Cryopreserved callus: a source of protoplasts for rice transformation |
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Authors: | Maria-Jesús Cornejo Virginia L Wong Ann E Blechl |
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Institution: | (1) United States Department of Agriculture, Western Regional Research Center, Agricultural Research Service, 800 Buchanan Street, 94710 Albany, CA, USA;(2) Present address: Departamento de Biología Vegetal, Facultad de Farmacia, Universidad de Valencia, Avda. Vicente Andres Estelles s/n, 46100 Burjassot, Valencia, Spain;(3) Present address: SUNY Health Science Center at Brooklyn, 450 Clarkson Ave., Box 651, 11203 Brooklyn, NY, USA |
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Abstract: | We cryopreserved whole rice calli (Oryza sativa L cv Taipei 309) to investigate the ability of the surviving cells to regenerate plants and yield protoplasts competent for genetic transformation. Four out of six callus lines cryopreserved after four months in culture contained small sectors able to continue cell division and subsequently regenerate fertile plants. Both cryopreservation efficiency and regeneration ability decreased when using eight month old cultures. High yields of protoplasts were obtained from different cryopreserved callus lines. Protoplasts were transfected with chimeric genes consisting of the maize ubiquitin 1 promoter, first exon and first intron fused to the coding region of either the GUS or BAR marker genes. Levels of transient gene expression from both marker genes were similar to those previously obtained using protoplasts derived from callus that had not been frozen. Stable transformants were selected by their resistance to Bialaphos and could be identified with the pH indicator chlorophenol red. Southern blot analysis confirmed the integration of the BAR gene into the rice genome. Therefore, cryopreservation does not affect the ability of rice cells to integrate and express foreign genes.Abbreviations BA
6-benzylaminopurine
- BAR
Bialaphos-resistance
- CaMV
cauliflower mosaic virus
- CPS
cryoprotectant solution
- CR
chlorophenol red 2,4-D 2,4-dichlorophenoxyacetic acid
- DMSO
dimethyl sulfoxide
- FW
fresh weight
- GUS
-glucuronidase
- IOD
interoptical density
- MS
Murashige and Skoog
- MU
methyl umbelliferone
- NAA
naphthaleneacetic acid
- PAT
phosphinothricin acetyl transferase
- PEG
polyethylene glycol
- TTC
2,3,5, triphenyltetrazolium chloride
- UBI
maize ubiquitin 1 promoter, first exon and first intron |
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