,15.
The indeterminate flowering growth habit of S. parvula, combined with its narrow leaf morphology has made it challenging to adopt the standard Agrobacterium-mediated floral-dip transformation method. In this study, we report the modified protocol we have developed for reproducible transformation of S. parvula.

<table border="0" cellpadding="0" cellspacing="0" style="width:1303px;" width="1302">
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Agar</td>
			<td style="width:293px;">VWR International, Radnor, PA</td>
			<td style="width:127px;">90000-762</td>
			<td style="width:541px;">Bacto Agar Soldifying Agent, BD Diagnostics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B5 vitamins</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G1019</td>
			<td>Gamborg&#8217;s Vitamin Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Desiccant</td>
			<td>W A Hammond Drierite, Xenia, OH</td>
			<td style="width:127px;">22005</td>
			<td>Indicating DRIERITE 6 mesh</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:341px;">Destination vector for plant transformation</td>
			<td>TAIR</td>
			<td style="width:127px;">Vector:6531113857</td>
			<td>pKGWFS7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electroporation cuvette</td>
			<td>USA Scientific</td>
			<td>9104-5050</td>
			<td>Electroporation cuvette, round cap, 0.2 cm gap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electroporator</td>
			<td>BIO-RAD Laboratories, Hercules, CA</td>
			<td>1652100</td>
			<td>MicroPulser Electroporator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fertilizer beads</td>
			<td>Osmocote Garden, Marysville, OH</td>
			<td>N/A</td>
			<td>Osmocote Smart-Release Plant Food&#160;Flower &#38; Vegetable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel extraction kit</td>
			<td>iNtRON Biotechnology, Boston, MA</td>
			<td>17289</td>
			<td>MEGAquick-spin Total fragment DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G1914-5G</td>
			<td>Gentamicin sulfate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Glufosinate-ammonium (11.3%) herbicide (BASTA)</td>
			<td>Bayer environmental science, Montvale, NJ</td>
			<td>N/A</td>
			<td>FINALE herbicide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td>VWR International, Radnor, PA</td>
			<td>200004-444</td>
			<td>Kanamycin monosulfate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES</td>
			<td>Bioworld, Dublin, OH</td>
			<td>41320024-2</td>
			<td>MES, Free Acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS salt</td>
			<td>MP Biomedicals, Santa Anna, CA</td>
			<td>092621822</td>
			<td>Hoagland&#39;s modified basal salt mixture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N6-benzylaminopurine (BA)&#160;</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>B3274</td>
			<td>6-Benzylaminopurine solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Alrich</td>
			<td>S7653</td>
			<td>Sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-ionic detergent</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>9005-64-5</td>
			<td>TWEEN 20&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Plasmid isolation kit</td>
			<td>Zymo Research, Irvine, CA</td>
			<td>D4036</td>
			<td>Zyppy Plasmid Kits</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinase enzyme mix kit</td>
			<td>Life Technology</td>
			<td>11791-020</td>
			<td>Gateway LR Clonase II Enzyme mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>R3501-1G</td>
			<td>Rifampicin, powder, &#62;= 97% (HPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>ThermoFisher Scientific, Waltham, MA</td>
			<td>SHKE4450</td>
			<td>MaxQ 4450 Benchtop Orbital Shakers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soil mix</td>
			<td>Sun Gro</td>
			<td>SUN239223328CFLP</td>
			<td>Sun Gro Metro-Mix 360 Grower Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectinomycin</td>
			<td>VWR International, Radnor, PA</td>
			<td>IC15206705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile 50ml conical tubes</td>
			<td>USA Scientific, Ocala, FL</td>
			<td>1500-1811</td>
			<td>50 ml conical screw cap tubes, copolymer, racks, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>VWR International, Radnor, PA</td>
			<td>57-50-1</td>
			<td>Sucrose, ACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surfactant solution</td>
			<td>Lehle seeds, Round Rock, TX</td>
			<td>VIS-02</td>
			<td>Silwet L-77</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Topoisomerase-based cloning kit</td>
			<td>Life Technologies, Carlsbad, CA</td>
			<td>K240020</td>
			<td>pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>VWR International, Radnor, PA</td>
			<td>90000-282</td>
			<td>BD Bacto Tryptone, BD Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR International, Radnor, PA</td>
			<td>90000-722&#160;</td>
			<td>BD Bacto Yeast Extract, BD Biosciences</td>
		</tr>
	</tbody>
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plant growth and agrobacterium-mediated floral-dip transformation of the extremophyte schrenkiella parvula

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 &#176;C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 &#181;mol m-2s-1 light intensity, at 22 &#176;C to 24 &#176;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&#8211;4 transgenic plants per 10,000 T1 seeds propagated.

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 &#8211;80 flowers in multiple inflorescences at 60&#8211;80 days after germination a...

Watch this Scientific Journal Video about Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula at JoVE.com

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

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.

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

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic ...

This method, a reproducible transformation protocol for Schrenkiella parvula, can help answer questions in the field of plant comparative and functional genoming, especially studies investigating plant adaptations to harsh environments. This technique enables investigations of routine functions in Schrenkiella parvula on the extremophyte adapted to multi-ion salt stresses. It also enables comparative studies which also looks at its close relative, the mother parent Aribidopsis Thaliana.Comparing Schrenkiella Parvula with Aribidopsis Thaliana can provide insight into variances in gene regulation and function that enable the unique adaptation of Schrenkiella parvula. The transformation protocol for Arabidopsis Thaliana has been already well-established. The method now enables comparative studies using transgenic Schrenkiella Parvula plants.Generally researchers new to this method will struggle because of the flowering habit and the leaf morphology of Schrenkiella Parvula plants which make effective transformation using a flora dip as well as the selection of true transformants difficult. To begin, use a wet toothpick to transfer about 20 to 25 S.Parvula seeds onto wet soil. Then stratify the seeds at four degrees celsius for five to seven days.After stratification, return the seeds to the growth chamber for seven to 10 days. Once all of the seedlings have germinated, leave only one seedling in each of the four corners and at the center of the pots. Grow the plants for eight to 10 weeks in the growth chamber until they flower.On the day of transformation, select S.Parvula plants according to the text protocol. Use forceps and small scissors to remove any pollinated inflorescences that are already developing into siliques. After plants are ready, prepare an agrobacterium infiltration solution.After this, dip the S.Parvula inflorescence in agrobacterium infiltration solution for 20 seconds. Shown here is the earliest stage of the plants that can be used for transformation, as long as already developed siliques are removed before transformation, older plants with more flowers can be used. After the transformation, place flower dipped plants horizontally in clean trays with domes to cover the plants.Then place the plants in a dark growth room for one to two days. Return the plants to an upright position and transfer them to the growth room. In the following week, monitor the dipped inflorescences.Ten days after the first floral dip, S.Parvula plants will keep producing new inflorescences and siliques. A second round of floral dip can be performed to further transform the newly emerging inflorescences. Grow the plants until seeds mature, and harvest the seeds around 21 weeks.Dry the seeds for two to three weeks at room temperature in an airtight container filled with desiccants. Plant the T one seeds according to the text protocol. Grow the plants until the first two to three true leaves develop.To perform the first selection for herbicide resistance, dilute glufosinate ammonium herbicide and spray it on the seedlings. Cover the seedlings with the domes overnight. Put the trays back to the growth room, and remove the dome.Wait until most of the seedlings are selected out by the herbicide. To begin the second selection, identify the plants that survive after being sprayed three to four times with the BASTA solution. After growing the plants for two to three more weeks, select the largest mature leaf on each plant.Next rub the surface of the leaf gently with a finger to remove the wax layer. Then place a drop of the diluted BASTA solution on the leaf. Monitor the leaves treated with the BASTA solution for signs of wilting for up to one week.Finally select the plants with leaves unaffected by the BASTA drops for further analysis. In this protocol, S.Parvula was transformed with agrobacterium using a floral dip method. Infection with agrobacterium resulted in abortion of some flowers.However the S.Parvula plants continued to produce new flowers after the first flower dipping, and could be transformed again. Using the BASTA spray and drop tests, true positive transformants can be accurately identified. The number of samples tested using PCR confirmation can be reduced.While this method is similar to the transformation of Arabidopsis Thaliana, it is important to remember the differences in growth and morphology of Schrenkiella Parvula. Modifying the protocol to reflect these differences is critical for successful transformation. This technique could help researchers explore the evolution of genomic variations leading to multiple salt stress adaptations in the extremophyte model, Schrenkiella Parvula.Combining this technique with the information on the functions of Aradopsis Thaliana, another plant closely related to Schrenkiella Parvula. Researchers can study the variations in the regulation and functions that these are two distinct structures between these two species. During the transformation process, it is important to contain all the agrobacterium infiltration solution in autoclaveable containers.Sterilize and safely discard all the materials that came in contact with agrobacterium including gloves, macro pipette tips, and paper towels.

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