Induction of variation in Gerbera (Gerbera jamesonii Bolus) and Chrysanthemum (Chrysanthemum morifolium Ramat) through gamma radiation and in vitro techniques

Abstract

Globally, the floral industry has experienced significant growth over the last few decades. Chrysanthemum (Chrysanthemum morifolium Ramat) and Gerbera (Gerbera jamesonii Bolus) are two very popular ornamental plants and widely used as cut flowers for decorative purposes all over the world. Gerbera and Chrysanthemums are popular because they have a lengthy vase life and a wide variety of colors and shapes. Creation of variation in colour and shape of the flowers in case of both the ornamental plants are very important for commercialization. Both plants are primarily reproduced vegetatively, which restricts the development of variants. Therefore, gamma radiation coupling with techniques of in vitro micropropagation was applied to create variation in flower characters of these two economically important ornamental plants. Specifically, the objectives of the present investigation were to develop elite mutant lines of Chrysanthemum and Gerbera through gamma radiation and in vitro techniques. For this purpose, an efficient in vitro regeneration system was established for both locally grown Chrysanthemum and Gerbera varieties, namely, BARI Chrysanthemum-1, BARI Chrysanthemum-2, BARI Gerbera-1 and BARI Gerbera-2. Three different types of explants, namely, shoot tip, internode (IN) and leaf (L) segments of two varieties of Chrysanthemum were used for in vitro regeneration. Maximum responses of multiple shoot regeneration (94.73% for BARI Chrysanthemum-1 and 90% for BARI Chrysanthemum-2) in both the varieties were achieved when the leaf explants were cultured on MS medium supplemented with 2.0 mg/l IAA and 0.5 mg/l BAP (T6), followed by sub-culturing on hormone free MS medium. It took about six weeks for the regeneration of multiple shoots. On the other hand, three different types of Gerbera explants, namely, flower bud (FB), flower stalk (FS) and leaf segments (LS) were used for in vitro regeneration of both the varieties. Highest percentage of multiple shoot regeneration (70.00% for Gerbera-1 and 73.30% for Gerbera-2) was achieved in both Gerbera varieties when the flower bud explants were cultured on MS medium supplemented with 6.0 mg/l BAP and 1.0 mg/l NAA (T6), followed by two subsequent cultures on half the strength MS medium with 2.0 mg/l BAP. It took about 7-8 weeks for the regeneration of multiple shoots in this case. During elongation more than 90-95% of the shoots from both the varieties of Chrysanthemum produced healthy roots on hormone free MS medium within 6 to 7 weeks of culture. While the best responses towards root induction in two varieties of Gerbera was recoded on half strength of MS medium containing 0.5 mg/l of IAA. Fully developed in vitro regenerated plantlets of both Chrysanthemum and Gerbera were successfully established in soil for further growth and development of flowers. For the induction of desired mutation in both Chrysanthemum and Gerbera five doses of gamma radiation (5Gy, 10Gy, 15Gy, 20Gy and 25Gy) were applied to the in vitro grown micro shoots. The survivability of the irradiated shoots (60 days after irradiation) was evaluated following determination of LD50 (50% lethal dose). The highest survival percentage was observed for 5 Gy irradiated shoots for both Chrysanthemum varieties (57.29% for BARI Chrysanthemum-1 and 64.08% for BARI Chrysanthemum-2). LD50 (50% lethal dose) was found at 9.25 Gy for BARI Chrysanthemum-1 and 11.19 Gy for BARI Chrysanthemum-2 variety. In case of Gerbera the highest survival percentage was observed for 5 Gy irradiated shoots for both varieties (72.77% for BARI Gerbera-1 and 68.09% for BARI Gerbera-2). The LD50 for BARI Gerbera -1 and BARI Gerbera-2 variety was found at 11.17 Gy and 9.32 Gy respectively. Among the five irradiation doses used, 15 Gy produced highest percentage of mutation frequency (75-80%) regarding the changes in leaf shape, nature of pigmentation, and internode size and plant height in BARI Chrysanthemum-1. Whereas in case of BARI Chrysanthemum-2, three different doses (5Gy, 10 Gy and 15 Gy) produced high frequency of mutation regarding the same factors as discussed for BARI Chrysanthemum-1. In the case of BARI Gerbera-1, irradiation doses of 5 Gy and 20 Gy caused 20% and 30%, respectively, of mutation frequency in the case of the flower's shape and color. On the other hand, in the instance of BARI Gerbera-2, irradiation doses of 5 Gy and 10 Gy respectively caused a mutation frequency of 45% and 20% for flower morphologies and color. Following the morphological studies, it was recorded that BARI Chrysanthemum-1 produced four mutant lines (YM1, Y1, Y5 and Y6) while BARI Chrysanthemum-2 produced three mutant lines (M1, M2 and M6) finally. Based on morphological differences BARI Gerbera-1 produced three mutant lines (WV1, WV2 and WV3) and BARI Gerbera-2 produced three mutant lines (RV1, RV2 and RV3). No variation was recorded on the plant developed from non-irradiated in-vitro culture of both Chrysanthemum and Gerbera varieties used. Further characterization of induced mutants was confirmed through ISSR (Inter-simple sequence repeats) molecular marker analysis. High levels of polymorphism (90.90% for chrysanthemum and 89.06% for Gerbera) were recorded among the mutants developed in these two plants. Moreover, this study revealed that mutants exhibited a broad range of diversity among themselves as well as different from their control plants. The mutants that developed through this study can be cultivated as new variants of Chrysanthemum and Gerbera.