Optimization of Protoplasts Preparation System and Establishment of Transient Transformation System in Brassica oleracea var. capitata L.

doi:10.7668/hbnxb.20191112

Abstract

Abstract: The effects of mannitol concentration, enzymatic hydrolysis time, centrifugal speeds and different explants on the yield and vitality of protoplasts were studied by orthogonal experiment to optimize the protoplasts preparation system. The transient transformation system of Brassica oleracea protoplasts from different explants was also established. The transient transformation system was also used for preliminary study of the CRISPR/Cas9 system. The results showed that the optimal enzyme solution for protoplasts isolation was 1% CelluseR-10+0.5% MacerozymeR-10+0.6 mol/L mannitol +10 mmol/L MES+1 mmol/L CaCl₂ + 0.1% BSA+5 mmol/L β-mercaptoethanol. The hypocotyl of Brassica oleracea was incubated in enzyme solution for 6 h at 26℃ in a dark rotary shaker at 60 r/min. The isolated protoplasts were centrifuged at 2 115 r/min for 5 minutes. The protoplasts yield amounted to 3.0×10⁶/g fresh weight and the vitality was about 90.9%. In addition, the PYBA 1132 vector with green fluorescent protein was successfully transformed into the purified protoplasts from hypocotyls, cotyledons and head leaves of Brassica oleracea by 20% PEG4000. The fluorescence signals were detected under the microscope. The transformation efficiency was about 43%, 35% and 19%, respectively. The protoplasts isolated from hypocotyls were the best suitable for transient transformation. The mitochondrion and plastid localization vectors COX and 969 were also successfully expressed in the protoplasts from hypocotyls. These results indicated that the transient transformation system had been established. It provided a platform for analyzing the gene localization and function in Brassica oleracea. At last, the editing capability of PBSE401-BoPDS was tested in the system. The results showed that the base substitution and insertion happened in the sequences near the target 1 and target 2. It meant that the CRISPR/Cas9 worked in this transient transformation system, which established an important platform for studying the gene functions and generating the germplasm resources of Brassica oleracea.

Key words: Brassica oleracea var. capitata L., Protoplast, Transient transformation, Orthogonal design, CRISPR/Cas9

CLC Number:

S635.03

WANG Fei, ZHONG Xionghui, CHEN Denghui, LI Hailong, KANG Jungen, XIE Jianming. Optimization of Protoplasts Preparation System and Establishment of Transient Transformation System in Brassica oleracea var. capitata L.[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(3): 69-78. doi: 10.7668/hbnxb.20191112.

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