Name: Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 (K599), Selection, and Harvesting of Hairy Roots.
Uploaded: 2023-05-05T14:40:00
Description: Watch this Scientific Journal Video about Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 K599, Selection, and Harvesting of Hairy Roots. at JoVE.com

infection of cotyledons with agrobacterium rhizogenes strain ncppb2659 (k599), selection, and harvesting of hairy roots.

Transgenik Soya Fasulyesi Tüylü Köklerinin Yüksek Verimli Üretimi

Soybean (Glycine max) is among the most valuable crops in agriculture. It has thousands of commercial and industrial uses, such as food, animal feed, oil, and as a source of raw materials for manufacturing1. Its ability to form a symbiotic relationship with nitrogen-fixing soil microorganisms, namely rhizobia, further elevates the importance of studying soybean genetics2. For instance, fine-tuning nitrogen fixation properties in soybean roots can lead to the reduction of carbon emissions and greatly reduce requirements for nitrogen fertilizer3. Thus, understanding the genetics that controls aspects of soybean root biology, in particular, has wide applications in agriculture and industry. Considering these benefits, it is important to have a reliable protocol to analyze the function of soybean genes.
Agrobacterium tumefaciens is perhaps the most commonly used tool for plant genetic transformation as it has the capability of integrating transfer DNA (T-DNA) into the nuclear genome of many plant species. When Agrobacterium infects a plant, it transfers the tumor-inducing (Ti) plasmid into the host chromosome, leading to the formation of a tumor at the infection site. The Agrobacterium-mediated transformation has been widely used for decades for gene functional analysis and in order to modify crop traits4. Although any gene of interest can be readily transferred into host plant cells through A. tumefaciens-mediated transformation, this method has several drawbacks; it is time-consuming, expensive, and requires extensive expertise for many plant species, such as soybean. Although a few varieties of soybean can be transformed by the cotyledonary node approach using A. tumefaciens, the inefficiency of this approach necessitates the need for an alternative genetic transformation technology that is rapid and highly efficient4,5. Even a non-expert can use this Agrobacterium rhizogenes-mediated hairy root (HR) transformation method to overcome these disadvantages.
HR transformation is a relatively fast tool, not only for analyzing gene function, but also for biotechnological applications, such as the production of specialized metabolites and fine chemicals, and complex bioactive glycoproteins6. The production of soybean HRs does not require extensive expertise, as they can be generated by wounding the surfaces of cotyledons, followed by inoculation with Agrobacterium rhizogenes7. A. rhizogenes expresses virulence (Vir) genes encoded by its Ti plasmid that transfer, carry, and integrate its T-DNA segment into the genome of plant cells while simultaneously stimulating ectopic root growth8.
Compared to other soybean gene&#160;expression systems, such as biolistics or A. tumefaciens-based transformation of tissue, cell, and organ culture, the HR expression system exhibits several advantages. Firstly, HRs are genetically stable and produced quickly on hormone-free media1,9,10. Additionally, HRs can produce specialized metabolites in amounts equivalent to or greater than native roots11,12. These advantages make HRs a desirable biotechnological tool for plant species that are incompatible with A. tumefaciens or that require special tissue culture conditions to form compatible tissues. The HR method is an efficient approach for analyzing protein-protein interactions, protein subcellular localization, recombinant protein production, phytoremediation, mutagenesis, and genome wide effects using RNA sequencing13,14,15. It can also be used to study the production of specialized metabolites that have value in the industry, including glyceollins, which medicate soybean&#39;s defense against the important microbial pathogen Phytophthora sojae and have impressive anticancer and neuroprotective activities in humans16,17.
This report demonstrates an easy, efficient protocol to produce soybean HRs. Compared to previous HR transformation methods, this protocol provides a significant (33%-50%) improvement in the rate of HR formation by prescreening A. rhizogenes transformants for the presence of the Ti plasmid prior to inoculating soybean cotyledons. We demonstrate the applicability of this protocol by transforming several binary vectors that overexpress or silence soybean transcription factor genes.

Yazar Spotlight: Gen Fonksiyonunun Analizi için Soya Fasulyesi Tüylü Kök Dönüşümü  

Gen fonksiyonunun analizi için soya fasulyesi tüylü kök transformasyonu

Our soybean hairy root transformation method is sufficient to simultaneously study the functions of several genes or networks, and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches. Soybean hairy root transformation is a useful tool for the analysis of gene function in soybean plants. The technology that are currently used, include CRISPR-Cas9 genome editing, RNA interference, transcriptomics and proteomics, and imaging technologies.The major challenge in the model era of functional genomics is sufficient analyzing the function of numerous genes. Most genes are polygenic, being achieved by the interaction of many genes. Our hairy root protocol will facilitate gene function analysis with sufficient throughput, so that polygenic networks can begin to be understood.We have established the importance of the Ti plasmid in agrobacterium rhizogene strains. We found that without screening for the Ti plasmid, it increased the risk for the transformed soybean cotyledon, to fail to produce any colors. As a result, screening for the plasmid became a vital step before the transformation.Compared to other gene expression techniques, our hair root expression system is easy to handle, less time consuming, and cost effective. Also, our new protocol has improved hairy root transformation rate up to 50%by checking for the Ti plasmid and agrobacterium prior to cotyledon transformation. By characterizing the function of more than a dozen transcription factor genes, our recent results have paved the way for multi-gene engineering studies, aimed at unlocking the biosynthesis of valuable phytoalexin metabolites in plants.In the future, we will use this hairy root transformation method to dissect the transcription factor gene networks that regulate the biosynthesis of phytoalexins. The approach could be used to enhance the production of these specialized metabolites for pharmaceutical and agricultural industries.

Watch this Scientific Journal Video about Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 K599, Selection, and Harvesting of Hairy Roots. at JoVE.com

Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 (K599), Selection, and Harvesting of Hairy Roots.  

Kotiledonların Agrobacterium rhizogenes Suşu NCPPB2659 (K599) ile Enfeksiyonu, Tüylü Köklerin Seçimi ve Hasadı.

Soya fasulyesi kotiledonlarını Agrobacterium rhizogenes ile enfekte etmek için, Agrobacterium rhizogenes PRI2659 plazmid dizisini kullanarak TI plazmid geni virD2'yi tespit etmek için primerler tasarlayın. Dönüşümü takiben, bir PCR kiti kullanarak virD2'nin PCR ile tutulması için Agrobacterium kolonilerini test edin. Hem virD2 hem de ilgili geni içeren Agrobacterium rhizogenes kolonilerini seçtikten sonra, bazı kolonileri, ilgilenilen plazmid için litre spektinomisin başına 100 miligram içeren LB plakalarına çizin.Ertesi gün, bir P200 pipet ucu kullanarak, Agrobakterinin 1,5 santimetre uzunluğundaki bir uzunluğunu LB plakasından kazıyın ve bir mililitre fosfat tamponunda yeniden askıya alın. Yeniden askıya alınmış hücre süspansiyonunu ve sterilize edilmiş ultra saf suyu asetosiringonda seyreltin. 600 nanometre optik yoğunlukta bir küvet tüpü kullanarak absorbansı ölçün.Daha sonra, bir biyogüvenlik kabininde, sterilize edilmiş bir neşteri Agrobacterium çözeltisine batırın ve kotiledonun iç yüzeyi boyunca bir milimetre derinliğinde bir kesim yapın. Altı ila sekiz kotiledonu, çimlenme ve asetosisingon ile yetiştirme ortamına doymuş filtre kağıdı içeren bir Petri kabına yan yana kesilmiş olarak yerleştirin. Plakaları 16 saatlik bir fotoğraf süresi altında üç gün boyunca oda sıcaklığında inkübe edin.Üç gün sonra, enfekte kotiledonları tüylü kök büyümesine veya HRG plakalarına aktarın. Plakaları, 22 santigrat dereceye ayarlanmış büyüme odalarında ve 16 saatlik bir fotoğraf periyodunda 100 mikromol ışık yoğunluğunda, iki ila üç santimetre uzunluğunda ikincil köklere sahip birincil kökler gözlenene kadar inkübe edin. Üç ila dört hafta sonra, nasırdan büyüyen ve steril bir neşter ve forseps kullanarak ikincil kökler içeren birincil kökleri hasat edin.Kökleri uygun antibiyotikler içeren seçilmiş HRG plakalarına aktarın ve beş gün daha büyümelerine izin verin. Beşinci günde, üç ila altı santimetre uzunluğunda ikincil kökleri olan transgenik tüylü kökleri hasat edin. Floresan proteinleri gözlemliyorsanız, ikincil köklerin çok az otofloresansa sahip olduğundan emin olun.Daha sonra, elicitor veya kimyasal işlemler yapmak için, ikincil kökleri bir santimetre parçalara ayırın ve bir yığın halinde HRG agar üzerine yaklaşık 100 miligram yerleştirin. Daha sonra yığını 80 mikrolitre uygun arıtma çözeltisi ile doyurun ve plakanın oda sıcaklığında inkübe etmesine izin verin. 24 saat sonra, RNA ekstraksiyonu için, kökleri sterilize edilmiş bir kağıt havlu üzerinde hızla kurutun ve doğrudan iki mililitrelik bir mikrosantrifüj tüpüne toplayın.Hemen parafilm kullanarak tüpün üstünü kapatın ve sivri forseps kullanarak iki küçük delik açın. Transforme Agrobakterinin koloni PCR sonuçları gösterilmiştir. PCR'deki pozitif koloniler ilgilenilen geni gösterdi.Bununla birlikte, kolonilerin üçte biri ila yarısı virD2 gen taraması için negatifti. Floresan mikroskopisi GFP-GmJAZ1-6'nın hücre altı lokalizasyonunu gösterir. Gen ekspresyon analizi, Williams 82 harry köklerinde gliseollin transkripsiyon faktörü GmHSF6-1'in aşırı ekspresyonunu ve GmMYB2912'nin RNAi susturulmasını doğruladı.

Research

JoVE Journal

Bioengineering

Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 (K599), Selection, and Harvesting of Hairy Roots.

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...