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Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco

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Abstract

It is known that protoplasts derived from either leaves or suspension cultures of a citrus genotype vary greatly in their regeneration capacities; however, the underlying physiological mechanisms are not well known. In this study, oxidative stress and antioxidant systems during in vitro culture of callus-derived protoplasts and leaf mesophyll-derived protoplasts of Ponkan (Citrus reticulata Blanco) were analyzed to gain insights into observed physiological differences. Morphological observations using light microscopy and scanning microscopy have shown that new cell wall materials appeared within 2–3 days, and the integrate cell walls were regenerated approximately after 6 days of culture of the callus protoplasts, whereas no cell wall formation was observed in the mesophyll protoplasts after culture. During the culture, higher levels of H2O2 and malondialdehyde were detected in the mesophyll protoplasts as compared with the callus ones. On the contrary, the callus protoplasts possessed higher activities of antioxidant enzymes (SOD, POD and CAT) and larger amount of glutathione and ascorbic acid (at one time point) than the mesophyll protoplasts during the culture process. The current data indicate that the mesophyll and callus protoplasts displayed remarkable difference in the degree of oxidative stress and the antioxidant systems, suggesting that high levels of antioxidant activities might play an important role in the regeneration of protoplasts.

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Abbreviations

AsA:

Ascorbic acid

CAT:

Catalase

CFW:

Calcofluor White M2R

CPW:

Cell and protoplast washing salts

EME:

Embryogenic callus culture media

FDA:

Fluorescein diacetate

FESEM:

Field emission scanning electron microscopy

GSH:

Reduced glutathione

MDA:

Malondialdehyde

MT:

Murashige and Tucker

NBT:

Nitro blue tetrazolium

POD:

Peroxidase

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

TBA:

Thiobarbituric acid

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Acknowledgments

This research was financially supported by the National Natural Science Foundation of China, China Postdoctoral Science Foundation (20090451251), the open projects of the National Key Laboratory of Crop Genetic Improvement (ZK201003) and the open funds of Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology (K09002). The authors are grateful to the Testing Center of Yangzhou University for scanning electron microscopy.

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Authors and Affiliations

  1. School of Horticulture and Plant Protection, Yangzhou University, Yangzhou, 225009, China

    Xiaoyong Xu, Li He, Jiaojiao Zhang, Xiaoling Xu & Rong Qian

  2. Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, China

    Gaisheng Xie & Ji-Hong Liu

  3. Key Laboratory of Ministry of Education for Plant Functional Genomics, Yangzhou University, Yangzhou, 225009, China

    Guohua Liang

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  1. Xiaoyong Xu
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  2. Gaisheng Xie
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Correspondence to Xiaoyong Xu or Ji-Hong Liu.

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Xu, X., Xie, G., He, L. et al. Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco. Plant Cell Tiss Organ Cult 114, 161–169 (2013). https://doi.org/10.1007/s11240-013-0312-4

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  • DOI: https://doi.org/10.1007/s11240-013-0312-4

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