regeneration and selection methods for brassica napus dh12075 regenerants and t1 plants

Indução e regeneração de raízes pilosas mediadas por Agrobacterium em plantas

Plant transformation is the bottleneck of any genetic study in plant biology. A soil-borne bacterium, Agrobacterium tumefaciens, is widely used as a means for gene delivery by floral dip or tissue culture to generate transformants. A. tumefaciens infects plants at a wounding site and causes tumors due to the transfer and integration of T-DNA from a Tumor-inducing (Ti) plasmid into the host plant genome. Engineered A. tumefaciens strains with modified Ti plasmid without the wild-type T-DNA and a binary vector with artificial T-DNA and cloning sites for inserting a gene of interest are commonly used as an efficient plant transformation system1. However, many model species and crops are recalcitrant to floral dip or in vitro plant regeneration or have long growth cycles, impacting the efficiency of this transformation system.
Agrobacterium rhizogenes induces the formation of adventitious roots, or hairy roots, at the wounding site after infecting a host plant. Similar to A. tumefaciens, A. rhizogenes transfers a T-DNA from a Root-inducing (Ri) plasmid to the host plant genome, causing the development of transgenic hairy roots. This process is controlled mainly by the root oncogenic loci (rol) genes2,3. Using agrobacterial strains carrying both the Ri plasmid and an artificial binary vector encoding a gene of interest, hairy root cultures have been used to produce recombinant proteins, analyze the function of promoters or genes, or edit genomes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)/CRISPR-associated protein 9 (Cas9)4,5,6.
Our protocol uses the transconjugant strain Ti-less A. tumefaciens C58C1 carrying the Ri plasmid pRiA4b7. The T-DNA of the Ri plasmid consists of two regions, right and left T-DNA (TR-DNA and TL-DNA, respectively), that can independently integrate into the plant genome8. Exploiting this system, the lengthy explant transformation process in Brassica napus DH12075 cultivar was optimized9. The protocol detailed below allows for the regeneration of selected hairy root lines and obtaining T1 plants carrying the transgene of interest and free of rol genes in roughly 1 year. The injection-based hairy root transformation can be used in other Brassicaceae species, as shown by transforming Arabidopsis thaliana Col-0. While hypocotyl is used to transform B. napus, A. thaliana is injected into the primary inflorescence stem.

Autor em destaque: Otimizando protocolos de transformação baseados em raízes peludas para maior eficiência em Brassicaceae

Transformação e regeneração de raízes pilosas em Arabidopsis thaliana e Brassica napus

Our research aimed at developing a protocol for an efficient transformation in Brassicaceae. Using a hair root-based transformation, we improved dramatically transformation efficiency in Brassica napus, which is crucial for our research. We also tested the protocol in model species Arabidopsis thaliana.We have optimized the protocol for spring cultivars DH12075 in Brassica napus. The next challenge is to optimize the protocol of transformation and regeneration of winter cultivars in Brassica napus, as well as for Brassica rapa and Brassica oleracea. Hairy roots can be readily propagated, and their rapid growth enables the prompt analysis of the gene of interest shortly after the transformation.Consequently, it becomes feasible to selectively regenerate specific hairy root lines that exhibit the desired trait. For example, a gene mutation introduced by CRISPR-Cas.

Métodos de Regeneração e Seleção de Plantas Regenerantes DH12075 Brassica napus e T1

Em condições estéreis, usando pinças, transfira de cinco a 10 linhas de raízes pilosas de Brassica napus em um prato contendo meio de regeneração. Sele o prato com fita permeável a gás e cultura a 21 graus Celsius em um fotoperíodo de dias longos. A cada três ou quatro semanas, transfira as raízes peludas para uma placa de Petri com meio de regeneração fresco.Observe a formação calosa após aproximadamente duas semanas e brote do caloso após mais duas semanas de formação calosa. Individualizar os brotos e transferi-los para a caixa de cultura vegetal contendo o meio de alongamento da parte aérea. Após duas a três semanas, cortar os restos do calo da parte aérea e transferir os brotos alongados para a caixa de cultura de plantas contendo meio de indução radicular.Agora transfira a planta enraizada para o solo e aclimate-a primeiro nos fitótrons Em seguida, transfira a planta para uma estufa para floração. Para acelerar a produção de plantas T1, colete síliquas contendo sementes verdes maduras dos regenerantes T0. Ao trabalhar em uma caixa de fluxo estéril, esterilize superficialmente as siliques com etanol 70%.Coloque as siliques em uma tampa de placa ou em uma lâmina. Usando uma agulha de calibre 24 montada em uma seringa de um mililitro, corte as síliquas ao longo das margens da válvula. Abra as carpotas e coloque-as de lado.Coletar as sementes imaturas e transferi-las para uma placa contendo meio de germinação. Sele a placa e coloque-a em uma sala de cultivo até a germinação. Em comparação com o tipo selvagem, a regeneração radicular pilosa de B.napus exibiu um fenótipo anão com sistemas radiculares densos, folhas enrugadas e tempo de floração alterado.

regeneration-selection-methods-for-brassica-napus-dh12075-regenerants

Research

JoVE Journal

Biology

Regeneration and Selection Methods for Brassica napus DH12075 Regenerants and T1 Plants

320 visualizações.  Masaryk University. Em condições estéreis, usando pinças, transfira de cinco a 10 linhas de raízes pilosas de Brassica napus em um prato contendo meio de regeneração. Sele o prato com fita permeável a gás e cultura a 21 graus Celsius em um fotoperíodo de dias longos. A cada três ou quatro semanas, transfira as raízes peludas para uma placa de Petri com meio de regeneração fresco.Observe a formação calosa após aproximadamente duas semanas e brote do caloso após mais duas semanas de forma?...

Vídeo: Métodos de Regeneração e Seleção de Plantas Regenerantes DH12075 Brassica napus  e T1

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a ...

Biologia

Agrobacterium-Mediated Hairy Root Transformation in Brassica napus DH12075

To begin, place the seeds of Brassica napus DH12075 in micro-tubes. To degrease the seeds, add water and 0.1%detergent to the micro-tube, and shake for 60 seconds. Now rinse the seeds with water followed by 70%ethanol, each for 60 seconds.Next, place the seeds in 10%sodium hypochlorite solution and shake for 20 minutes. After removing the bleach, wash the seeds four times with sterile water for 60 seconds each. Place the seeds on a Petri dish containing the seed germination medium.Cold stratify the seeds at four degrees celsius overnight before moving the dish to a cultivation room. After five days, transfer the seedlings to a plant culture box containing a plant growth medium. Inoculate Ti less agrobacterium tumefaciens C58C1 carrying a hairy root-inducing plasmid PRIA 4B in five milliliters of LB medium.Grow the culture overnight at 28 degrees Celsius until it reaches an optical density at 600 nanometers of 0.9 to one. Transfer the bacterial inoculum into the syringe. Using a 26-gauge needle mounted on the syringe, puncture the hypocotyl of an 18-day-old seedling at about one centimeter above the growth medium.Inject approximately 50 microliters of inoculum into the wound and scratch the tissue on the surface of the hypocotyl. Return the plant to the cultivation room at 21 degrees Celsius for two to four weeks. Next, cut off the callus with emerged hairy roots from the hypocotyl.Place the callus with hairy roots on a Petri dish containing hairy-root growth medium and antibiotics to suppress agrobacterial growth. Seal the dish with gas-permeable tape before incubating it at 24 degrees Celsius. After incubation, isolate the hairy roots from the callus, and transfer the individual roots to a hairy-root growth medium.Alternatively, to generate a composite plant consisting of wild-type shoots and transgenic hairy roots, remove the native roots of the plant. Then transfer the plant with emerging hairy roots to a culture box containing plant-growth medium and antibiotics to suppress agrobacterial growth.

Transformação de raízes pilosas mediada por agrobactérias em Brassica napus DH12075

Under sterile conditions, using tweezers, transfer five to 10 hairy root lines of Brassica napus on a dish containing regeneration medium. Seal the dish with gas permeable tape and culture at 21 degrees Celsius in a long-day photoperiod. Every three to four weeks, transfer the hairy roots to a Petri dish with fresh regeneration medium.Observe the callous formation after approximately two weeks and shoot from the callous after two more weeks of callous formation. Individualize the shoots and transfer them to the plant culture box containing the shoot elongation medium. After two to three weeks, cut off the remnants of the callous from the shoot and transfer the elongated shoots to the plant culture box containing root induction medium.Now transfer the rooted plant to the soil and acclimatize it first in the phytotrons Then transfer the plant to a greenhouse for flowering. To speed up the production of T1 plants, collect siliques containing mature green seeds from the T0 regenerants. While working in a sterile flow box, surface sterilize the siliques with 70%ethanol.Place the siliques on a plate lid or a slide. Using a 24 gauge needle mounted on a one milliliter syringe, slit the siliques along the valve margins. Open the carpals and put them aside.Collect the immature seeds and transfer them to a plate containing seed germination medium. Seal the plate and place it in a cultivation room until germination. Compared to the wild-type, hairy root regenerative of B.napus displayed a dwarf phenotype with dense root systems, wrinkled leaves, and altered flowering time.

Hairy Root Transformation and Regeneration in Arabidopsis thaliana Col-0

To begin, place the surface-sterilized Arabidopsis thaliana Col-0 seeds onto a plate containing seed germination medium. After two days of cold stratification, move the plate to a cultivation room set at 21 degrees Celsius with a long day photoperiod and 50%humidity. Transfer one-week old seedlings to a plant culture box with a plant growth medium.Grow the Agrobacterium tumefaciens culture in LB medium until it reaches an optical density at 600 nanometers of 0.9 to 1. Using a needle mounted on an insulin syringe, inject approximately 50 microliters of bacterial culture at the base of the primary inflorescent stem of a one-month old Arabidopsis plantlet. Scratch the tissue on the surface of the primary stem with the syringe.After two to four weeks, excise the emerging hairy roots and cultivate them on a Petri dish with the hairy root growth medium supplemented with antibiotics to suppress agrobacterial growth. Culture the hairy root at 24 degrees Celsius in the dark. After one to two weeks, individualize the hairy roots on the plate with hairy root growth medium and transfer selected hairy root lines to a fresh medium every four to five weeks.Transfer the hairy roots to a plate with a regeneration medium to induce callus formation, and culture at 21 degrees Celsius in a long day photoperiod. Once the shoots emerge from the callus, cut the shoots and transfer them to a shoot elongation medium for two to three weeks to promote growth and elongation. Then transfer the elongated shoots to the root induction medium.Finally, transfer the rooted plant to the soil to perform transgenic selection. In A.thaliana, yellow calli were induced within 14 days in all tested hairy root lines. First shoot primordia, visible as dark green spots, emerged within three weeks after transfer to the regeneration medium.After four weeks, shoots covered the hairy roots in 90%of the lines, with some cases showing adventitious root formation. However, one line did not regenerate even after three months. Compared to the wild type, hairy root regenerants of A.thaliana displayed a dwarf phenotype with dense root systems, wrinkled leaves, and altered flowering time.