A protocol of homozygous haploid callus induction from endosperm of Taxus chinensis Rehd. var. mairei

Abstract

Obtainment and characterization of the novel endosperm callus of Taxus chinensis Rehd. var. mairei are valuable for haploid breeding, genome, and functional genome in Taxus. Callus was obtained by hydropriming with sterile water for 3 days and suitable medium composition. The highest callus induction (70.89 %) and lower browning ratio (7.95 %) were obtained from Gamborg (B5) medium supplemented with 30 g l−1 of sucrose, 2.5 mg l−1 of 2,4-dichlorophenoxyacetic (2,4-D), 0.5 mg l−1 of 6-benzylademine (6-BA) and 7 g l−1 of agar under dark conditions. The auxin of 2,4-D had a better efficiency of callus induction than naphthylacetic acid, and over 1 mg l−1 of 6-BA was inhibitory to the callogensis of endosperm. The endosperm callus was haploid which was detectable by the flow cytometry. The genome block of homozygosity of callus was homozygous which was indicated by PCR-based SNP marks. The homozygous haploid of endosperm callus in vitro culture may be useful tools for taxoid-metabolism of gene engineering and bio-fermentation engineering.

The Taxus genome provides insights into paclitaxel biosynthesis

“…mairei was approximately 10 Gb (Extended Data Fig. 1a ), which is consistent with the results from the flow cytometry tests 23 . A de novo assembly of the Taxus genome was achieved by PacBio continuous long reads (318.05 Gb) and augmented with Illumina whole-genome sequencing reads (693.73 Gb) (Supplementary Table 1 ).…”

“…This feature of continuous insertion in the Taxus genome was distinctly different from that in the rice genome, where almost 95% of LTR insertions occurred within the last 5 million years 26 . Considering that LTR insertion in Norway spruce and ginkgo mainly occurred 12–24 and 16–24 Ma 23 , 25 , the continuous insertion of LTRs might be a common phenomenon in gymnosperms.…”

“…mairei were collected from the natural range of Taxus (113° 89′ 55′′ N, 28° 26′ 32′′ E) in the Liuyang region, Changsha city, Hunan Province, China, in November 2015. Single embryos and endosperm were induced as calli 23 , 38 .…”

“…For sequencing of the haploid tissue, DNA was extracted from the endosperm callus of T. chinensis var. mairei 23 . The DNA quality was checked by agarose gel electrophoresis and a Qubit fluorimeter (Thermo Fisher).…”

TheTaxus genome provides insights into paclitaxel biosynthesis

“…This feature of continuous insertion in the Taxus genome was significantly different from that in the rice genome, where almost 95% of LTR insertions occurred within the last 5 million years 26 . Considering that LTR insertion in Norway spruce and ginkgo mainly occurred 12-24 and 16-24 MYA 23,25 , the continuous insertion of LTRs might be a common phenomenon in gymnosperms.…”