ISSN:
1432-203X
Keywords:
Key words Orchid
;
Particle bombardment
;
PLB
;
Transformation
;
Key terminologyProtocorm A small storage organ formed from the germinating embryo
;
possessing an apical meristem and a leaf primordium
;
protocorm-like body (PLB) A somatic protocorm derived from in vitro culture of apical or axillary bud meristems
;
primary PLBs PLBs induced by culturing apical meristem-tips aseptically
;
secondary PLBs PLBs formed on the surface of a primary PLB in culture
;
proliferate PLBs PLBs proliferating on the surface of either primary or secondary PLBs
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002990050694
