Development of T-DNA Insertion Molecular Markers to Assess Pollen Flow of Transgenic Papaya

ASIA unversity > 醫學暨健康學院 > 生物科技學系 > 博碩士論文 > Item 310904400/2521

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题名:	Development of T-DNA Insertion Molecular Markers to Assess Pollen Flow of Transgenic Papaya
作者:	Marita Anggaran
贡献者:	Department of Biotechnology
关键词:	Pollen flow;PRSV/PLDMV;Flanking sequence
日期:	2009
上传时间:	2009-11-06 06:11:45 (UTC+0)
出版者:	Asia University
摘要:	Transgenic papayas conferring the resistance to PLDMV/PRSV (Papaya Leaf-Distortion Mosaic Virus/Papaya Ringspot Virus) had been developed. This study aims to develop molecular markers T-DNA insertion flanking sequences which are used to measure the pollen flow of transgenic papaya. The experimental plot of 27.5 m x 27.5 m consisted of 9 plants of 12-4 which are hermaphrodite transgenic papaya as the pollen donor in the center and surrounded by 112 plants of 10-4 which are female transgenic papaya as the pollen recipient. The first part of this study is to monitor the pollination rate based on the seed number collected from the female fruits, and the latter is to monitor the pollen flow by PCR analysis of the progeny genotypes. The pollination rate implies the pollen that fertilized the female plants, which tends to decrease as the distance increase except the similar result in the 5 and 10 m. Instead of wind which was known as pollinator, this result also supports the presence of insect as another pollinator such as the honeybee and moth. Flanking sequences of T-DNA insertion have been generated by adaptor ligation PCR which can distinguish these two different transgenic lines. Specific sets of primer for PCR analysis have been developed as molecular markers to detect the presence of transgene in the progenies of those female plants which might be contributed from the 12-4 which has 1 insert of two copies of T-DNA and 10-4 which has 2 inserts of T-DNA in the progenies. The pollen flow on the progenies was measured by detecting the flanking sequences that located in the border of T-DNA insertion. A total of 816 progenies of 28 female plants in each three different distances have been assayed by PCR for their T-DNA segregation ratio. The gene flow frequency was measured as the number of detected flanking from pollen donor of 12-4 RB flanking to the number of tested progenies. The result showed that the gene flow frequency for 2, 5 and 10 m were 48.33, 54.55 and 49.17, respectively. The shortcoming of this research is the limited size of experimental field, where 10 m is not sufficient to precisely measure the gene flow distance. Therefore, a larger experimental field to know the actual distribution distance is recommended. Nevertheless, this study has offered valuable tools, the molecular markers derived from T-DNA insertion flanking sequence, to assess the pollen flow in the opened area.
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