Articles
EVALUATION OF ENDOGENOUS AND EXOGENOUS EFFECT OF TREHALOSE ON REGENERATION OF CRYOPRESERVED CHRYSANTHEMUM (DENDRANTHEMA GRANDIFLORUM KITAM.) SHOOT-TIPS
Article number
1039_11
Pages
99 – 105
Language
English
Abstract
In the present work the endogenous and exogenous effect of trehalose on plant regeneration of cryopreserved shoot-tips of chrysanthemum (Dendranthema grandiflorum Kitam.) cultivar Indianapolis was evaluated.
The cryopreserved shoot-tips of in vitro plantlets representing the same cultivar were isolated from a non-tranformed line (Ctrl), and two transgenic lines (TL1 and TL2) with different intracellular trehalose contents.
Shoot-tips were subjected to different preculture conditions using trehalose and/or sucrose, and cryopreserved following the standard vitrification procedure.
After dissection, explants were precultured on semi-solid MS medium containing 0.3 M sucrose or trehalose or with both, 0.15 M sucrose and 0.15 M trehalose for four days.
Precultured shoot-tips were loaded in 0.4 M sucrose + 2 M glycerol solution for 20-30 min, and then exposed to PVS2 or PVS3 vitrification solutions for 0, 10, 20, 30, 40, 50 or 60 min at room temperature prior to rapid immersion in liquid nitrogen.
After cryopreservation, the highest shoot regeneration (45, 63 and 71% for Ctrl, TL1 and TL2, respectively) was achieved after preculture on medium with 0.15 M sucrose + 0.15 M trehalose, and exposure to PVS3 for 40 min.
Cryopreserved shoot-tips of both transgenic lines displayed higher plant regeneration than shoot-tips of non-transformed plantlets when preculture was performed using 0.3 M sucrose or 0.15 M sucrose + 0.15 M trehalose, irrespectively of the PVS solution used for 30 or 40 min.
However, plant regeneration of non-transgenic shoot-tips was higher than regeneration of transgenic tissues when preculture was performed with 0.3 M trehalose.
The 0.3 M trehalose preculture was also the only condition, which allowed survival of transgenic and non-transgenic cryopreserved samples without any exposure to PVS solutions.
These results underline the cryoprotective effect provided by the exogenous application of trehalose, and the importance of its endogenous accumulation to improve tolerance to cryopreservation.
The cryopreserved shoot-tips of in vitro plantlets representing the same cultivar were isolated from a non-tranformed line (Ctrl), and two transgenic lines (TL1 and TL2) with different intracellular trehalose contents.
Shoot-tips were subjected to different preculture conditions using trehalose and/or sucrose, and cryopreserved following the standard vitrification procedure.
After dissection, explants were precultured on semi-solid MS medium containing 0.3 M sucrose or trehalose or with both, 0.15 M sucrose and 0.15 M trehalose for four days.
Precultured shoot-tips were loaded in 0.4 M sucrose + 2 M glycerol solution for 20-30 min, and then exposed to PVS2 or PVS3 vitrification solutions for 0, 10, 20, 30, 40, 50 or 60 min at room temperature prior to rapid immersion in liquid nitrogen.
After cryopreservation, the highest shoot regeneration (45, 63 and 71% for Ctrl, TL1 and TL2, respectively) was achieved after preculture on medium with 0.15 M sucrose + 0.15 M trehalose, and exposure to PVS3 for 40 min.
Cryopreserved shoot-tips of both transgenic lines displayed higher plant regeneration than shoot-tips of non-transformed plantlets when preculture was performed using 0.3 M sucrose or 0.15 M sucrose + 0.15 M trehalose, irrespectively of the PVS solution used for 30 or 40 min.
However, plant regeneration of non-transgenic shoot-tips was higher than regeneration of transgenic tissues when preculture was performed with 0.3 M trehalose.
The 0.3 M trehalose preculture was also the only condition, which allowed survival of transgenic and non-transgenic cryopreserved samples without any exposure to PVS solutions.
These results underline the cryoprotective effect provided by the exogenous application of trehalose, and the importance of its endogenous accumulation to improve tolerance to cryopreservation.
Authors
A. Osorio-Saenz, J.O. Mascorro-Gallardo, M.T. González-Arnao, G. Iturriaga, F. Engelmann
Keywords
chrysanthemum, trehalose, sucrose, transgenic plantlets, shoot-tips, cryopreservation
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