Abstract
Key message
Suspension cell cultures of rice minichromosomes were established. The minichromosomes in suspension cultured cells were mitotically stable and had active gene expression, thus have the potential to be used as gene expression vectors to produce valuable bioactive products.
Abstract
The plant artificial chromosome (PAC) is a novel vector for plant genetic engineering to produce genetically modified crops with multiple transgenes, or to produce valuable bioactive products through the expression of multiple genes or biochemical pathways as a bioreactor. PAC is mainly constructed by engineered minichromosomes through telomere-mediated chromosome truncations. We have constructed rice minichromosomes in a previous study. Thus, the understanding of rice minichromosome inheritance under different culture conditions has potential importance for their utility in future studies and applications. In this study, we performed suspension cultures of three rice minichromosome-containing cell lines, 1004-111, 1008-100 and 1004-011. Two cell lines, 1004-111 and 1008-100, showed typical S growth pattern consisting of a lag phase, an active growing exponential phase and a stationary phase, whereas cell line 1004-011 grew very slowly and eventually died. Both 1004-111 and 1008-100 minichromosomes were stably transmitted in cell suspension cultures without any abnormality. Foreign gene expression was verified from 1004-111 and 1008-100 minichromosomes in suspension cultures. The stable mitotic inheritance of minichromosomes and gene expression from them indicated that rice minichromosomes could be maintained and propagated in cell suspension cultures. This study tested key parameters for suspension cultures of rice cell lines with minichromosomes, and proved in concept the potential for industrial use of PAC vectors as bioreactors.
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Abbreviations
- 2,4-D:
-
2,4-Dichlorophenoxyacetic acid
- BFB:
-
Breakage-fusion-bridge
- BIBAC:
-
Binary bacterial artificial chromosome
- BSA:
-
Bovine serum albumin
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- DNA:
-
Deoxyribonucleic acid
- dUTP:
-
2′-Deoxyuridine 5′-triphosphate
- EB:
-
Ethidium bromide
- FISH:
-
Fluorescence in situ hybridization
- FW:
-
Fresh weight
- HAC:
-
Human artificial chromosome
- HPTII:
-
Hygromycin phosphotransferase II gene
- MES:
-
4-Morpholinoethanesulfonic acid
- MTR:
-
Minichromosome transmission rate
- PAC:
-
Plant artificial chromosome
- PAGE:
-
Polyacrylamide gel electrophoresis
- PBS:
-
Phosphate buffered saline
- PBST:
-
Phosphate buffered saline solution with the detergent Tween-20
- PVDF:
-
Polyvinylidene difluoride
- RFW:
-
Relative fresh weight
- RFWI:
-
Relative fresh weight increase
- RT-PCR:
-
Reverse transcription polymerase chain reaction
- SDS:
-
Sodium dodecyl sulfate
- SSC:
-
Saline sodium citrate
- TAC:
-
Transformation-competent artificial chromosome
- YAC:
-
Yeast artificial chromosome
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Acknowledgments
This work was supported in part by National Natural Science Foundation of China (Project No. 31271422), a Shenzhen Peacock Innovation Team Project (No. KQTD201101), Shenzhen Commission of Science and Technology Innovation (Project No. CXZZ20120619150627261) and the Hong Kong RGC AoE Project AoE/M-05/12.
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Communicated by Z. Y. Wang.
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Yang, X., Li, J., Chen, L. et al. Stable mitotic inheritance of rice minichromosomes in cell suspension cultures. Plant Cell Rep 34, 929–941 (2015). https://doi.org/10.1007/s00299-015-1755-3
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DOI: https://doi.org/10.1007/s00299-015-1755-3