JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

Published: August 18th, 2023

DOI:

10.3791/65799

1Key Laboratory of State Forestry and Grassland Administration/Beijing on Bamboo & Rattan Science and Technology, 2Institute of Gene Science and Industrialization for Bamboo and Rattan Resources, International Centre for Bamboo and Rattan

In this study, a novel in planta gene expression and gene editing method mediated by Agrobacterium was developed in bamboo. This method greatly improved the efficiency of gene function validation in bamboo, which has significant implications for accelerating the process of bamboo breeding.

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and regeneration processes. This method involves Agrobacterium-mediated gene expression via wounding and vacuum for bamboo seedlings. It successfully demonstrated the expression of exogenous genes, such as the RUBY reporter and Cas9 gene, in bamboo leaves. The highest transformation efficiency for the accumulation of betalain in RUBY seedlings was achieved using the GV3101 strain, with a percentage of 85.2% after infection. Although the foreign DNA did not integrate into the bamboo genome, the method was efficient in expressing the exogenous genes. Furthermore, a gene editing system has also been developed with a native reporter using this method, from which an in situ mutant generated by the edited bamboo violaxanthin de-epoxidase gene (PeVDE) in bamboo leaves, with a mutation rate of 17.33%. The mutation of PeVDE resulted in decreased non-photochemical quenching (NPQ) values under high light, which can be accurately detected by a fluorometer. This makes the edited PeVDE a potential native reporter for both exogenous and endogenous genes in bamboo. With the reporter of PeVDE, a cinnamoyl-CoA reductase gene was successfully edited with a mutation rate of 8.3%. This operation avoids the process of tissue culture or callus induction, which is quick and efficient for expressing exogenous genes and endogenous gene editing in bamboo. This method can improve the efficiency of gene function verification and will help reveal the molecular mechanisms of key metabolic pathways in bamboo.

The investigation of gene function in bamboo holds great promise for the advanced understanding of bamboo and unlocking its potential for genetic modification. An effective way of this can be achieved through the process of Agrobacterium-mediated infection in bamboo leaves, whereby the T-DNA fragment containing exogenous genes is introduced into the cells, subsequently leading to the expression of the genes within the leaf cells.

Bamboo is a valuable and renewable resource with a wide range of applications in manufacturing, art, and research. Bamboo possesses excellent wood properties such as high mechanical strength, toughness, mo....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Preparation of bamboo seedlings

  1. Prepare moso bamboo (Phyllostachys edulis) seedlings using seeds harvested in Guilin, Guangxi, China. Begin by soaking the seeds in water for 2-3 days, making sure to change the water daily. Next, create a substrate by mixing soil and vermiculite in a ratio of 3:1.
  2. Sow the soaked seeds into the substrate for germination. Maintain the seedlings under laboratory conditions, keeping the temperature between 18-25 °C. Ensure a 16 h light/8.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Agrobacterium-mediated in planta gene expression in bamboo leaves
The RUBY reporter gene has been demonstrated to be effective in visualizing transient gene expression due to its ability to produce vivid red betalain from tyrosine10. In this study, Agrobacterium-mediated transformation was utilized to transiently express the exogenous RUBY gene in bamboo leaves (Figure 1). At  the 3rd .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This method significantly reduces the time required compared to traditional genetic transformation methods, which typically take 1-2 years, and achieves transient expression of exogenous genes and gene editing of endogenous genes within 5 days. However, this method has limitations as it can only transform a small proportion of cells, and the gene-edited leaves are chimeric and lack the ability to regenerate into complete plants. Nevertheless, this in planta gene expression and gene editing technology provides a .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The authors would like to thank the National Key Research and Development Program of China (Grant No. 2021YFD2200502), the National Natural Science Foundation of China (Grant No. 31971736) for the financial support.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
35S::RUBY Addgene, United States 160908 Plamid construct
Agrobacterium competent cells of GV3101, EHA105,LBA4404, and AGL1 Biomed, China BC304-01, BC303-01, BC301-01, and BC302-01 For Agrobacterium infection
CTAB Sigma-Aldrich, United States 57-09-0 DNA extraction
Imaging-PAM fluorometer Walz, Effeltrich, Germany Detect chlorophyll fluorescence of bamboo leaves
ImagingWin Walz, Effeltrich, Germany Software for Imaging-PAM fluorometer
Paq CI or Aar I NEB, United States R0745S Incorporate the target sequence onto the CRISPR/Cas9 vector.
PrimeSTAR Max DNA polymerase Takara, Japan R045Q For gene cloning
T4 DNA ligase NEB, United States M0202V Incorporate the target sequence onto the CRISPR/Cas9 vector.

  1. Jiang, Z. H. . World Bamboo and Rattan (in Chinese). , (2002).
  2. Ye, S., et al. An efficient plant regeneration and transformation system of ma bamboo (Dendrocalamus latiflorus Munro) started from young shoot as explant. Frontiers in Plant Science. 8, 1298 (2017).
  3. Ye, S., et al. Robust CRISPR/Cas9 mediated genome editing and its application in manipulating plant height in the first generation of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro). Plant Biotechnology Journal. 18 (7), 1501-1503 (2020).
  4. Xiang, M., et al. Production of purple Ma bamboo (Dendrocalamus latiflorus Munro) with enhanced drought and cold stress tolerance by engineering anthocyanin biosynthesis. Planta. 254 (3), 50 (2021).
  5. Huang, B., et al. An efficient genetic transformation and CRISPR/Cas9-based genome editing system for moso bamboo (Phyllostachys edulis). Frontiers in Plant Science. 13, 822022 (2022).
  6. Lee, M. W., Yang, Y. Transient expression assay by agroinfiltration of leaves. Methods in Molecular Biology. 323, 225-229 (2006).
  7. Canto, T. Transient expression systems in plants: potentialities and constraints. Advances in Experimental Medicine and Biology. 896, 287-301 (2016).
  8. Chen, L., et al. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants. Horticulture Research. 5, 13 (2018).
  9. Sun, H., et al. A new biotechnology for in-planta gene editing and its application in promoting flavonoid biosynthesis in bamboo leaves. Plant Methods. 19 (1), 20 (2023).
  10. He, Y., Zhang, T., Sun, H., Zhan, H., Zhao, Y. A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research. 7 (1), 152 (2020).
  11. Wang, C., Shen, L., Fu, Y., Yan, C., Wang, K. A simple CRISPR/Cas9 system for multiplex genome editing in rice. Journal of Genetics and Genomics. 42 (12), 703-706 (2015).
  12. McCormick, S., et al. Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Reports. 5 (2), 81-84 (1986).
  13. Lou, Y., et al. a violaxanthin de-epoxidase gene from moso bamboo, confers photoprotection ability in transgenic Arabidopsis under high light. Frontiers in Plant Science. 13, 927949 (2022).
  14. Zhou, R., et al. Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula. Proceedings of the National Academy of Sciences of the United States of America. 107 (41), 17803-17808 (2010).
  15. De Roeck, A., et al. Deleterious ABCA7 mutations and transcript rescue mechanisms in early onset Alzheimer's disease. Acta Neuropathologica. 134 (3), 475-487 (2017).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved