This method can help answer key questions in miRNA response to ability to stress in plant grafts such as how miRNA are regulated under cold stress in watermelon bottle gourd grafts. The main advantage of this technique is that it is highly efficient and reproducible method to make homo and heterografts. It requires no specific equipment, it is very easy to perform, and typically results in a very high survival rate of grafting.
Through this method, can provide insight into miRNA response to cold stress in the watermelon bottle gourd graft system. It can also be applied to other modern organisms for revealing the mechanisms of local and long distance miRNA transportation. Visual demonstration of this method is critical as the grafting steps are difficult to learn.
This step required advanced skills and are key to the survival of the grafted plants. To begin, soak bottle gourd seeds in a 500 milliliter beaker of 58 degrees Celsius water. Stir the seeds occasionally until the water temperature drops to 40 degrees Celsius.
While the water cools, put three kilograms of peat soil into a nylon bag and autoclave it. Once the water reaches room temperature, rinse the seeds two to three times in distilled water and drain the excess water. Allow the seeds to sprout in a gauze bag at 28 degrees Celsius in the dark growth chamber.
After germination, sow the seeds into plastic pots filled with the sterilized peat soil. When the bottle gourd seedlings develop two flattened cotyledons, repeat this process with watermelon seeds. Grow the bottle gourd and watermelon seedlings in a growth chamber.
Add water to the seedlings once per day in the afternoon. Next, cut the hypocotyls of the watermelon seedlings two to three centimeters below the cotyledons. Cut the top of the bottle gourd seedlings at the side, immediately above the cotyledons.
Then use a toothpick to make a hole in the top of the trimmed bottle gourd seedlings. Insert the trimmed watermelon seedlings into the hole to make heterografts. After this, prepare homografts using the previously described method.
First, wrap the grafted seedlings in transparent polyethylene bags to maintain a relatively high humidity. Then maintain the wrapped seedlings in a growth chamber for seven days. After the seventh day, remove the bags and allow the plants to grow under the same conditions for seven to 10 days.
Divide the seedlings into two groups, one for cold treatments and one for control. Return the control seedlings to the same environmental conditions. Place the cold treated seedlings into a growth chamber set to six degrees Celsius with the same light dark conditions as the control group.
After 48 hours, leave the samples of the scion and rootstocks from the grafts. Freeze the samples immediately in liquid nitrogen and store them at minus 70 degrees Celsius. Transfer the frozen sample to a two milliliter microcentrifuge tube in liquid nitrogen.
Add a stainless steel bead to each sample tube and homogenize the tissues to a fine powder. For each grafting combination, take equal amounts of ground sample from 10 seedlings and mix them in a 10 milliliter centrifuge tube. Add an appropriate amount of guanidium hydrochloride reagent.
Next, add RNA-free DNase one to 150 units per milliliter at 37 degrees Celsius for one hour. Then determine the total RNA quantity on a microcapillary electrophoresis system. After this, thaw the library normalization reagents and adapters.
Then ligate small RNase with the five prime and three prime adapters, and elute and purify them. Reverse transcribe the five prime and three prime ligated small RNase following the manufacturer's guidelines. Next, perform PCR amplification following the manufacturer's protocol.
Then use the microcaillary electrophoresis system to ensure that the RNA integrity number is greater than seven. Load one microliter of an RNA library on the microcapillary electrophoresis system to ensure that the RNA integrity number is greater than seven. Finally, sequence the small RNA libraries on a high throughput sequencing instrument.
Using this protocol, a 98%survival rate for grafting and the phenotypes for room temperature and cold stress conditions were obtained. sRNAs of 24 nucleotides made up the biggest class of sRNAs in all grafting combination, regardless of temperature treatment. After 48 hours of cold treatment, 30 and 268 microRNAs were up and down-regulated, respectively.
In the leaves of the scion in heterografts, conversely, in the leaves of the rootstock, 31 and 12 microRNAs were up and down-regulated, respectively. In the watermelon-watermelon homografts, 64 and 83 microRNAs were up and down-regulated, respectively. This demonstrated that heterografting caused profound reprogramming of the microRNA expressions.
While attempting this procedure, it's important to remember the relative size and age of the daughter stalk and the scion is critical to making a successful graft. Following this procedure, other methods like lncRNA sequencing, proteomic profiling can be performed in order to investigate the regulation of lncRNA and protein and the coding system in the grafting system. After its development, this technique paved the way for research in the field of plant abiotic tolerance to explore the mechanism of plant grafting advantage in Cucurbitaceae and beyond.
Don't forget that working with guanidine hydrochloride can be extremely hazardous and appropriate precaution should always be taken with performing this procedure.
