Journal Article

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9

Zhang, Hanguo, Wang, Junhui, Cao, Qing, Wang, Chen, Yan, Shanshan, Zhang, Lei, Zhang, Sufang, An, Peiqi

2021

Overview

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG 4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium -mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD 600 of the infection solution(suspension of A . tumefaciens ) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: 2,4-D / Abscisic acid / Analysis / Biology and Life Sciences / Breeding / Callus / Cones / Conifers / Dichlorophenoxyacetic acid / Embryonic growth stage / Embryos / Forestry / Genetic aspects / Genetic engineering / Genetic transformation / Germplasm / Growth regulators / Hygromycin / Hygromycin B / Inositol / Kinetin / Laboratories / Larch / Larix gmelinii / Larix kaempferi / Physical Sciences / Plant growth / Plant hormones / Propagation / Research and Analysis Methods / Seeds / Somatic embryogenesis / Sucrose / Transformations / Trees