Введення в культуру in vitro тису ягідного (Taxus baccata L.) і отримання таксол-продукуючих калюсних ліній - Автореферат

в культуру in vitro тисса ягодного (Taxus baccata L.) и получение таксол-продуцирующих каллусных линий. Институт клеточной биологии и генетической инженерии НАН Украины, Киев, 1999. Цель данной работы - изучение закономерностей каллусообразования и продуцирования противоракового препарата - таксола в культуре клеток тисса ягодного (T. baccata L.). Изучено распределение содержания таксола между отдельными частями растений в 15-ти клонах T. baccata и 3-х клонах T. cuspidata. Filonova L. G. Introduction in vitro culture of Taxus baccata L. and obtaining of taxol produced callus lines. - Manuscript. Thesis for gaining a scientific degree of Candidate of Biological Sciences on speciality 03.00.22 - Cell Biology, Institute of Cell Biology and Genetic Engineering, National Academy Science of Ukraine, Kiev, 1999. The diterpenoid taxol belongs to the class of efficient antineoplastics drugs, taxoids. Taxol promotes abnormal microtubule assembly and stabilizes the previously formed microtubules. Further spindle formation is arrested. Due to this unique antimitotic mechanism, this natural compound has become a well known anticancer drug. The only known source of taxol is from Taxus species. Family Taxaceae is represented by ancient slowly growing species with narrow natural habitats, and the levels of taxol production by intact plants do not exceed 0.1% on the dry weight basis. Large-scale exploitation of natural stands of Taxus spp. for taxol production will eventially lead to complete disappearance of these species. Development of alternative ways of taxol production is required to meet current demands of medicine. Cell culture of Taxus spp. attracts much attention as a promising alternative tool to produce sufficient amounts of taxol. In Ukraine and other European countries, the only indigenously growing Taxus spp. is Taxus baccata L. Cell culture of T. baccata was studied less deeply, than other species. The aim of the present study is to describe peculiarities of callusogenesis and taxol production in vitro cultured cells of Common yew (Taxus baccata L.). Distribution of taxol content in different parts of the plant has been analysed for 15 genotypes of T. baccata and 3 genotypes of T. cuspidata. The highest levels of taxol have been found in the bark, what is 10-fold higher than in the needles and one-year-old twigs. Concentration of taxol varies significantly depending on the genotype. The least variation has been demonstrated for the bark. Also, both T. baccata and T. cuspidata exhibited high interclonal variation of taxol content in three types of samples analysed. A veraged values of taxol content in two Taxus spp. differed significantly for needle samples only. The culture protocols have been developed to establish callus cultures from different types of explants. The temporal pattern and frequency of callus formation from shoot segments, vegetative buds and pollen have been shown to be independent on the time of collecting plant material. In contrast, degree of seed maturity determines both parameters with the highest callusogenic activity occuring in September. Primary seed-originated callus was less susceptible to necrotic process than shoot-originated callus which, however, grew faster. Five basal nutrient compositions routinely used for culturing coniferous species have been tested. The best medium was SH (Shenk, Hilderbrandt, 1972) showing 2 to 2.5 times higher frequency of callus formation (91%) compared to other media. Мета роботи - введення в культуру in vitro T. baccata і вивчення динаміки накопичення таксолу в калюсі. В одному з експериментів використовували річні пагони двох чітко фенотипово відмінних генотипів T. baccata - хлорофіл-дефектного (CD) і нормального щодо синтезу хлорофілу (N). Поживні середовища і методи культивування Для визначення оптимального мінерального складу поживного середовища пагони культивували на середовищах SH (Schenk, Hilderbrandt, 1972); МS (Murashige, Skoog, 1962); LP (von Arnold, Eriksson, 1982); B5 (Gamborg et al., 1968); DBM2 - РР (Gresshoff, Doy, 1972) з додаванням інозиту (50 мкМ), нікотинової кислоти (0,8 мкМ), піридоксину-HCl - В6 (0,5 мкМ), тіаміну-HCl - В1 (3 мкМ), 2,4-дихлорфеноксиоцтової кислоти (2,4-Д; 10 мкМ), кінетину (5 мкМ), 3% сахарози і 0,7% агару.