preparation of bamboo seedlings and agrobacterium-mediated in planta transformation system for gene expression

竹の遺伝子形質転換・編集法の開発

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, moderate stiffness, and flexibility1, which is now widely used in a variety of household and industrial supplies, including toothbrushes, straws, buttons, disposable tableware, underground pipelines, and cooling tower fillers for thermal power generation. Therefore, bamboo breeding plays a crucial role in obtaining bamboo varieties with excellent wood properties for replacing plastics and reducing plastic usage, protecting the environment, and tackling climate change, as well as generating significant economic value.
However, traditional bamboo breeding faces challenges due to the lengthy vegetative growth stage and uncertain flowering period. Although molecular breeding techniques have been developed and applied to bamboo breeding, the process of bamboo gene transformation is time-consuming, labor-intensive, and complicated due to the callus induction and regeneration processes2,3,4,5. Stable genetic transformation often requires Agrobacterium-mediated methods, which involve tissue culture processes such as callus induction and regeneration. However, bamboo has a low ability for callus regeneration, greatly limiting the application of stable genetic transformation in bamboo. After Agrobacterium infects plant cells, the T-DNA fragment enters the plant cells, with the majority of T-DNA fragments remaining non-integrated in the cells, resulting in transient expression. Only a small portion of T-DNA fragments randomly integrate into its chromosome, leading to stable expression. The transient expression levels show an accumulation curve that can vary for each gene expressed from an Agrobacterium-delivered T-DNA. In most cases, the highest expression levels occur 3-4 days after infiltration and quickly decrease after 5-6 days6,7. Previous studies have shown that more than 1/3rd of mutations in gene-edited plants obtained without selection pressure for resistance come from the transient expression of CRISPR/Cas9, while the remaining less than 2/3rd come from stable expression after DNA integration into the genome8. This indicates that T-DNA integration into the plant genome is not necessary for gene editing. Moreover, selection pressure for resistance significantly inhibits the growth of non-transgenic cells, directly affecting the regeneration process of infected explants. Therefore, by using transient expression without selection pressure for resistance in bamboo, it is possible to achieve non-integrated expression of exogenous genes and study gene function directly in plant organs. Hence, an easy and time-saving method can be developed for exogenous gene expression and editing in bamboo9.
The developed exogenous gene expression and gene editing method is characterized by its simplicity, cost-effectiveness, and the absence of expensive equipment or complex procedures9. In this method, the bamboo endogenous violaxanthin de-epoxidase gene (PeVDE) was used as the reporter for exogenous gene expression without selection pressure. This is because the edited PeVDE in bamboo leaves reduces the photoprotection ability under high light and demonstrates a decrease in the non-photochemical quenching (NPQ) value, which can be detected through chlorophyll fluorescence imaging. To demonstrate the effectiveness of this method, another bamboo endogenous gene, the cinnamoyl-CoA reductase gene (PeCCR5)9, was knocked out using this system and successfully generated mutants of this gene. This technique can be used for the functional characterization of genes that have functions in bamboo leaves. By overexpressing these genes transiently in bamboo leaves, their expression levels can be enhanced, or by gene editing, their expression can be knocked down, allowing for the study of downstream gene expression levels, leaf phenotypes, and product contents. This provides a more efficient and feasible approach for gene function research in bamboo. This technique can be applied to the functional characterization of genes that function in bamboo leaves. By overexpressing these genes transiently in bamboo leaves, their expression levels can be enhanced, or by gene editing, their expression can be knocked down, allowing for the study of downstream gene expression levels, leaf phenotypes, and product contents. Additionally, it is important to note that, due to extensive polyploidization, the majority of commercially important genes in bamboo genomes are present in multiple copies, resulting in genetic redundancy. This poses a challenge for performing multiplex genome editing in bamboo. Prior to the application of stable genetic transformation or gene editing techniques, it is crucial to quickly validate gene functions. In addressing the issue of multiple gene copies, one approach is to analyze transcriptome expression profiles to identify genes that are actively expressed during specific stages. Furthermore, targeting the conserved functional domains of these gene copies allows for the design of common target sequences or the incorporation of multiple target sites into the same CRISPR/Cas9 vector, enabling the simultaneous knockout of these genes. This provides a more efficient and feasible approach for gene function research in bamboo.

著者スポットライト:持続可能なプラスチック代替品のための竹の葉の遺伝子編集の進歩 

都市: Planta(プランタ) 孝宗竹葉の遺伝子発現と遺伝子編集

In this study, we used Agrobacterium as a vector to introduce foreign DNA fragments into bamboo leaves. Most DNA fragments are not integrated into chromosomes and undergo transient expression. We took advantage of this transient expression to carry out gene editing.The main challenge is the limitation of gene editing of bamboo leaves, which cannot regenerate. This propose difficulties in performing editing in regenerative organs, like young shoots, and obtaining edited branches or subsequent generations with desired genetic challenges. Its main advantage is the time-saving aspect of the principle.By being able to edit bamboo genes directly in bamboo leaves, it allows for quick verification of gene fractions. This eliminates the needs for time-consuming procedures, like tissue culture or transformation, significantly speeding up research on bamboo gene fractions. The primary research will be focused on investigating the molecular mechanism of bamboo biomass formation.This will involve understanding why bamboo grows rapidly, and using that knowledge to develop bamboo varieties which are suitable for plastic substituent.

Watch this Scientific Journal Video about Preparation of Bamboo Seedlings and Agrobacterium -Mediated In Planta Transformation System for Gene Expression at JoVE.com

Preparation of Bamboo Seedlings and Agrobacterium -Mediated In Planta Transformation System for Gene Expression  

竹苗と アグロバクテリウム媒介Inプランタ形質転換系の遺伝子発現のための調製

まず、あらかじめ浸した竹の種を土とバーミキュライトの基質に蒔いて発芽させます。アグロバクテリウムに感染する場合は、土が根付着した生後15日の苗木を基質から慎重に取り除きます。苗木をアルミホイルで包んで水分を維持し、土壌の剥離を防ぎます。包んだ苗木を、湿度が高く、照明の低い植物成長チャンバーに2時間移します。次に、注射器の鋭利な針を使用して、丸まった未熟な葉の上部を1〜2回巻きます。葉の傷ついた部分をアグロバクテリウム懸濁液にすばやく浸します。次に、すぐに苗木を25〜27ミリメートルの水銀柱の圧力の真空チャンバーに2分間移します。掃除機をかけた後、苗木を慎重に開封し、基材に植え直します。湿度の高い薄暗い場所で2日間、植物成長室で苗木を成長させます。次に、通常の成長条件下で苗木を培養し、5〜7日ごとに水をやります。

preparation-bamboo-seedlings-agrobacterium-mediated-planta

Research

JoVE Journal

Biology

Preparation of Bamboo Seedlings and Agrobacterium-Mediated In Planta Transformation System for Gene Expression

183 ビュー.  Key Laboratory of State Forestry and Grassland Administration/Beijing on Bamboo & Rattan Science and Technology. まず、あらかじめ浸した竹の種を土とバーミキュライトの基質に蒔いて発芽させます。アグロバクテリウムに感染する場合は、土が根付着した生後15日の苗木を基質から慎重に取り除きます。苗木をアルミホイルで包んで水分を維持し、土壌の剥離を防ぎます。包んだ苗木を、湿度が高く、照明の低い植物成長チャンバーに2時間移します。次に、注射器の鋭利な針?...

ビデオ: Preparation of Bamboo Seedlings and Agrobacterium -Mediated In Planta Transformation System for Gene Expression  

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

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.

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 ...

生物学

To begin, sow the pre-soaked bamboo seeds into the substrate of soil and vermiculite for germination. For agrobacterium infection, carefully remove the 15 day old seedlings with soil attached roots from the substrate. Wrap the seedlings with tinfoil to maintain moisture and prevent soil detachment.Transfer the wrapped seedlings to the plant growth chamber with higher humidity and lower illumination for two hours. Next, using a sharp needle from a syringe, wound the upper part of curled immature leaves once or twice. Quickly dip the wounded part of the leaves into agrobacterium suspension.Then immediately transfer the seedlings to a vacuum chamber with a pressure of 25 to 27 millimeters of mercury for two minutes. After vacuuming, carefully unwrap the seedlings and replant them into the substrate. Allow the seedlings to grow in a plant growth chamber in dim light with high humidity for two days.Then culture the seedlings under normal growth conditions, watering them every five to seven days.

In Planta Gene Expression and Measurement of Chlorophyll Fluorescence of Non-Photochemical Quenching Values in Bamboo Leaves

Begin by extracting the genomic DNA from the wild-type and Agrobacterium-infected bamboo leaves. Amplify the genomic DNA containing the target site of the target genes from wild-type and Agrobacterium-infected bamboo leaves using the following PCR conditions. After PCR, perform endonuclease enzyme digestion by preparing a reaction mixture containing one microliter of age-1, one microgram of PCR products, and five-microliter 10X buffer.Then, add water to a final volume of 50 microliters. Incubate the digestion mixture at 37 degrees Celsius for one hour. Using gel electrophoresis, analyze the proportion of digested DNA fragments.Compare the digested fragments from the wild-type and Agrobacterium-infected samples to assess gene editing efficiency. Expose the bamboo seedlings to high light intensity conditions to increase the amount of absorbed light quantum and activation of the leaf photoprotection system. Next, turn on the IMAGING-PAM fluorometer and set the actinic light intensity to measure in vivo photosystem II chlorophyll fluorescence of bamboo leaves.The red beta lane color produced by the RUBY gene indicated successful Agrobacterium-mediated gene expression in bamboo leaves. Out of the four Agrobacterium strains, GV3101 strain caused the most significant beta lane accumulation in infected bamboo leaves. After infection with the CRISPR-Cas9 constructs and high light treatments, certain areas of the leaf blades showed lower non-photochemical quenching values.Furthermore, enzyme digestion and sequencing analysis confirmed the successful mutation of the PeVDE gene in the edited regions. Sanger sequencing results of the PeVDE fragment after editing by both sgRNA1 and sgRNA2 showed deletion of a long fragment in the PeVDE gene. After 30 days of Agrobacterium infection, the leaf areas transfected with sgRNAs for both PeVDE and PeCCR5 exhibited lower non-photochemical quenching values.