| Summary: | 博士 === 臺灣大學 === 農藝學研究所 === 96 === Pangolagrass A254 (Digitaria decumbens) is a forage species and has a wide adaption in Taiwan. Male sterility is a major limiting factor in traditional breeding program of pangolagrass. The objective of this study was to develop an efficient plant regeneration system from immature inflorescences and cell suspension culture of pangolagrass to accelerate crop improvement of this species by genetic transformation.
Explants were cultured in MS medium supplemented with different concentrations of 2,4-D, KN, BA and CPPU. The effects of cytokinins on callus induction frequency and morphology were examined. The frequency of white and compact callus induced were KN 0.5 mg/l (100.0 %)，BA 0.5 mg/l (95 %), CPPU 0.5 mg/l (90.0 %) with 2,4-D 2 mg/l was supplied. Calli induced with different cytokinins and cultured on MS medium supplemented with TDZ 0.05 mg/l. The plant regenerated from white and compact callus with 2,4-D 2.0 mg/l and BA 0.5 mg/l were 96.7 %, KN 0.5 mg/l (68.9 %) and CPPU 0.5 mg/l (80.0 %) respectively. There was no plant regeneration from friable and transparent callus. The optimal medium for callus induction was 2,4-D 2 mg/l with BA 0.5 mg/l and plant regenerated with BA 0.5 mg/l. The high frequency of plant regeneration was very essential and beneficial for cell suspension cultures and genetic transformation studies.
The transparent and friable calli were used to establish and maintain the suspension cultures with 2,4-D 2 mg/l for at least 6 months. For plant regeneration, the embryogenic calli were induced by adding BA 0.5 mg/l in suspension medium for one week and then transferring to solid medium with 2,4-D 2 mg/l and BA 0.5 mg/l. The addition of BA in the suspension medium enhanced embryogenic callus formation and the ability of plant regeneration. The plant regeneration frequency of the embryogenic calli derived from cell suspension system reached up to 88.9 % and 87.8 % cultured with BA 0.5 mg/l and TDZ 0.05 mg/l, respectively. The results showed that the calli could keep proliferating and regenerating into the plantlets with high frequency under control.
Embryogenic callus, initiated from immature inflorescence of pangolagrass, was bombarded with a vector mainly containing the CaMV35S promoter, GUS or GFP reporter gene and HPT (hygromycin phosphotransferase) selectable marker gene. Stable calli with GUS stained were visualized for 2 weeks after bombardment. Plantlets regenerated from callus with hygromycin selection medium for four months were analyzed by PCR, and indicate the reporter gene integration into the plant genome. The transformation efficiency was 3.2 % which was estimated with 16 plantlets regenerated with hygromycin selection medium from 496 calli after particle bombardment. These results would be played a very important base of polyploid grass and it is the first report on transgenic plants production of triploid pangolagrass using particle bombardment technique.
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