Abstract
An efficient protocol for plant regeneration was developed from protoplasts of Gentiana macrophylla Pall. through somatic embryogenesis. Viable protoplasts were isolated from cell suspensions derived from young seedling leaves in an enzyme solution containing 2 % Cellulase Onozuka R-10, 0.5 % Macerozyme R-10, 0.5 % Hemicellulase, and 0.4 M sorbitol with a yield of 6.2 × 106 protoplasts g−1 fresh weight. Liquid, solid–liquid double layer (sLD) and agar-pool (aPL) culture systems were used for protoplast culture. The frequency of protoplast cell divisions and colony formations in aPL culture were significant (p < 0.05) higher than those in liquid and sLD cultures. The highest division frequency and plating efficiency were 37.7 % and 16.5 %, respectively, in aPL culture supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.5 mg l−1 6-benzylaminopurine (BA). Protoplast-derived microcalli obtained from aPL culture system were transferred to solid MS medium with a reduced concentration of 2,4-d (0.5 mg l−1) to promote the formation of embryogenic calli. Somatic embryos developed into plantlets on MS medium supplemented with 2 mg l−1 BA at a rate of 51.3 %. RAPD analysis of G. macrophylla revealed a low variation among regenerants.
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Abbreviations
- aPL:
-
Agar-pool culture
- BA:
-
6-Benzylaminopurine
- CH:
-
Casein hydrolysate
- 2,4-d :
-
2,4-Dichlorophenoxyacetic acid
- FDA:
-
Fluorescein diacetate
- MES:
-
2-(N-morpholino) ethanesulfonic acid
- MS:
-
Murashige and Skoog (1962) medium
- sLD:
-
Solid-liquid double layer culture
- RAPD:
-
Random amplified polymorphic DNA
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We thank the anonymous referees for their helpful comments on the manuscript. This work was financially supported by the National Natural Science Foundation of China (30960161, 31360095).
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Xuemin Hu and Yao Yin have contributed equally to this work.
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Hu, X., Yin, Y. & He, T. Plant regeneration from protoplasts of Gentiana macrophylla Pall. using agar-pool culture. Plant Cell Tiss Organ Cult 121, 345–351 (2015). https://doi.org/10.1007/s11240-014-0705-z
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DOI: https://doi.org/10.1007/s11240-014-0705-z