Patent RU-2757318-C1 (RU)
Title: Method for obtaining microplants of medicinal plant stephania glabra (roxb.) miers
Inventor: Gorpenchenko Tatyana Yurevna
IPC: A01H4/00
Filed: 2021-04-12
Granted: 2021-10-13
Abstract: 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 (NH 4 NO 3 ) to 400 mg/l. As a result, it contains, mg/l, respectively: 400 - NH 4 NO 3,  1900 - KNO 3 , 170 - KH 2 PO 4 , 440 - CaCl 2  2H 2 O, 370 - MgSO 4  7H 2 O, 37,3 - Na 2 EDTA 2H 2 O, 27,8 – FeSO 4  7H 2 O, 6,2 - H 3 BO 3 , 22,3 - MnSO 4  4H 2 O, 0,025 - CuSO 4  5H 2 O, 0,025 - CoCl 2  2H 2 O, 8,6 - ZnSO 4  7H 2 O, 0,25 - Na 2 MoO 4  2H 2 O, 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 - NH 4 NO 3,  1900 - KNO 3, , 170 - KH 2 PO 4 , 440 - CaCl 2  2H 2 O, 370 - MgSO 4  7H 2 O, 37,3 - Na 2 EDTA 2H 2 O, 27,8 – FeSO 4  7H 2 O, 6,2 - H 3 BO 3 , 22,3 - MnSO 4  4H 2 O, 0,025 - CuSO 4  5H 2 O, 0,025 - CoCl 2  2H 2 O, 8,6 - ZnSO 4  7H 2 O, 0,25 - Na 2 MoO 4  2H 2 O, 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 - NH 4 NO 3 , 1900 - KNO 3 , 170 - KH 2 PO 4 , 440 - CaCl 2  2H 2 O, 370 - MgSO 4  7H 2 O, 37,3 - Na 2 EDTA 2H 2 O, 27,8 – FeSO 4  7H 2 O, 6,2 - H 3 BO 3 , 22,3 - MnSO 4  4H 2 O, 0,025 - CuSO 4  5H 2 O, 0,025 - CoCl 2  2H 2 O, 8,6 - ZnSO 4  7H 2 O, 0,25 - Na 2 MoO 4  2H 2 O, 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 (W 0  (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