Method for obtaining regenerated plants from common camellia oleifera in somatic embryogenesis mode

[0034] Example

[0035] The experimental steps are as follows:

[0036] In mid-July, good individual tea fruits were selected from mature camellia forests in Meihuashan Village, Shunhe Town, Macheng, Hubei, and stored in a refrigerator at 4°C. The sterilization inoculation was completed within 3 days.

[0037] Under the aseptic condition of the ultra-clean workbench, peel off the peel of Camellia oleifera fruit with your bare hands, disinfect the surface in 75% alcohol for 2 minutes, then disinfect with 0.1% HgCl2 for 10 minutes, rinse with sterile water 4 times, and peel off the inside Seed coat. Under aseptic conditions, the immature embryos were cut into pieces, and each immature embryo was cut into 3-5 pieces and inoculated into 90mm petri dishes (callus induction medium), and 7-9 explants were inoculated per dish.

[0038] The medium used for callus induction (callus induction medium) is a modified MSB medium supplemented with plant hormones. The composition of MSB medium is: potassium nitrate 1.9g/L, ammonium sulfate 1.65g/L, diphosphate Potassium hydrogen 0.17g/L, magnesium sulfate heptahydrate 0.37g/L, calcium chloride dihydrate 0.44g/L, manganese sulfate tetrahydrate 22.3mg/L, boric acid 6.2mg/L, zinc sulfate heptahydrate 8.6mg/L, Potassium iodide 0.83mg/L, sodium molybdate dihydrate 0.25mg/L, copper sulfate pentahydrate 0.025mg/L, cobalt chloride hexahydrate 0.025mg/L, sodium ethylenediaminetetraacetate 37.3mg/L, sodium sulfate heptahydrate Iron 27.8mg/L, Glycine 2mg/L, Niacin 1mg/L, Thiamine Hydrochloride 10mg/L, Pyridoxine Hydrochloride 1mg/L, Inositol 100mg/L, additional 3% (W/V) sucrose, 0.9 %(W/V) agar, pH is 5.8~6.0. In addition, the two added plant hormone combinations are 2,4-dichlorophenoxyacetic acid 2.0mg/L and kinetin 1.0mg/L (2,4-D+KT), and naphthalene acetic acid 5.0mg/L and kinetin 1.0mg/L (NAA+KT). Use high-pressure steam sterilization, sterilization pressure is 1.2kg/cm2, temperature is 121℃, sterilization time is 15min.

[0039] Place the petri dish (callus induction medium) inoculated with immature embryos in a constant temperature incubator at 28°C for 16 hours of light per day at a light intensity of 2000 lux. Obvious callus formation can be seen after 30 days of induction culture ( figure 1 ). Subculture the well-grown callus to differentiation medium (MSB medium with 1.0mg/LNAA+1.0mg/LKT, pH adjusted to 5.8) for differentiation regulation, subculture twice, after 2 months of culture, it is obvious Mature embryoid bodies and embryogenic callus of cotyledons ( figure 2 ), transfer the explants with their embryoid bodies into the embryoid body development and plant regeneration medium (MSB medium 1.0mg/LNAA+3.0mg/L6-BA, pH adjusted to 5.8) and continue to culture, Generation 1 time, culture for 2 months. After obtaining embryoid bodies with obvious cotyledons, transfer to rooting medium (1/2MS+3.0mg/L6-BA, pH adjusted to 5.8) and culture for 1 month ( image 3 ). After reaching the root system, the seedlings are cultivated by hydroponics, and after acclimation, they are transplanted into a soil bowl mixed with nutrient soil and vermiculite (3:1). Complete regenerated plants can be obtained within 6 months from the extraction of young embryos of Camellia oleifera, which are domesticated and transferred to the nutrient bowl to continue growing. The above treatment was repeated 3 times for each 18 dishes, and subcultured once for 30 days. Tissue culture of immature embryos was carried out according to this method. The callus induction rate of 2,4-D+KT and NAA+KT treatments were 83.23% and 45.34%, respectively; the embryo emergence rate was 15.25%. The callus induction rate was counted after 30 days of induction culture, and the embryo rate was counted after 60 days of differentiation-regulated culture. The culture conditions in this example are all cultures in a constant temperature incubator at 28°C, with 16 hours of light per day, and light intensity above 2000 lux.

[0040] Callus induction rate (%)=number of anthers forming callus/number of anthers inoculated×100%.

[0041] Results: The average callus induction rate under the 2,4-D+KT and NAA+KT plant hormone combinations were 83.23% and 45.34%, respectively.

[0042] Embryo emergence rate (%)=number of differentiated embryoid bodies/number of inoculated callus×100%.

[0043] Results: The embryo emergence rate was 15.25%.

[0044] Normal seedling rate (%) = normal seedling number / cotyledon embryo number × 100%.

[0045] Results: The average normal seedling rate of 4 varieties under the 2,4-D+KT and NAA+KT phytohormone combinations were 52.18% and 72.65%, respectively.