No abstract

A plant tissue culture protocol from stevia was optimized for the production of planting materials and the natural sweetener, rebaudioside A. The highest survivability (88.90% ± 5.55) of explants was achieved at 15 and 30 days after culture initiation (DACI) on Murashige and Skoog (MS) media by sterilization with 30% Clorox (5 min) and 10% Clorox (10 min), respectively. Supplementation of MS with 0.50 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.10 mg/L zeatin produced 50% callus at 15 DACI while 1.50 mg/L 2,4-D and 0.10 mg/L zeatin at 30 DACI increased callus production to 76.67%. The highest shoot proliferation per callus was achieved with 10.00 mg/L 6-benzyl amino purine (BAP) in MS at 15 DACI (5.80) and 30 DACI (12.33). The longest shoots of 4.31 cm and 6.04 cm at 15 and 30 DACI, respectively, were produced using BAP (10.00 mg/L) and 1.00 mg/L naphthalene acetic acid (NAA). MS media (0.50 strength) induced 2.86 and 6.20 roots per shoot and produced 3.25 cm and 7.82 cm long roots at 15 and 30 DACI, respectively. Stevia grown on 0.25 MS accumulated the highest concentration of rebaudioside A (6.53%), which correlated with the expression level of its biosynthetic gene uridine-diphosphate-dependent (UDP)-glycosyltransferase (UGT76G1).

The UDP-glycosyltransferase 76G1 (UGT76G1) is responsible for the conversion of stevioside to rebaudioside A. Four single guide RNAs (sgRNAs) were designed from the UGT76G1 proximal promoter region of stevia by using the online-based tool, benchling. The dCas9 fused with VP64 as a transcriptional activation domain (TAD) was produced and purified for the formation of ribonucleoproteins (RNPs) by mixing with the in vitro transcribed sgRNAs. Protoplast yield was the highest from leaf mesophyll of in vitro grown stevia plantlets (3.16 × 106/g of FW) using ES5 (1.25% cellulase R-10 and 0.75% macerozyme R-10). The RNPs were delivered into the isolated protoplasts through the Polyethylene glycol (PEG)-mediated transfection method. The highest endogenous activation of the UGT76G1 gene was detected at 27.51-fold after 24 h of transfection with RNP30 consisting of CRISPR/dCas9-TAD with sgRNA30 and a similar activation level was obtained using RNP18, RNP33, and RNP34, produced using sgRNA18, sgRNA33, and sgRNA34, respectively. Activation of UGT76G1 by RNP18 led to a significant increase in the expression of the rate-limiting enzyme UGT85C2 by 2.37-fold and there was an increasing trend in the expression of UGT85C2 using RNP30, RNP33, and RNP34. Successful application of CRISPR/dCas9-TAD RNP in activating specific genes can avoid the negative integration effects of introduced DNA in the host genome.