Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional ...Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.展开更多
Embryonic axis and cotyledon of immature and mature embryos were induced as explants for embryogenesis in Ginkgo biloba. Results showed somatic embryos could be induced only through embryonic axis and cotyledon of imm...Embryonic axis and cotyledon of immature and mature embryos were induced as explants for embryogenesis in Ginkgo biloba. Results showed somatic embryos could be induced only through embryonic axis and cotyledon of immature embryos. MK + NAA 1 mg·L -1 + 6_BA 1 mg·L -1 was the best medium for somatic embryogenesis from embryonic axis, and somatic embryogenesis rate was 45.28%. MK + NAA 1.5 mg·L -1 + 6_BA 1 mg·L -1 was the best medium for somatic embryogenesis from cotyledon, and somatic embryogenesis rate was 12.9%. The growth and development of somatic embryos were promoted by adding 10% coconut milk into MK, and 34.48% somatic embryos could develop into plants.展开更多
基金supported by Biological Breeding of Early Maturing and Disease Resistant Cotton Varieties (NO.2023ZD04041)the Project of China Agriculture Research System (Grant No. CARS-15-06)+2 种基金Natural Science Foundation of Henan Province (Grant No. 232300421041 and 222300420382)National Natural Science Foundation of China (Grant No. U21 A20213)the Central Public-interest Scientific Institution Basal Research Fund (Grant No. 1610162023017 and 1610162023028)。
文摘Background Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.Results To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5%(w/v) cellulase, 0.75%(w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L^(-1) mannitol. After 5 h of dark incubation at 25℃, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10^(6) protoplasts per gram(fresh weight) and 95% viability. We incubated 100 μL protoplasts(2.5 × 10^(5)·m L^(-1)) with 15 μg plasmid in the solution of 0.4 mol·L^(-1) mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.Conclusions This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation(Bi FC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.
文摘Embryonic axis and cotyledon of immature and mature embryos were induced as explants for embryogenesis in Ginkgo biloba. Results showed somatic embryos could be induced only through embryonic axis and cotyledon of immature embryos. MK + NAA 1 mg·L -1 + 6_BA 1 mg·L -1 was the best medium for somatic embryogenesis from embryonic axis, and somatic embryogenesis rate was 45.28%. MK + NAA 1.5 mg·L -1 + 6_BA 1 mg·L -1 was the best medium for somatic embryogenesis from cotyledon, and somatic embryogenesis rate was 12.9%. The growth and development of somatic embryos were promoted by adding 10% coconut milk into MK, and 34.48% somatic embryos could develop into plants.
