METHOD FOR OBTAINING MICROPLANTS OF MEDICINAL PLANT STEPHANIA GLABRA (ROXB.) MIERS Russian patent published in 2021 - IPC A01H4/00 

FIELD: plant biotechnology.

SUBSTANCE: invention relates to the field of plant biotechnology, involves the use of the method for plant tissue culture for microclonal reproduction of the medicinal species Stephania glabra. The method is carried out as follows: a heterogeneous culture is cultivated from the primary explant on a liquid nutrient medium, after which the obtained heterogeneous cell culture is cultivated on a nutrient medium for the formation of somatic embryos, followed by the formation of microplants from the obtained somatic embryos on a nutrient without hormonal solid medium. As a primary explant, young leaves of the Stephania glabra plant are used, which are pre-sterilized in a laminar box in a 0.1-0.2% diacid solution for 3-5 minutes and after thorough washing in 5 changes of sterile water are cultivated at least 3 passages on a nutrient medium to obtain a heterogeneous culture on an orbital shaker at 14-day intervals in the dark at a temperature of 25°С and a relative humidity of 50-70%. A liquid nutrient medium for obtaining a heterogeneous culture (W_2,4D liquid) is prepared on the basis of macro- and microsalts according to the recipe of Murashige and Skoog with a reduced content of ammonium nitrate (NH₄NO₃) to 400 mg/l. As a result, it contains, mg/l, respectively: 400 - NH₄NO_3, 1900 - KNO₃, 170 - KH₂PO₄, 440 - CaCl₂ 2H₂O, 370 - MgSO₄ 7H₂O, 37,3 - Na₂EDTA 2H₂O, 27,8 – FeSO₄ 7H₂O, 6,2 - H₃BO₃, 22,3 - MnSO₄ 4H₂O, 0,025 - CuSO₄ 5H₂O, 0,025 - CoCl₂ 2H₂O, 8,6 - ZnSO₄ 7H₂O, 0,25 - Na₂MoO₄ 2H₂O, 0,83 – KI, 0.2 - thiamine hydrochloride, 1 - cysteine, 25,000 - sucrose and 0.5 - 2,4-dichlorophenoxyacetic acid. As a nutrient medium for the formation of somatic embryos, a nutrient medium is used, which contains, mg/l, respectively: 400 - NH₄NO_3, 1900 - KNO_3,, 170 - KH₂PO₄, 440 - CaCl₂ 2H₂O, 370 - MgSO₄ 7H₂O, 37,3 - Na₂EDTA 2H₂O, 27,8 – FeSO₄ 7H₂O, 6,2 - H₃BO₃, 22,3 - MnSO₄ 4H₂O, 0,025 - CuSO₄ 5H₂O, 0,025 - CoCl₂ 2H₂O, 8,6 - ZnSO₄ 7H₂O, 0,25 - Na₂MoO₄ 2H₂O, 0,83 – KI, 100 - meso-inositol, 100 - peptone, 0.5 - nicotinic acid, 0.5 - pyridoxine hydrochloride, 0.2 - thiamine hydrochloride, 1 - cysteine, 25,000 - sucrose, 0.5 - 6-benzylaminopurine and 2.0 - α-naphthylacetic acid. The cultivation is carried out at least 2 passages with 20-day intervals on an orbital shaker in the dark at a temperature of 25°С and a relative humidity of 50-70%. Then the formed somatic embryos for growing and regenerating full-fledged microplants in the amount of 30-50 pieces per flask are placed in 100 ml Erlenmeyer flasks on a hormone-free solid nutrient medium and grown in the light for at least 4 weeks (Fig. Б) until separate microplants are formed, while the hormone-free solid nutrient medium includes, mg/l, respectively: 400 - NH₄NO₃, 1900 - KNO₃, 170 - KH₂PO₄, 440 - CaCl₂ 2H₂O, 370 - MgSO₄ 7H₂O, 37,3 - Na₂EDTA 2H₂O, 27,8 – FeSO₄ 7H₂O, 6,2 - H₃BO₃, 22,3 - MnSO₄ 4H₂O, 0,025 - CuSO₄ 5H₂O, 0,025 - CoCl₂ 2H₂O, 8,6 - ZnSO₄ 7H₂O, 0,25 - Na₂MoO₄ 2H₂O, 0,83 – KI, 100 - mesoinositol, 100 - peptone, 0.5 - nicotinic acid, 0.5 - pyridoxine hydrochloride, 0.2 - thiamine hydrochloride, 1 - cysteine, 25000 - sucrose, 7000 - agar.

EFFECT: in the claimed method, the entire process from the stage of obtaining the primary callus from the leaf explant to planting the formed microplants in the ground takes no more than 21-24 weeks, includes the change of three variants of nutrient media: induction (W_2,4D (liquid)), morphogenic (W_B/А (liquid)) and hormone-free (W₀ (solid)), which more than 3 times accelerates the process of obtaining microplants and significantly reduces labor costs, in addition, ensures the stable formation of S. glabra microplants by preserving the embryogenicity of cell cultures throughout the entire technological process.

1 cl, 2 tbl, 5 dwg