Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Our research aimed at developing a protocol for an efficient transformation in brassica C.Using a food-based transformation, we improved dramatically transformation efficiency in brassica napus, which is crucial for our research. We also tested the protocol in model species Arabidopsis Thaliana. We have optimized the protocol for spring cultivar DH 12075 in Brassica Napus.

The next challenge is to optimize the protocol of transformation and regeneration of winter cultivar in Brassica Napus, as well as for brassica napa and brassica oleracea. Hair roots can be readily propagated and the rapid growth enables the prompt analysis of the gene of interest shortly after the transformation. Consequently, it becomes feasible to selectively regenerate specific hairy root lines that exhibit the desire trait.

For example, a gene mutation introduced by crisp percuss. To begin place the seeds of brassica napus DH 12075 in micro tubes. To degrease the seeds, add water and 0.1%detergent to the micro tube and shake for 60 seconds.

Now, rinse the seeds with water followed by 70%ethanol, each for 60 seconds. Next, place the seeds in 10%sodium hypochlorite solution and shake for 20 minutes. After removing the bleach, wash the seeds four times with sterile water for 60 seconds each.

Place the seeds on a Petri dish containing the seed germination medium. Cold stratify the seeds at four degrees Celsius overnight before moving the dish to a cultivation room. After five days, transfer the seedlings to a plant culture box containing a plant growth medium.

Inoculate, Ti-less agrobacterium tumefacins, C58C1, carrying a hairy root inducing plasmid, PRIA 4B in five milliliters of LB medium. Grow the culture overnight at 28 degrees Celsius until it reaches an optical density at 600 nanometers of 0.9 to one. Transfer the bacterial inoculum into the syringe.

Using a 26 gauge needle mounted on the syringe. Puncture the hypocotyl of an 18 day old seedling at about one centimeter above the growth medium. Inject approximately 50 microliters of inoculum into the wound and scratch the tissue on the surface of the hypocotyl.

Return the plant to the cultivation room at 21 degrees Celsius for two to four weeks. Next, cut off the callus with emerged hairy roots from the hypocotyl. Place the callus with hairy roots on a Petri dish contain hairy root growth medium and antibiotics to suppress agrobacterial growth.

Seal the dish with gas permeable tape before incubating it at 24 degrees Celsius. After incubation, isolate the hairy roots from the callus and transfer the individual roots to a hairy root growth medium. Alternatively, to generate a composite plant consisting of wild type shoots and transgenic hairy roots, remove the native roots of the plant, then transfer the plant with emerging hairy roots to a culture box containing plant growth medium and antibiotics to suppress agrobacterial growth.

Under sterile conditions, using tweezers, transfer five to 10 hairy root lines of brassica napus on a dish containing regeneration medium. Seal the dish with gas permeable tape and culture at 21 degrees Celsius in a long day photo period. Every three to four weeks, transfer the hairy roots to a Petri dish with fresh regeneration medium.

Observe the callous formation after approximately two weeks and shoot from the callus after two more weeks of callus formation. Individualize the shoots and transfer them to the plant culture box containing the shoot elongation medium. After two to three weeks, cut off the remnants of the callus from the shoot and transfer the elongated shoots to the plant culture box containing root induction medium.

Now, transfer the rooted plant to the soil and acclimatize it first in the phytotrons. Then, transfer the plant to a greenhouse for flowering. To speed up the production of T one plants, Collect siliques containing mature green seeds from the T zero regenerates.

while working in a sterile flow box, surface sterilized the siliques with 70%ethanol. Place the sliliques on a plate lid or a slide. Using a 24 gauge needle mounted on a one milliliter syringe, slit the silique along the valve margins.

Open the carpals and put them aside. Collect the immature seeds and transfer them to a plate containing seed germination medium. Seal the plate and place it in a cultivation room until germination.

Compare to the wild type hairy root regenerates of B.napus displayed a dwarf phenotype with dense root systems, wrinkled leaves, and altered flowering time. To begin, place the surface sterilized Arabidopsis thaliana col-0 seeds onto a plate containing seed germination medium. After two days of cold stratification, move the plate to a cultivation room set at 21 degrees Celsius with a long day photo period and 50%humidity.

Transfer one week old seedlings to a plant culture box with a plant growth medium. Grow the agrobacterium tumefacins culture in LB medium until it reaches an optical density at 600 nanometers of 0.9 to one. Using a needle mounted on an insulin syringe, inject approximately 50 microliters of bacterial culture at the base of the primary influorescent stem of a one month old Arabidopsis plantlet.

Scratch the tissue on the surface of the primary stem with the syringe. After two to four weeks, excise the emerging hairy roots and cultivate them on a Petri dish with the hairy root growth medium, supplemented with antibiotics to suppress agrobacterial growth. Culture the hairy root at 24 degrees Celsius in the dark.

After one to two weeks, individualize the hairy roots on the plate with hairy root growth medium and transfer selected hairy root lines to a fresh medium every four to five weeks. Transfer the hairy roots to a plate with a regeneration medium to induce callous formation and culture at 21 degrees Celsius in a long day photo period. Once the shoots emerged from the callus, cut the shoots and transfer them to a shoot elongation medium for two to three weeks to promote growth and elongation.

Then transfer the elongated shoots to the root induction medium. Finally transfer the rooted plant to the soil to perform transgenic selection. In A.thaliana, yellow Cali were induced within 14 days in all tested hairy root lines.

First shoot primordia, visible as dark green spots, emerged within three weeks after transfer to the regeneration medium. After four weeks, shoots covered the hairy roots in 90%of the lines with some cases showing adventitious root formation. However, one line did not regenerate even after three months.

Compared to the wild type hairy root regenerates of A.thaliana displayed a dwarf phenotype with dense root systems, wrinkled leaves, and altered flowering time.