Programmed cell death and in vitro culture for regeneration in endosperm tissue of diploid bananas(Musa balbisiana and M. formosana)
Date Issued
2008
Date
2008
Author(s)
Sun, Chong-Gin
Abstract
Diploid bananas (2n=22) Musa balbisiana and M. formosana were used, for studying programmed cell death (PCD), rescuing culture, and regeneration in endosperm tissue. The developmental stages of endosperm in M. balbisiana could be categoried into liquid stage(1-50 days after pollination, DAP), glutinous stage(62-70 DAP), solid stage(74-83 DAP) and starchy stage (>83DAP). The initiation of PCD in developing endosperm was studied using Evans blue staining method and DNA fragmentation markers. We demonstrated that dense blue staining took place in late glutinous stage about 72 DAP, that was also coincided with the timing of genomic DNA degradation by electrophoresis, about 74 DAP during fall season. The various development stage endosperm were excised and cultured on MS medium supplemented containing 1mg/L 2,4-D, 0.5 mg/L BA, 2 mg/L TDZ and 0.5 mg/L GA. Callus formation rate in glutinous stage 64 DAP was 53%. Callus formation decreased in solid stage( 74 DAP) due to PCD. The addition of 200 mg/L malt extract increased callus formation. Glutinous endosperm of M. formosana cultured on MS medium containing 1.0mg/L 2,4-D, 0.5 mg/L BA, 0.1 mg/L TDZ, 1.0 mg/L GA showed higher callus formation rate, about 35.0 %. Increasing GA concentration delayed the embryogenic callus formation. The endosperm-derived embryogenic callus of M. formosana were recorded 3 months after cultivation, which is longer than what is recorded in M. balbisiana. The endosperm-derived callus of M. formosana formed adventitious bud and embryoids on SH3 medium. The endosperm-derived calli of M. balbisiana and M. formosana were inoculated on TB5 liquid medium containing 1.0 mg/L 2,4-D, 100 mg/L glutamine, 1.0 mg/L biotin and 100 mg/L malt extract with110rpm shaking. Homogenous cell population of M. balbisiana were obtained after 3 months in suspension cell culture, while homogenous suspension cell of M. formosana were obtained after 4 months in suspension culture. The size of suspension cell cluster was separated with <60, 60-30 and >30 meshes and were diluted 1/30 c.c PCV proceeding to plat on SH3 modified medium. The size classes cell clusters of 30-60 and <60 mesh respectively generated 1285 and 1533 embryoids/0.033 ml PCV in 60 DAC. Cell clusters on gelrite semisolid medium had greater plating efficiency than that on paper cushion enriched with liquid medium. Before plating, the suspension prembryogenic cells of M. balbisiana were subcultured on TB5, SH3 medium with or without was consistent 10 g/L MES. That the extracellular pH of SH3 was remaining in the rage of 4.55-5.48 and SH3 + MES solution between 5.35 and 5.56 during 14 subculture period. Furthermore, two pretreated cells regenerated larger and higher quality of somatic embryos on plating medium than those on TB5 or TB5+MES pretreated. omatic embryos inoculated with 1/2MS solid medium adding 0.1 mg/L NAA induced 81.0% and 75.9% of rooting and shooting respectively. The plantlets grew well in medium added auxin. However the plantlets had exceptional discoloration and elongation on medium adding GA. Endosperm-derived adventitious buds of M. formosana inoculated on paper bridge adding 1/2MS and 0.1 mg/L NAA liquid medium induce rooting sooner than that in agar medium. The plantlets were transplanted into soilless mix, and obtain more than 90% survival. Endosperm- derived calli, adventitious buds, suspension cells, and plantlets of M. balbisiana were analyzed by flow cell cytometry for measurement of nuclear DNA content which were range 1.74-1.01 times of diploid bananas. The endosperm-derived calli or plantlets exhibit karyotype instability. It is required to select the homogenous triploids in future.
Subjects
Banana
programmed cell death
DNA fragmentation
in vitro culture
suspension culture
Type
thesis
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