Research on in vitro germination and micropropagation of Centaurea zeybekii Wagenitz

SUMMARYIn this study, in vitro germination and micropropagation of Centaurea zeybekiihad been investigated. Achenes from the capitula were used as initial explants in theexperiments. The experiments were divided into two steps. At the first, effects ofdifferent in vitro media with gibberellic acid, illumination, temperature ongermination were investigated. In these experiments, determination of optimum invitro germination conditions for seeds of C. zeybekii and reinforcement of naturalpopulation with a support from seedlings acclimated to natural conditions have beenaimed. At the second step, determination of appropriate medium for sterile seedlingsfrom in vitro germinated seeds and induction of axillary and adventitous shootformation by using some parts of these seedlings have been strived out.Viability tests were applicated to the seeds and results were shown as % of totalnumber. According to tetrasolium test results of seeds which were collected in Julyand August, seeds of July were less (pale red) tinged (some parts were not tinged)and the percentage was 30 %. The seeds of August were almost completely tinged(red) and the percentage was 98 %. According to TTC tests, seeds collected inAugust were almost completely alive (survive long time under 20ºC temperature 50-60 % humidity conditions)In the first experiment, two different in vitro media (with GA3 and not) were usedto describe the most proper medium for the germination. Achenes were sterilized byusing a procedure of 70 % ethanol for 10 min. 4,5 % hypochlorite for 15 min. andthen they were washed three times with distilled water after the first washing undertap water for 30 minutes. Sterilized seeds were cultured under 24±2 ºC 16/8 hphotoperiod conditions by transferring them to germination media (liquid MS, B5,vitamine supported distilled water) containing gibberellic acid in differentconcentrations and without gibberellic acid. At the result of this experiment, thehighest germination rate (80 %) was in distilled water containing 1 mg/L GA3 andfollowed by White (45 %) containing 2 mg/L GA3 and B5 (45 %). In the later stepsof the experiment, distilled water containing 1 mg/L GA3 presented the maximumgermination rate was used as main medium.In the second experiment, effect of illumination on germination was investigated.A group of seeds cultured in media containing 1 mg/L GA3 was exposed to 16/8 hphotoperiod while the another group was kept in dark under 24±2 ºC condition.Finally, germination rate of C. zeybekii seeds was 80 % under dark condition and itwas 78 % under photoperiod condition showing no important difference between thegroups.At the third experiment, effect of temperature on germination was investigated. Itwas found that the highest germination rate was held (79 %) in seeds incubated under24±2 ºC and germination rate was decreased below and above this temperature.Some of in vitro germinated seeds were transferred to pots and they survived 8months under growth chamber conditions. Remaining germinated seeds weretransferred to B5, MS and White media to determine the in vitro medium foroptimum development. Approximately 8 weeks later (subculturing in 4 weeksinterval), development of seedlings (shoot length, shoot number, leaf number, leafwidth, root length, root number) were evaluated. All of the seedlings which weretransferred to White medium died one month later. MS was selected as the mostproper medium for seedling development depending on a comparison with B5medium using morphological criteria after 8 weeks.4 weeks old seedlings, that were developed on MS media which was determinedas the optimum media for seedling development, were separated from primary rootsand transferred to MS basal media with different types of cytokinin andconcentrations (0.2, 0.5, 1 ve 2 mg/L BA, 0.2, 0.5, 1 ve 2 mg/L KIN and 0.001,0.005, 001 and 0.02 mg/L TDZ) for axillary propagation. At the end of 4 week-period, increase in shoot number and shoot length were determined and optimumcytokinin type and concentration for axillary shoot propagation were determined.Inspite of induction of axillary shoot regeneration in seedlings growing in TDZmedia, the seedlindgs were developed in short groups having hyperhydricity (nearlyin all). The highest propagation rate was obtained in the medium containing 1 mg/LBA with 14.85 shoot per explant. The highest average of the maximum shoot lengthwas obtained in MS media without plant growth regulators but containing 2 mglKIN. There were decrease in shoot number which is negatively correlated withincrease in shoot length in MS media containing KIN and BA.Leaves of in vitro germinated steril seedlings were used as explants in directadventitous shoot regeneration experiments. Leaves of sterile seedlings (two monthsold) without axillary buds were cultured in the media with or without BA (0.2, 0.5, 1and 2 mg/L), KIN (0.2, 0.5, 1 and 2 mg/L) and TDZ (0.001, 0.005, 0.01 and 0.02mg/L) as their abaxial sides down and contact with the medium.. The highest callusformation rate was observed in the media containin TDZ. Although callus formationrates were 90 % in 0.001 mg/ L TDZ, 100 % in 0.005 mg/ L and 0.01 mg/L TDZ , itwas decreased as 48 % in 0.02 mg/L TDZ. In BA containing media, callus formationrates were 52,5 % in 0.5 mg/L, 27,5 % in 1mg/L BA and 35 % in 2 mg/L BA.Although the low percentage of callus formation in 0.2 mg/L BA added medium (5%), this cytokinin type is found non-efficient for direct adventitious shootproliferation as organogenic answer appears with only shoot buds and this casecontinues in the subcultures. Callus formation was also observed in mediacontaining KIN. Callus formation rates were %5 in 0.2 mg/L KIN, 2,5 % in 0.5 mg/LKIN, 4 % in 1 mg/L KIN and no callus formation was observed in the mediumcontaining 2 mg/L KIN. In all concentrations, initiations of shoots (small leafinitiations) close to midrib and well developed shoots were observed at the end ofsixth week. In the media having direct shoot regeneration, calli formation in explantson their surfaces having contact with the medium were observed but these callibecame brownish in time and showed no activity. The highest number of shoots perexplant (6,2 shoot per explant) was obtained from the medium containing 1 mgl KIN.The highest average of maximum shoot length was also observed in this medium.Finally, it is possible to stress that medium containing 1 mgl KIN is the best mediumby obtaining maximum shoot length and highest number of shoots per explant fordirect adventitous shoot regeneration with explants from in vitro germinated shootleaves.The shoots from axillary and adventitous shoot formation experiments weretransferred to MS and ½ MS media containing 0.5, 1, 2 and 5 mg/L IAA, IBA, NAAfor rooting. There was no difference between MS and ½ MS media in rooting but itwas very low and only 15 % of total shoots could rooted in medium containing 0.5mg/L IBA. Rooted plantlets were gradually acclimated to the natural conditions.