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

A technology of embryogenesis and regeneration of plants, applied in the field of plant cell engineering, can solve the problems of low plant regeneration efficiency, self-sufficiency rate of less than 40%, affecting grain and oil safety, etc., and achieve the effect of more normal regeneration plants, good social and economic benefits

Active Publication Date: 2020-08-07
HUANGGANG NORMAL UNIV
4 Cites 5 Cited by

AI-Extracted Technical Summary

Problems solved by technology

In addition, my country's edible oil consumption is huge, but the current self-sufficiency rate is less than 40%, which seriously affects my country's grain and oil security
[0003] With the development of production and the continuous improvement of social needs, the production has put forward higher and higher requirements for the yield, quality, and resistance of Camellia oleifera varietie...
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Abstract

The invention provides a method for obtaining regenerated plants from common camellia oleifera in a somatic embryogenesis mode. The method comprises the following steps of storing young embryos of thecommon camellia oleifera in the middle ten days of July at 4 DEG C, cutting the young embryos into blocks after disinfection, and inoculating the young embryos into a callus induction culture mediumfor callus induction culture; selecting well-growing calluses, and subculturing the calluses in a differential culture medium for embryoid differentiation; transferring explants with embryoids into anembryoid development and plant regeneration culture medium for embryoid development and plant regeneration; transferring the embryoids with obvious cotyledons to a rooting culture medium for rootingculture; and finally, domesticating and transplanting obtained complete plants into a nutritional bowl for continuous growth. According to the method provided by the invention, the common camellia oleifera can be subjected to somatic embryogenesis within 6 months to obtain the regenerated plants, a convenient, rapid and effective method is provided for obtaining the regenerated plants through thesomatic embryogenesis of the common camellia oleifera, and the method has relatively good social benefits and economic benefits.

Application Domain

Technology Topic

Somatic embryogenesisTransplanting +5

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

Examples

  • Experimental program(1)

Example Embodiment

[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.
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