Abstract
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 PEG4000 (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 OD600 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.
Highlights
MethodsPlant materialsThe plant material, the immature cones of hybrid larch Larix kaempferi 3x Larix gmelinii 9 freely pollinatedis(ultivated and open pollination in an orchard) without damaging the plant population and comply with relevant institutional, national, and international guidelines and legislation, selected from seedling seed orchard, were collected on June 27, July 1, July 4, July 7, and July 16, 2017(about 58, 62, 65, 68 and 77 days after pollination, respectively), from the Qingshan seed orchard, Linkou County, Heilongjiang Province and the plant materials in this study are available from the Qingshan seed orchard upon reasonable request
Larch is among the main genus in the representative cold-humid coniferous forest
The best sterilisation method was as follows: first, after peeling the seed coat, the explants were placed in a 4 ̊C refrigerator for 12 hours and washed with sterile deionized water 3 times; second, the samples were disinfected with 75% alcohol for 1 min and washed with sterile deionized water 3 times; the samples were disinfected with 3% NaClO for 10 min and washed with sterile deionized water 3 times
Summary
Plant materialsThe plant material, the immature cones of hybrid larch Larix kaempferi 3x Larix gmelinii 9 freely pollinatedis(ultivated and open pollination in an orchard) without damaging the plant population and comply with relevant institutional, national, and international guidelines and legislation, selected from seedling seed orchard, were collected on June 27, July 1, July 4, July 7, and July 16, 2017(about 58, 62, 65, 68 and 77 days after pollination, respectively), from the Qingshan seed orchard, Linkou County, Heilongjiang Province and the plant materials in this study are available from the Qingshan seed orchard upon reasonable request. The collected cones were packed and marked in self-sealed bags, stored in ice boxes and taken to the laboratory. The cones were stored in a 4 ̊C refrigerator until use within 14 days. The water on the surface of the explant was blotted dry with sterile filter paper. The following two disinfection methods were carried out:(1) the seeds were cut longitudinally, and the embryo and downward incised endosperm were inoculated in embryogenic callus induction medium, or (2) an incision was made from the top of the seed, and the embryo was removed from the endosperm, placed on sterile filter paper until the water on the surface was blotted dry, and inoculated in embryogenic callus induction medium
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