Genetics
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Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula
Chapters
Summary January 7th, 2019
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.
Transcript
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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