In this study, we use the as a vector to introduce foreign DNA fragments into bamboo leafs. Most DNA fragments are not integrated into chromosomes and good transferred expression. We took activation of this transferred expression to carry out gene editing.
The main challenge is the limitation of gene editing of bamboo leaves, which cannot regenerate. These propose difficulties in performing editing in regenerative organs, like young shoots, and opportunity added benches are subsequent generations with desired genetic challenges. The main advantage is the time saving aspect of the principle.
By being able to edit bamboo genes directly in bamboo leaves, it allows for quick verification of gene factions. This eliminates the needs for time consuming procedures, like tissue culture or transformation, significantly speeding up research on bamboo gene infections. The primary research will be focused on investigating the molecular mechanism of bamboo biomass formation.
This will involve understanding why bamboo grows rapidly, and using that knowledge to develop bamboo varieties which are suitable for plastic substitute. To begin, sow the pre-soaked bamboo seeds into the substrate of soil and vermiculite for germination. For agrobacterium infection, carefully remove the 15 day old seedlings with soil attached roots from the substrate.
Wrap the seedlings with tinfoil to maintain moisture and prevent soil detachment. Transfer the wrapped seedlings to the plant growth chamber with higher humidity and lower illumination for two hours. Next, using a sharp needle from a syringe wound, the upper part of curled immature leaves once or twice.
Quickly dip the wounded part of the leaves into agrobacterium suspension. Then immediately transfer the seedlings to a vacuum chamber with a pressure of 25 to 27 millimeters of mercury for two minutes. After vacuuming, carefully unwrap the seedlings and replant them into the substrate.
Allow the seedlings to grow in a plant growth chamber in dim light, with high humidity for two days. Then culture the seedlings under normal growth conditions, watering them every five to seven days. Begin by extracting the genomic DNA from the wild type and agrobacterium infected bamboo leaves.
Amplify the genomic DNA containing the target site of the target genes from wild type and agrobacterium infected bamboo leaves, using the following PCR conditions. After PCR, perform endonuclease enzyme digestion by preparing a reaction mixture containing one microliter of AGE1, one microgram of PCR products, and five microliter 10 times buffer. Then add water to a final volume of 50 microliters.
Incubate the digestion mixture at 37 degrees Celsius for one hour. Using gel electrophoresis, analyze the proportion of digested DNA fragments. Compare the digested fragments from the wild type and agrobacterium infected samples to assess gene editing efficiency.
Expose the bamboo seedlings to high light intensity conditions to increase the amount of absorbed light quantum and activation of the leaf photo protection system. Next, turn on the imaging pam fluorimeter and set the actinic light intensity to measure in vivo photo system two, chlorophyll fluorescence of bamboo leaves The red beta lane color produced by the ruby gene indicated successful agrobacterium, mediated gene expression in bamboo leaves. Out of the four agrobacterium strains, GV3101 strain caused the most significant, beta lane accumulation in infected bamboo leaves.
After infection with the CRISPR CAS9 constructs and high light treatments, certain areas of the leaf blades showed lower, non-photo chemical quenching values. Furthermore, enzyme digestion and sequencing analysis confirmed the successful mutation of the PeVDE gene in the edited regions. Sanger sequencing results of the PeVDE fragment, after editing by both, sgRNA1 and sgRNA2 showed deletion of a long fragment in the PeVDE gene.
After 30 days of agrobacterium infection, the leaf areas transfected with sgRNAs for both PeVDE and PeCCR5 exhibited lower, non-photo chemical quenching values.
