Identification and evaluation of tobacco cytoplasmic male sterile line Nta(gla)S K326 generated from asymmetric protoplast fusion of Nicotiana glauca and N. tabacum followed by backcrossing with N. tabacum K326

Abstract

Cytoplasmic male sterile (CMS) lines are valuable resources in plant breeding for heterosis utilization, guaranteeing quality of hybrid seeds, and genetic integrity. Nta(sua)S K326, which contains the nuclei of Nicotiana tabacum cv. K326 and cytoplasm of N. suaveolens, is currently the dominant CMS source used in tobacco leaf production in Yunnan, China. Therefore, the alternative CMS lines are required. Here, the Nta(gla)S K326 with cytoplasm of N. glauca has been generated from asymmetric somatic hybridization between protoplasts of K326 and N. glauca followed by backcrossing with K326. Flow cytometry analysis revealed that Nta(gla)S has almost identical ploidy level as that in K326. Moreover, Nta(gla)S, Nta(sua)S, and K326 plants showed similar relative growth rate, yield, and contents of several chemical components in the field. Further scaning electron microscopic observation of floral organs revealed that the Nta(gla)S has shortened filaments and shrivelled stamens, whereas the Nta(sua)S has severe degenerated and carpel-liked stamens, compared with fertile K326 plants. In addition, the expression patterns of several mitochondrial genes in Nta(gla)S, Nta(sua)S, and K326 plants were compared. Furthermore, the cytoplasm source of Nta(gla)S and Nta(sua)S could be distinguished by cleaved amplified polymorphic sequence (CAPS) analysis with chloroplast-specific universal primers. Taken together, these results showed the development of a Nicotiana CMS hybrid via asymmetric somatic hybridization, suggesting that flow cytometry analysis at early stage could facilitate and accelerate the selection process of generating CMS lines. Importantly, the Nta(gla)S provides a valuable germplasm as an alternative CMS line instead of Nta(sua)S in Nicotiana hybrid breeding.

Key message

The Nicotiana CMS hybrid Nta(gla)S has been generated by asymmetric protoplast hybridization. The Nta(gla)S provides a valuable germplasm as an alternative CMS line instead of Nta(sua)S in Nicotiana hybrid breeding.

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Abbreviations

2, 4-D:

2,4-Dichlorophenoxyacetic acid

BAP:

6-Benzylaminopurine

CMS:

Cytoplasmic male sterility

CAPS:

Cleaved amplified polymorphic sequence

CPW:

Cell and protoplast washing solution

IBA:

Indole-3-butyric acid

IOA:

Iodoacetic acid

MS:

Murashige and Skoog

NAA:

Naphthalene acetic acid

Kn:

Kinetin

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Acknowledgements

This study was funded by the National Natural Science Foundation of China (Nos. 31760072 and 31860413) and the Yunnan Academy of Tobacco Agricultural Sciences (Nos. CNTC-110201302009 and YNTC-2017YN05).

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X-J C, D-H Y, and Y-P L designed the experiments. D-H Y and X-J C wrote the manuscript. X-J C, B-G X and F-C J carried out asymmetric fusion. D-H Y, Z-J T, D-H F, HX and GB carried out the analyses of nucleotide sequences, CAPS, and the expression patterns of several mitochondrial genes. X-F W and Y-H Z carried out the agronomic traits analysis for Nta(gla), Nta(sua), and MF K326 in the field. X-J C carried out flow cytometry analysis and the SEM observation. YL and TP carried out the quantification of chemical components. All authors read and approved the final manuscript.

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Correspondence to Xue-Jun Chen or Da-Hai Yang.

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Communicated by Sergio J. Ochatt.

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Chen, X., Tong, Z., Xiao, B. et al. Identification and evaluation of tobacco cytoplasmic male sterile line Nta(gla)S K326 generated from asymmetric protoplast fusion of Nicotiana glauca and N. tabacum followed by backcrossing with N. tabacum K326. Plant Cell Tiss Organ Cult 142, 269–283 (2020). https://doi.org/10.1007/s11240-020-01855-w

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Keywords

  • Somatic hybrid
  • Nicotiana tabacum
  • Nicotiana glauca
  • Flow cytometry
  • Cytoplasmic male sterility (CMS)
  • Protoplast fusion