Efficient Protoplast Regeneration Protocol and CRISPR/Cas9-Mediated Editing of Glucosinolate Transporter (GTR) Genes in Rapeseed (Brassica napus L.)

Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3–4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l−1) and 2,4-D (0.5 mg l−1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l−1 TDZ in combination with auxin 0.5 mg l−1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.

Effect of Enzyme Treatments on Protoplast Isolation from Leaves of Vetiver (Vetiveria spp.)

Protoplast isolation is a first and important step for establishing a new plant with desired traits through protoplast fusion technology. This experiments were conducted to evaluate various concentration of enzymes and incubation time on protoplast yield and viability in two vetiver ecotypes, Kamphaeng Phet 2 (Vetiveria zizanioides Nash) and Prachuap Khiri Khan (V. nemoralis A.Camus). The results revealed that protoplast yields were significantly affected by different enzyme treatments. The highest protoplast yield (6.12x105 protoplasts/ml) and high viability (98.61%) in Kamphaeng Phet 2 was obtained through the process of cell wall digestion when treated with enzyme solution containing 0.5% (w/v) cellulase onozuka R-10 and 0.5% (w/v) macerozyme R-10 in combination. While, the optimal enzyme solution for protoplast isolation from leaves of Prachuap Khiri Khan was the combination of 1.0% (w/v) cellulase onozuka R-10 and 0.4% (w/v) macerozyme R-10, resulting in the highest yield (6.80x105 protoplasts/ml) and viability (96.56%) of protoplasts. Meanwhile, incubation time of 24 h with the optimal enzyme solution resulted in the highest protoplast yields of both ecotypes. Our findings have the potential to generate an efficient protocol to isolate the protoplast from leaves of vetiver which can be used for further research studies in protoplast culture and fusion for vetiver improvement. Keywords: Cellulase onozuka R-10, Macerozyme R-10, Protoplast isolation, Vetiver

Plant Breeding Through Protoplast Fusion

Protoplast culture (protoplast fusion) is one method of tissue culture that is widely used in plant breeding programs in a relatively short time. This method is used to overcome the problem of plants that are difficult or impossible to cross conventionally as well as used for species improvement by transferring the desired gene from the donor plant to the target plant via protoplast fusion. Protoplast fusion makes it possible to produce plants that are resistant to a disease and various abiotic stresses, rapid growth rates and have a better quantity and quality of metabolites than their parents. Various factors affect the success of fusion and regeneration of protoplasts into whole plants, including the source of explants, the composition of the enzyme solution and the duration of incubation, fusagen type and culture media for regeneration.

ACTIN7 Is Required for Perinuclear Clustering of Chloroplasts during Arabidopsis Protoplast Culture

In Arabidopsis, the actin gene family comprises eight expressed and two non-expressed ACTIN (ACT) genes. Of the eight expressed isoforms, ACT2, ACT7, and ACT8 are differentially expressed in vegetative tissues and may perform specific roles in development. Using tobacco mesophyll protoplasts, we previously demonstrated that actin-dependent clustering of chloroplasts around the nucleus prior to cell division ensures unbiased chloroplast inheritance. Here, we report that actin-dependent chloroplast clustering in Arabidopsis mesophyll protoplasts is defective in act7 mutants, but not act2-1 or act8-2. ACT7 expression was upregulated during protoplast culture whereas ACT2 and ACT8 expression did not substantially change. In act2-1, ACT7 expression increased in response to loss of ACT2, whereas in act7-1, neither ACT2 nor ACT8 expression changed appreciably in response to the absence of ACT7. Semi-quantitative immunoblotting revealed increased actin concentrations during culture, although total actin in act7-1 was only two-thirds that of wild-type or act2-1 after 96 h culture. Over-expression of ACT2 and ACT8 under control of ACT7 regulatory sequences restored normal levels of chloroplast clustering. These results are consistent with a requirement for ACT7 in actin-dependent chloroplast clustering due to reduced levels of actin protein and gene induction in act7 mutants, rather than strong functional specialization of the ACT7 isoform.

Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus is Affected by Phytosulfokine (PSK)

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.