In vitro clonal micropropagation of lentisk (Pistacia lentiscus L.)

A method was developed for micropropagation of lentisk, Pistacia lentiscus L., and using shoot tips from seedlings of four cloned genotypes. An effective surface sterilization method for the production of sterile explants from mature seeds of lentisk was achieved. The factors controlling the effect of immersion time, the effect of NaOCl concentrations on surface sterilization, the clonal shoot proliferation, rooting and acclimatization of plantlets derived from mature fruit explants were investigated. The following surface sterilization, multiplication, rooting and acclimatization parameters were monitored: Contaminated and decontaminated seeds, mean shoot number, mean shoot length, % of rooting, adventive root number, root length, % of viable regenerants after five and 8 weeks. DNAs from leaves of regenerated plants along with parental controls were isolated and analyzed through PCR using 5 Interretrotransposon Amplified Polymorphism (IRAP) Primers. A 20% NaOCl x 20 min treatment was the optimum combination in NaOCl to sterilize lentisk seeds because there were 88.30% germination success and no oversterilized or malformed seeds. Analysis of the effect of all 4 medium types on multiplication phase of lentisk infers that MS gives the best results with the maximum shoot proliferation (100%) together with the highest number of shoots (3.44) per explant. After 28 days of explanting, the best response in terms of number (3.60?0.13) and length of (16.80?0.20) healthy shoots in Clone-II was obtained with 1/2x MS and 1/4x MS together with 1 mgl-1 BA, and the poorest responses with the QL medium in Clone-I, and the WPM with Clone-II. BA was found to be optimum for shoot morphogenesis in terms of the number and length of shoots among the cytokinins tested, while the highest shoot length was also yielded by BAP and 2iP each at 1.0 mg/l. Regardless of cytokinins applied, Clone-II induced rhizogenesis to some extent. BA produced the greatest number of plantlets (2.92±0.36) with optimal multiplication at approximately at 2 mgl-1 among the different concentrations tested with Clone-II. Shoot tips grown on 3% fructose medium produced more and longer shoots than those on sucrose, lactose or glucose again for Clone-II. The maximum number of shoots per explant (3.62) for Clone-II was recorded on MS medium containing 1 mgl-1 BA and 30 g/l fructose but the general appearance and growth habit of shoots were better on medium with sucrose than fructose, lactose and glucose. IBA induced strong rooting at the highest concentration (4 mgl-1) and it produced the greatest percentages of rooting (94%) with Clone-II, and 25% of explants cultured also rooted in the control treatment. In this context, the result of Clone-II was superior to those of the rest of the clones tested. The method developed for plant acclimatization was satisfactory because a high percentage of plant survival (95%) in the growth room was obtained and the regenerated plantlets resumed growth after 2 months. It was observed that Clone-II has interesting variation when the dendograms were constructed. Index of genetic variation for Clones I, II and III was lower in the mother plants and the regenerants of 6, 9 and 12 times subcultured cultures. The six times subcultured regenerants of the clones I and 6 (I-6, III-6) was the most closest to the mother plants whereas the regenerants of the clone I was the furthest from the mother plant when they were subcultured 12 times. The correlation coefficient between similarity matrices based on IRAP was 80% and this was found by using the IRAP which is dominant, multiplex marker system that examine variation in retrotransposon insertion sites. Our study, the mean value of PIC, 0.331 indicates that tissue culture generates a low level of variation, which is related with incubation time and is clone specific. Moreover, IRAP can successfully be used to explore such polymorphism within and among different clones of lentisk. Reported results undoubtedly suggest that the cytokinins, auxins, media and carbohydrate type and concentration suitable for micropropagation of lentiks are genotype-dependent. The protocol developed holds also good model system for in vitro cloning of mature elite lentisk trees. However, the exploitation of organogenesis for cloning requires developing protocols to regenerate and/or rejuvenate mature elite-trees under in vitro conditions. It will not be possible to realize the potential without further extensive research on organogenesis. Keywords: P.lentiscus L., Mastic Tree, Clone, Micropropagation, IRAP