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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 132, Issue 1, pp 213–217 | Cite as

Construction of genetic transformation system of Salix mongolica: in vitro leaf-based callus induction, adventitious buds differentiation, and plant regeneration

  • Qingjie Guan
  • Mingling He
  • Haiyan Ma
  • Xu Liao
  • Zhenjuan Wang
  • Shenkui LiuEmail author
Research Note
  • 493 Downloads

Abstract

Songnen meadow grassland is a typical saline-alkaline land majorly comprised of carbonate soil. Salix mongolica, a woody species with high adaptability to carbonate soil, is an important supplementary feed in the grassland. Therefore, it is necessary to cultivate new varieties of S. mongolica by using genetic engineering methods to reveal the functions of the plant’s related genes and to construct a plant regeneration and genetic transformation system. In this study, we used leaves of S. mongolica as the explants for induction of leaf-based callus, differentiation of adventitious buds and rooting of adventitious by adding different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine and naphthaleneacetic acid into the Murashige and Skoog medium. Under the screening conditions of 7.5 mg L−1 hygromycin B and transformation period of 2–5 min using a specific Agrobacterium containing pCXSN-gus plasmids infection concentration (ODλ600 = 0.5), we obtained transgenic strains. PCR detected exogenous gus gene integrated into the chromosome of S. mongolica, Southern blot analysed the T0 transgenic strains single copy inserted into the chromosome, Northern hybridization signals indicated that gus gene mRNA was expressed in the five contemporary transgenic strains. The infected callus, adventitious buds, and regenerated plants displayed a blue color through detection by GUS staining, which reflected the activity of ß-glucuronidase enzyme. This result demonstrated the successful establishment of an Agrobacterium-mediated genetic transformation system from the callus (S. mongolica leaf as a transformation receptor).

Keywords

Salix mongolica Agrobacterium Leaf Callus Genetic transformation 

Notes

Acknowledgements

This work was supported by the National High Technology Research and Development Program of China (Grant No. 2013AA102701-7), The National Key Research and Development Program of China (2016YFC0501203), Program for Chang Jiang Scholars and Innovative Research Team in University (IRT13053), and Heilongjiang Postdoctoral Scientific Research Developmental Fund (LBH-Q15004).

Supplementary material

11240_2017_1265_MOESM1_ESM.doc (34 kb)
Supplementary material 1 (DOC 34 KB)
11240_2017_1265_MOESM2_ESM.tif (12.5 mb)
Supplementary Figure 1—Induction of leaf-based callus and plantlet regeneration of S. mongolica. Optimization of culture medium for leaf-based callus induction (A) and differentiation of adventitious buds from the callus. (B); explant leaves (C); callus inducted from the leaves. Bar = 500 µm (D); regeneration buds differentiated from the callus (Bar = 2 mm E and F); rooting plantlets. Bar = 1 cm (G); regenerated seedlings under soil cultivation. Bar = 5 cm (H). (TIF 12819 KB)
11240_2017_1265_MOESM3_ESM.tif (2.3 mb)
Supplementary Figure 2—PCR amplification of the gus gene (573 bp) in randomly selected transformed plantlets maintained for non-transgenic and transgenic T0 line after propagation from cuttings. Lane M: 2000 bp plus DNA marker; Lane CK+:pCXSN-gus plasmid DNA as a CK+; Lane CK: genomic DNA from a non-transformed plantlet as a CK; 2000 bp plus DNA marker (Lanes 1, 12, and 21), CK+ (Lanes 2 and 13), CK (Lanes 2 and 14), #1 (Lane 4), #5(Lane 5), #6(Lane 6), #7 (Lane 7), #8 (Lane 8), #9 (Lane 9), #11 (Lane 10), #12 (Lane 11), #13 (Lanes 15), #14 (Lanes 16), #15 (Lanes 17), #18 (Lanes 18), #19 (Lanes 19), and #20 (Lanes 20) transgenic gus gene plants. (TIF 2319 KB)

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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Qingjie Guan
    • 1
  • Mingling He
    • 1
  • Haiyan Ma
    • 1
  • Xu Liao
    • 1
  • Zhenjuan Wang
    • 1
  • Shenkui Liu
    • 1
    Email author
  1. 1.Key Laboratory of Saline-alkali Vegetation Ecology Restoration (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC)Northeast Forestry UniversityHarbinChina

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