Studies on Protoplast culture and Agrobacterium-mediated transformation system of Chrysanthemum

• Main Authors: Chen-Chun Ou, 歐貞君
• Other Authors: Li-Jen Liao
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/13008580258686725543

碩士 === 國立高雄師範大學 === 生物科學研究所 === 88 === 　　Chrysanthemum（Chrysanthemum morifolium） is one of the important cut flowers and the flowering potted plants in the 20th century. It is also the largest cut-flower crop in the world and an important flower for export in Taiwan. In the early studies, shoot meristem is the major material for tissue culture of chrysanthemum explants. Since 1990, some researchers used leaf and stem for chrysanthemum’s micropropagation. This study tried to induce callus formation from petal or leaf tissue by different plant growth regulator（PGR）combinations in medium. Both materials showed a good response in growth with a better effect in leaf than petal. Medium supplemented with 10 ppm IAA、10 ppm BA and 0.1 ppm Kinetin is better in callus induction for purple variety and medium with 5 ppm IAA、5 ppm BA and 0.05 ppm Kinetin is better for yellow variety. A complete plantlet will be observed about one month. By the establishment of tissue culture system, the gene transformation system in chrysanthemum, including protoplast transformation system and Agrobacterium-mediated transformation system was approached. The results are listed as follows. 1、Isolation of protoplasts by 1/4 MS medium supplemented with 1% Cellulase RS、1% Macerozyme R-10 and 0.45 M mannitol as enzyme solution displays the best result. And then cultured in 1/2 MS medium supplemented with 2.0 ppm NAA and 1.0 ppm BA resulted in easy cell wall formation in protoplasts and the subsequent cell divided. 2、Compasion for growing condition between different sources of protoplasts we found that isolation of protoplasts from shoots showed better cell division and plantlet growth than that was derived from leave. It is possible to point out that the shoot is a more cell-dividing tissue and leaf is a completely differentiated organ. Therefore, leaf protoplasts cell divided slower than shoot protoplasts. However, protoplast isolation from shoot was relatively difficult to conduct. 3、The protolasts cultured under condition of 4℃ and darkness appeared a faster ability in cell wall synthesis and cell division. In contrast, leaf protoplasts exposed to 4℃ treatment only showed a active cell wall synthesis delay in cell division. 4、Protoplast fusion experiments resulted in forming cell colonies after several times of cell division. 5、The protoplasts showed cell division and formed microcalli easily, but shoot regeneration from microcalli is still not achieved. In the Agrobacterium-mediated transformation system, the regeneration ability of transform plants and infection ability of Agrobacterium strain have been discussed. Leaf discs were chosen as the explants of transformation and LBA4404 and EHA105 were infection strains. Infcetion time was 30 min and co-culture time was 2 days. Cefoxitin''s concentration is 500 μg/ml. The transformation of anti-telomere DNA was detected by determination of the product of report gene GUS. In this investigation, gene transformation using Agrobacterium-mediated system in chrysanthemum has been established and elementary selection of some transgenic plant tissues has been achieved. However, the influence of anti-telomere DNA fragments（RP primer）on plant life cycle is now under investigation.