Zhenlan Liu

Efficient culture protocol for plant regeneration from petiole explants of physiologically mature trees of Jatropha curcas L.

An efficient and reproducible protocol for induction of adventitious shoot buds and plant regeneration from petiole explant cultures of Jatropha curcas, an important biofuel crop, is described. Physiologically mature trees of three J. curcas genotypes were selected and explants were prepared from young petioles. Treating the explants with high concentrations (5 to 120 mg/L) of thidiazuron (TDZ) solution for short time periods (5 to 80 min) helped increase the regeneration frequency and improved the quality of the regenerated buds significantly. The age of the petioles and inoculation methods were found to influence the culture results. The best shoot buds induction (65.78%) and number of buds (6.77) per explant was seen in the second petiole explants of genotype M-1 treated with 20 mg/L TDZ solution for 20 min, followed by 35-day culture on hormone-free Murashige and Skoog medium. The regenerated buds could elongate to become shoots in a medium containing gibberellic acid. The elongated shoots initiated roots to become intact plantlets in rooting medium containing indole-3-butyric acid and L-glutamine (Gln), and supplementing 16 mg/L Gln into the rooting medium effectively stimulated the initiation and growth of roots, with the best rooting rate (51.72%). After acclimatization, these plantlets were transplanted to soil wherein normal growth was observed. Therefore, an intact plantlet could usually be obtained at 60 days of culture by using the culture protocol described in this study. This protocol can be used for mass production of true-to-type plants and the production of transgenic plants through Agrobacterium/biolistic-mediated transformation.

An Efficient Rice Mutagenesis System Based on Suspension‐Cultured Cells

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells, with rice (Oryza sativa L.) as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22 h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.

Functional identification of ELO-like genes involved in very long chain fatty acid synthesis in Arabidopsis thaliana

Very long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh. that AtELO1, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3, and AtELO4 were localized in the endoplasmic reticulum (ER), where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10–20% in seeds of atelo1 T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and might lay the foundation for further genetic manipulations to generate oil crops with the high oil content.