A liquid suspension culture method for promoting somatic embryo maturation of liquidambar plant
A technology of Liquidambar genus and plant body, which is applied to the field of cell engineering breeding in the forestry industry, can solve the problem that the liquid culture somatic embryo cannot be reflected in the mature stage, the Liquidambar plant somatic embryo cannot mature normally, and the Liquidambar genus somatic embryo seedling. Low efficiency and other problems, to achieve the effects of suitable large-scale production, easy culture and management, and synchronization of somatic embryo development
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Embodiment 1
[0071] 1. Test method
[0072] The embodiments of the present invention refer to the methods in the following published academic papers: W.A.Vendrame C.P.Holliday S.A.Merkle, etc., Clonal propagation of hybrid sweetgum (Liquidambarstyraciflua × L.formosana) by somatic embryogenesis, Plant Cell Rep (2001) 20:691- 695.
[0073] 2. Test materials
[0074] 1. Immature seeds of hybrid sweetgum
[0075] In the present invention, the immature seeds of the hybrid sweetgum (golden sweetgum as the female parent and the sweetgum eucalyptus as the male parent) are used as explant experimental materials.
[0076] In June and July of each year, immature spherical capsules of hybrid sweetgum (gum xanthus × liquidgum) were collected, and seeds were taken out from them as explant materials.
[0077] 2. Plant growth regulator
[0078] The plant growth regulator used in the present invention adopts 6-benzylaminoadenine (6-BA) and 2,4-dichlorophenoxyacetic acid (2,4-D).
[0079] 3. Medium
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Embodiment 2
[0099] 1. Sterilization of explants
[0100] The explants were sterilized according to the method of W.A.Vendrame·C.P.Holliday·S.A.Merkle et al., Clonal propagation of hybrid sweetgum (Liquidambar styraciflua×L.formosana) by somaticembryogenesis, Plant Cell Rep (2001) 20:691-695).
[0101] 2. Embryogenic callus induction culture
[0102] Embryogenic callus induction culture was carried out according to the method of W.A.Vendrame·C.P.Holliday·S.A.Merkle et al., Clonal propagation of hybrid sweetgum (Liquidambar styraciflua×L.formosana) by somaticembryogenesis, Plant Cell Rep (2001) 20:691-695). figure 1 Embryogenic callus induced from immature hybrid sweetgum seed embryos.
[0103] After the immature zygotic embryos of the explants were cultured on the induction medium for 3-4 weeks, the growth rate of the embryogenic tissues began to slow down and the explants began to brown. At this time, the embryogenic tissues were selected for subculture and transferred to Repeated subcu...
Embodiment 2A
[0131] In addition to step 3, during the embryogenic callus proliferation and culture process, the typical embryogenic callus of the subcultured hybrid sweetgum is inoculated in the embryogenic callus proliferation liquid medium for suspension culture, and the inoculation amount is every 100ml 2-4g in the embryogenic callus proliferation liquid medium; carry out the liquid proliferation culture of the hybrid sweetgum embryogenic callus under dark conditions, wherein, the culture temperature is (25±2)°C, 100-120 revolutions per minute , the 2,4-D used in the embryogenic callus proliferation liquid medium was 0.5mg / L, the 6-BA was 0.25mg / L, the enzymatically hydrolyzed casein was 1g / L, and the sucrose was 40g / L; In the process of sexual callus proliferation and culture, it was subcultured every 3-4 weeks, that is, a new culture was started every 3-4 weeks, that is, 90% fresh medium was added, and the inoculum was kept at 2-4%. The amount of cycle multiplication is 1.5-4 times of...
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