Otimização do cultivo in vitro visando a transformação genética das cultivares brasileiras de alho (Allium sativum L.) / Optimization of in vitro culture aiming at genetic transformation of Brazilian garlic (Allium sativum L.) cultivars

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Garlic (Allium sativum L.) breeding has been limited to clonal selection of mutant genotypes, since almost all the species germplasm is sterile, not flowering under standard culture conditions. Garlic sterility affects not only breeding but also directly influences production costs. Further, the requirement of vegetative propagation using small bulbs as propagules increases the risk of pest and viral disease transmission. Genetic manipulation of garlic introducing genes associated with flowering by Agrobacterium tumefaciens would allow the development of an efficient system for propagation and enable new genetic recombination. Thus, the objective of this work was to establish an in vitro regeneration protocol for commercial Brazilian cultivars of garlic, as well as genetic transformation by A. tumefaciens using calli derived from root segments. Eight garlic cultivars were used, derived from three groups: medium cycle semi-noble (Amarante-Embrapa; Roxinho 5063; IAC 75 - Gigante de Curitibanos; IAC 63 - Mexicano Br; and Lavínia 1632); common garlic with medium (Cateto Roxo), or early cycle (Cajuru 2315); and from vernalized noble (Jonas). From each cultivar, root segments from small bulbs cultivated in vitro were isolated. These explantes were maintained in MS media supplemented with 4,5 müM 2,4-D e 0,5 müM iP for 60 days to develop callus. The calli were then transferred to fresh MS media containing 8,8 müM BAP e 0,1 müM ANA, and evaluated for regeneration frequency. Initially, diverse factors were analyzed for each cultivar during the introduction of the small bulbs and during calli development. These results enabled to conduct test of callus regeneration potential, with Jonasshowing the best regeneration rate via indirect organogenesis (84%), followed by cultivar Amarante(52%), IAC 63and Cajuru(47%). The cultivar with less regeneration was Lavínia(30%). The lethal dosis of hygromycin or kanamycin were estimated, testing increasing concentrations to establish optimum levels to be adopted as selective agents for transformation. Doses above 50 mg/L hygromycin or 200 mg/L kanamycin were lethal to callus of all cultivars. Cultivar Jonaswas chosen for the establishment of the genetic transformation protocol via A. tumefaciens LBA4404 (pCAMBIA1303), using uidA as a reporter gene (beta-glucuronídase) to determine the transformation efficiency. Jonaswas the best cultivar in terms of callus and regeneration potential using the regeneration media proposed by Kondo, Hasegawa and Suzuki (2000). Moreover, the Brazilians garlic cultivars appeared to be amenable to transformation via A. tumefaciens

ASSUNTO(S)

segmentos radiculares allium sativum l. genetic transformation florescimento root segments plant regeneration regeneração de plantas allium sativum l. flowering transformação genética

ACESSO AO ARTIGO

Documentos Relacionados