MORPHOGENIC RESPONSE FROM LEAF EXPLANT OF Dendranthema grandiflora VAR MICROMARGARA

Background: Chrysanthemum is one of the most popular landscape plants worldwide. Introducing desirable traits into this plant by conventional breeding has limitations due to cross-incompatibility. An approach to this problem is the introduction of resistance or tolerance traits via plant genetic transformation. In vitro regeneration of plants is necessary for implementing genetic transformation systems; therefore, regeneration via morphogenesis is required before any genetic transformation attempt. Objective: To induce morphogenesis from the leaf explant of Dendranthema grandiflora var Micromargara. Methodology: Micropropagation from node cuttings was induced from node cuttings of 3 cm in length from 6 months-old plants that were used as explants to produce seedlings as source explants. Morphogenesis experiments were done using leaf segments of 1 cm2 from 8-weeks-old in vitro seedlings. The explants were transferred to basal medium Murashige and Skoog at 4.4 gL-1 supplemented with different plant growth regulators. During the morphogenic process, leaves samples were collected to detect which morphogenic process was induced. Results: Node cuttings culture on Murashige and Skoog medium at 3.3 gL-1 supplemented with benzylaminopurine at 2.2 µM yielded a vegetative growth increase from 1 to 7 shoots, 2 to 12 leaves per node cutting, and the stem length of 3 to 9.5 cm. The leaf explant induced three ways of in vitro morphogenesis: direct and indirect roots organogenesis (rhizogenesis) and, shoot organogenesis. Direct rhizogenesis was induced in plant growth regulator-free Murashige and Skoog medium (18 roots/explant). Indirect rhizogenesis from leaf explant was less efficient than direct rhizogenesis, get obtaining six roots per leaf explant cultured on Murashige and Skoog medium at 4.4 g L-1 supplemented with 0.4 µM thidiazuron and 4.5 µM 2,4-dichlorophenoxyacetic acid, and three roots per leaf explant cultured on Murashige and Skoog medium supplemented with 0.4 µM thidiazuron and 9.05 µM 2,4-dichlorophenoxyacetic acid. Indirect shoot organogenesis was induced from leaf explants cultured on Murashige and Skoog at 4.4 g L-1 supplemented with 13.32 µM benzylaminopurine and 4.83 µM naphthalene acetic acid; 50% of explants with callus formed shoots (2 shoots/leaf explant). Through histological analysis it was possible to verify that the morphogenic response obtained was organogenesis. Implications: Regeneration of Dendranthema grandiflora var. Micromargara established herein will allow improvement of the variety through techniques of genetic transformation mediated by Agrobacterium and any other strategy to incorporate or silence genes of interest in the plant. Conclusion: The high capacity to induce roots of Dendranthema grandiflora var Micromargara, may provide an efficient model for further investigating the mechanism of rhizogenesis from leaf explants cultured without plant growth regulators.