Transient Transformation of Oncidium Taka by Particle Bombardment
Oncidium Taka was used in this study for its importance as cut flower in Malaysia floriculture industry. Protocorm likes bodies (PLBs) that proved to have vigorous propagation and regeneration capacity were induced from shoot tips and flower buds in MS hormone free medium. Subsequently, callus in...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2006
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/111/1/548988_FBSB_2006_13.pdf |
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| Summary: | Oncidium Taka was used in this study for its importance as cut flower in Malaysia
floriculture industry. Protocorm likes bodies (PLBs) that proved to have vigorous
propagation and regeneration capacity were induced from shoot tips and flower buds in
MS hormone free medium. Subsequently, callus induction by using different
concentrations of auxins (2,4-D, dicamba, picloram, IAA and NAA) was investigated in
dark condition. Picloram in a concentration of 40 μM was found to be the most effective
for inducing callus from PLBs. However, the initiated callus was maintained and
proliferated in medium consisted of combination hormones of 50 μM picloram and 20
μM kinetin. Both PLBs and callus were used as target tissues in the genetic
transformation study.Regeneration study of these calli was carried out in half strength MS
medium under light condition. The calli regenerated by intervening
PLB stage and the shoot primordial was developed as early as 44 days
after culture. Besides that, media composition and different
concentrations of BAP were examined to enhance plant regeneration
and plant growth from PLBs. Half strength MS medium supplemented
with full strength B5 vitamin, 3 % sucrose and 20 μM BAP was found
to be efficient for plant regeneration from PLBs. Apparently, better
plant growth can be promoted in full strength MS medium
supplemented with full strength B5 vitamin, 2 % sucrose and 20 μM
BAP.
The effectiveness of hygromycin as selective agents to inhibit growth of
PLBs and callus was evaluated. Toxicity effect of hygromycin was
found rapid and effective to kill the untransformed PLBs and callus in
low concentration at 25 mg/L and 10 mg/L, respectively.
GFP as a non-destructive, early and rapid detection reporter system
was used in this transformation studies. The p35S-sgfp-TYG-nos GFP
plasmid was determined to give the highest transient expression in both target tissues among five different plasmids evaluated. The
fluorescent signals expressed with elevated level of green fluorescent
on nuclei were observed using Leica Stereo-fluorescence System MZ
FLIII with GFP 2 filter set that masked out chlorophyll. The highest
numbers of transient expression achieved on day two postbombardment
for both target tissues.
The physical and biological parameters affecting DNA delivery, based on the highest
scorable transient GFP expression and minimal tissue dislocation upon impact, were
optimised. The physical parameters optimised in helium pressure, distance from
stopping plate to target tissue, vacuum pressure, size of microcarrier, quantity of DNA
and effect of CaCl2 and Spermidine were found no different between PLBs and callus.
As for biological parameters tested, PLBs in sizes of 5 – 6 mm had significantly higher
transient expression compared to calli. Seven days old calli with one day pre-culture
period and fourteen days old PLBs that pre-culture on the same day gave the highest
transient expression. The quantity of DNA per bombardment used was 1.5 μg for both
target tissues to achieve the highest transient expression.
The bombarded tissues were subsequently transferred to hygromycin containing
medium for a minimum of six months selection. The survived and proliferated putative
transformed tissues were subjected to molecular analysis. Insertion of the transgenes
(gfp, gus A, hpt II and chs) into host genome was confirmed using PCR and Southern
vi
Blot analysis. Transformation efficiency was determined at 1.5 % to 1.67 % and 1.67 %
to 2.5 % when using PLBs and callus, respectively as target tissue. In addition, PCR
analysis showed co-transformation frequency of the un-linked genes from different
plasmids was 40 % to 67 %. |
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