Plant tissue culture and regeneration of transgenic plants from embryogenic callus and immature leaves of sugarcane / Cultura de tecidos e regeneração de plantas transgênicas a partir de calos embriogênicos e de folhas imaturas de cana-de-açúcar

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

Sugarcane is a polyploidy monocot and allogamous species that has low reproductive rate due to the difficulty of flowering. Because of these genetic and physiological characteristics breeding program takes long time and demand hard labor. Alternatively, modern biotechnology approaches contribute to the development of new cultivars. In this work we studied the methodology of plant tissue culture from immature leaf discs to establish callus culture and plant regeneration from those calli and from immature leaves, directly. The main objective was to contribute to the development of efficient methods to produce transgenic plants from callus and immature leaves, due to the growing need to produce new cultivars with specific agronomics traits. MS medium with different concentrations of 2,4-D and kinetin were tested to obtain highly embryogenic calli and to induce cellular dedifferentiation in the immature leaf discs prior to plant regeneration. Culture media without growth regulators (MS) and with the addition of BAP and NAA were tested for plant regeneration from leaf discs. Callus culture with 12 to 20 weeks resulted on average 3 to 5 plants on regeneration medium designed as MS. Immature leaves showed a high potential for plant regeneration when 2,4-D at concentrations of 5 and 8 mg/L in periods of 5 and 8 days in the dark. There were inducing of somatic embryos that resulted in average 12 to 16 plants per explant in the total period of 7 to 10 weeks. In addition, we tested the pre-treatment of leaf discs in MS3K medium which contain 2,4-D (3 mg/L) and kinetin (0.1 mg/L) before transfering to plant regeneration MS medium . The discs submitted to this pretreatment for 14, 21 and 28 days showed significant increase in the efficiency of plant regeneration, with on average of 41 to 50 plants per leaf disc in varieties RB835089 and RB855156. The reduction of time to obtain plants coupled with the increase of plants obtained is the basis for increasing the efficiency of plant genetic transformation. Co-transformation with genes neo and comt(AS), were performed by biolistics. Plants regenerated from immature leaves of the variety RB835486, PCR analysis confirmed the incorporation of the neo selection marker gene in 57 and 90% of the plants on selective medium with geneticin (30 mg/L), the higher efficiency of transgenic plants (90%) was obtained on pre-treatment in MS3K medium. Transgenic plants for the neo gene, 7 and 38% were also confirmed for the incorporation of comt (AS). PCR analysis of candidates transgenic plants from callus growing on selective medium, revelled only the insertion of the neo gene, which occurred in 52% of the analyzed plants. The results of this work showed that the approach of using immature leaf discs to obtain plant genetic transformation by biolistics methodology is a viable, cheaper and faster than using embryogenic callus.

ASSUNTO(S)

auxin cytokinin comt genes growth regulators caffeic acid-o-methyltransferase reguladores de crescimento vegetal. biolística cana-de-açúcar biolistics. polimorfismo cotransformation

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