Determination of In vitro Regeneration Potentials Through Thin Cell Layer Culture System and Essential Oil Composition of Laurel (Laurus nobilis L.) Plant

Project no. 22506 · Status: Kapanmış (Yayın bekleniyor) · 2020

Summary

Turkey is one of the leading countries in the production of Laurus nobilis L. (Laurel) plant. Alternative production techniques must be used to meet the increasing demand of the bay plant, which is produced by seed or vegetative methods, and to ensure homogeneous quality. Leaf and stem explants of seeds germinated under in vitro conditions and leaf, stem and petiole explants taken from a female laurel tree in the field were cut transversely and longitudinally with the Thin Cell Layer (TCL) system to determine the regeneration potential. After the regenerated laurel shoots were taken into micropropagation experiments, they were transferred to growth media to increase their length. Following the laurel shoots, which reached at least 3-4 cm in length, were rooted in rooting media, the amount and components of essential oils in their leaves were determined. Laurel plantlets regenerated in vitro were acclimatized and tried to get used to outdoor conditions.As a result of 6 different sterilization methods applied to seeds with and without pericarp, the highest percentage of sterile seeds for seeds with pericarp (%76.6) was obtained by soaking in %70 ethyl alcohol for 1 minute and % 80 bleach for 5 minutes and then rinsing with sterile water 5 times has been obtained. For seeds without pericarp, the highest percentage of sterile seeds (%100) was determined by treatment with % 70 ethyl alcohol for 1 minute and %0.1 HgCl2 for 5 minutes and rinsing with sterile water 5 times.As a result of the observations made 6 weeks after the seeds were cultured, although some seeds germinated, no shoot development was observed. Germination did not occur in any of the seeds with pericarp. The highest germination percentage (%33.33) and shoot formation percentage (%26.67) were obtained in laurel seeds without pericarp and with cuts on the seed coat. As a result of pre-treatments made to the seeds to break dormancy and increase germination; the highest germination percentage (%86.67), shoot formation percentage (%80) and shoot length (2.43 cm) were obtained in seeds soaked in 1000 mg/L GA3 solution for 24 hours. After determining the best pre-treatment to be applied to bay laurel seeds, nutrient media containing 1 mg/L BAP, NAA or GA3 were used to determine the effect of plant growth regulators on germination. The highest germination percentage (100%), shoot formation percentage (100%), average shoot length (5.13 cm), number of roots (2.07), length (7.11 cm), number of leaves (3.43) and leaf length (0.90). cm) values were obtained from seeds cultured in MS nutrient medium containing 1 mg/L GA3.Seven different sterilization methods were applied to leaf, stem and petiole explants taken from female laurel trees. The highest percentage of sterile explants was observed for leaf explants (%93.33) in the 5th method (soaking in %70 ethyl alcohol for 1 minute, soaking in % 25 bleach for 5 minutes and rinsing with sterile water 5 times); for petiole explants (%100) in the 1st method (rinsing in detergent water for 1 minute, waiting in running water for 10 minutes, waiting in % 0.1 HgCl2 for 1 minute and rinsing with sterile water 5 times), in the 3rd method (rinsing in detergent water for 1 minute, soaking in running water for 10 minutes, soaking in % 0.1 HgCl2 for 5 minutes and rinsing with sterile water 5 times) and in the 6th method (soaking in %70 ethyl alcohol for 1 minute, soaking in %50 bleach for 3 minutes and rinsing with sterile water for 5 minutes); for stem explants (%96.67) in the 3rd method (rinsing in detergent water for 1 min, soaking in running water for 10 min, soaking in %0.1 HgCl2 for 5 min and rinsing with sterile water 5 times) and the 5th method (soaking in %70 ethyl alcohol for 1 min, soaking in %25 bleach for 5 minutes, and rinsing with sterile water 5 times) were obtained. The highest percentage of non-browned explants was obtained for leaf explants (%83.33) and petiole explants (%56.67) in 1th method (rinsing in detergent water for 1 minute, waiting in running water for 10 minutes, waiting in % 0.1 HgCl2 for 1 minute and rinsing with sterile water 5 times). Browning was observed in all stem explants..Transversely and longitudinally cut TCL explants of the leaf and stem parts of seeds germinated in vitro and the leaf, stem and petiole parts of the female laurel tree in the field were cultured in different MS based nutrient media containing 1, 2 or 4 mg/L BAP, kinetin, NAA, TDZ or 2,4-D to ensure callus and shoot regeneration. No shoot regeneration was observed in any of the leaf and petiole explants. For bay seeds germinated under in vitro conditions, the highest shoot regeneration percentage (33.33%) was obtained in longitudinally cut stem TCL explants on ½MS nutrient medium containing 4 mg/L BAP; the highest shoot regeneration percentage (26.67%) for the female laurel tree in the field was observed in transversely cut stem TCL explants on ½MS nutrient medium containing 0.5 mg/L TDZ. Then, in order to determine the effect of sucrose and darkness on shoot regeneration, experiments were set up at different sucrose concentrations and dark conditions in the nutrient medium compositions that achieved the highest shoot regeneration. The highest shoot regeneration (16.67%) in stem TCL explants of seeds germinated in vitro was obtained in longitudinal TCL explants on nutrient medium containing 40 g/L sucrose. In the stem TCL explants of the female laurel tree in the field, the highest shoot regeneration (16.67%) was obtained in the transversely cut stem TCL explants in the nutrient medium containing 40 g/L sucrose. For both explant sources, shoot regeneration could not be observed in TCL explants cultured in dark conditions.As a result of micropropagation experiments established to increase the number of shoots regenerated from TCL explants, the highest multiplication coefficient (2.13) was obtained on MS nutrient medium containing 0.5 mg/L BAP and 0.5 mg/L IBA. The highest shoot length (7.15 cm), number of leaves (7.27 number) and average leaf length (1.63 cm) in laurel shoots transferred to growth medium were obtained on MS nutrient medium containing 0.5 mg/L GA3. The highest root formation percentage (%70), average number of roots (1.97 pieces) and root length (4.12 cm) in laurel shoots transferred to rooting media were achieved in ½ MS nutrient medium containing 0.25 mg/L IBA.In essential oil, %6.71 alpha-pinene, %1.90 camphene, %5.13 beta-pinene, %6.16 sabinene, %1.73 beta-myrcene, % 5.84 beta-phlandrene, %1,75 limonene, %18.42 1,8-cineole, % 3.98 beta-ocimene, %3.95 linalool, %1.96 bornyl acetate, %11.63 beta-elemen, % 0.41terpinene-4- ol, %3.41 beta-cartophyllene, % 1.04beta-farnesene, %0.60 alpha-humulene, %7.77 alpha-terpinyl acetate, %1.74 germasiren, %4.84 bicyclogermasiren, %0.43 delta-cadinene, %5.01 methyl eugenol, %0.65 cinnamyl acetate and %4.24 unidentified components were detected.As a result of acclimatization of laurel plantlets regenerated and rooted from TCL explants; none of the 60 plants could survive at the end of the 20th day. For this reason, the acclimatization process was carried out with two different systems in which stone wool was used instead of peat. In the first system; fresh shoot, leaf and root development was achieved in 22 of the 26 acclimatized plantlets (Success rate: %84.6). In the second system; new shoot, leaf and root development was observed in 26 of the 28 plants acclimatized (Success rate: %92.9). These plants were then transferred to pots containing peat.

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