Patent BG-50665-A1 (BG)
Title: Method for callus tissue inducing and cultivation from vinca herbacea producer of biologically active substances
Inventor: BAKLOVA BOJKA I
IPC: C12N5/00
Filed: 1990-07-05
Abstract: This invention relates to a method of cultivating callus tissue of the type Vinca Herbacea, producer of biologically active substances, which are used in the pharmaceutical industry.A method for extracting callus tissue from respective explants is already known. It was developed in 1934 by White and Guatheret. It relies exclusively on the use of meristematic materials. A number of methods for extracting calluses from various plant types have been developed. This particular method is not only plant-specific, but also variety-specific. Two varieties do not yield callus at identical conditions /1/. And while, in many cases, the attempt to define the conditions, at which various explants from a certain plant yield callus has proven successful (like in the case of Catharanthus rosaus /4/, Humulus lupulus /2/, Perilla sp. /3/), in many others no technical solution has been reached. In this way, despite the efforts of a number of scientific research units, the conditions for extracting callus from Papaver somniferum, producing morphine alkaloids, either from "Digitalis lanata", producing digoxin, are not determined. Nowhere does the scientific literature report about research exploring a method of extracting callus, producing alkaloids from Vinca herbacea.The task of the invention is to create a method for extracting and cultivating callus tissue, which will allow the extraction of a starting material of maximum induction response, as well as a yearlong cycle of production, while the extracted callous fabric preserves the genetic information about the biosynthesis of the alkaloids, contained in the parent plant. The task is solved by the cultivating non-damaged leaves, parts from stems, flowers and roots of Vinca herbacea in a Gamborg medium /B5/, containing in mg/l: 2.4 dichlorine phenoxyacetic acid between 0.1 and 0.3; kinetin between 0.9 and 1.1; α-naphthylacetic acid between 0.9 and 1.1; 2% Saccharose and agar-agar 7.0 g/l; pH 6.5, after which they are left in phytostatic medium at temperatures between 24-260 degrees C and 65 to 75% of humidity for about 30 days. Meanwhile, upon extraction of the primary callus, the cultivation is carried out at 2,000 luxes of light for 16 hours a day, and during the production of the secondary callus, the cultivation is made in dark conditions. According to the invented method, whole, non-damaged leaves, parts from stems, flowers and roots of Vinca herbacea are cultivated in a medium, containing in mg/l: (NH4)2SO4 - 133-135; MgSO4.7H2O - 249-251; NAH2PO4 - 149-151; KNO3 - 2,498-2,501; CaCL2.2H20 - 149-151; Kl - 0.74-0.76; MnSO4.H2O - 9.0-11.0; H3BO3 - 2.0-4.0; ZnSO4.7H20 - 1.0-3.0; NaMoO4.2H2O - 0.24-0.26; CuSO4.5H2O - 0.022-0.027; CoCl2.6H20 - 0.023-0.028; nicotine acid - 0.9-2.0; pyridoxine /B6/ - 0.9-1.9; thiamine (B1) - 9.0-11.0; sodium ethylenediaminetetraacetate /Na2 EDTA/ - 37.2-37.4; FeSO4.7H20 - 27.7-27.9; inosite - 99-101; casein hydrolysate - 1998-2002; 2,4-dichlorinephenoxyacetic acid - 0.1-0.3; kinetin - 0.9-1.9; α- naphthyl acetic acid - 0.9-1.9; Saccharose - 20.0 g; agar-agar - 7.0 g/l, under the above-mentioned conditions, after which the total alkaloid mixture is isolated. After each cultivation cycle, a tenth of the mixture is transferred in a fresh nutritional medium with the same composition of re-cultivation, while the remaining nine tenths are subjected to chemical analysis. The result of the analysis prove that, under the so-defined conditions, the explants do not only yield an almost 100% extraction response from the small quantities of starting material, but also extract a callus tissue, whose cells have preserved its genetic encoding for the biosynthesis of tabersonine and other alkaloids. The extracted callus tissue is subjected to chemical analysis aiming to identify its composition. The chromatographic data display presence of alkaloids.In Bulgaria, Vinca herbacea is scantily distributed, while the few places where it is found are poor and may not serve as a serious source of vegetable material. As a grass plant, its cultivat