Agrobacterium-Mediated Gene Transfer

Introduction

Introduction of additional DNA into plant cells or tissues is termed as plant transformation. A DNA of any source can be used for plant transformation. These transformation process has become an very important technique for both scientific and commercial purpose. These transgenic plants, which have a new set of DNA, aid in the understanding of plant gene regulation and also for identification and evaluation of useful traits in plants. Plant transformation is used for the introduction of these useful traits into other plants. Agrobacterium tumefaciens is the most efficient used vector to plant transformation. It has the advantage of evolved crown gall inducing mechanism of DNA transfer present in the common soil pathogen. Much has been learned about the mechanisms of this form of DNA movement and subsequent crown gall induction. A detailed protocol for Agrobacterium-mediated transformation of tobacco cells and their subsequent selection and regeneration into transgenic plants is discussed below.

The production of transgenic plants involves two major process. The first step is the introduction of new genetic material into plant cells, a process known as transformation. The second step uses tissue culture techniques for proliferation and to regenerate the transformed cells into a transgenic plants. Of various methods used for plant transformation, transformation mediated by Agrobacterium tumefaciens is the most commonly used method.

Agrobacterium tumefaciens is a rod shaded, gram-negative bacteria found in soil and it is the natural causative agent for crown gall disease. The bacteria enters the plant through cuts or wounds present in its root or stem. The bacteria then inserts its DNA and stimulates the plant to grow swollen galls. Agrobacterium tumefaciens is capable of interkingdom DNA transfer, thus making it a potential vector in the production of transgenic plants. Transformation using this organism has its advantage in identification of transgenic expression. The presence of gall represents the manifestation of transfer and expression of additional DNA in plant cells.  The application of this method is found both in scientific and commercial purpose.

Agrobacterium tumefaciens is attracted to the amino acids, sugars and organic acids released by the wounded plants. It binds the wounded tissue by polar attachment mechanism. During this attachment switches on the genetic operons critical to the start of gene transfer expression. The vir regulon is regulated and starts expression during the attachment stage. The wound phenolics and monosaccharides directly or indirectly cause the autophosphorylation of the virA transmembrane  receptor kinase. This in turn activates the soluble cytoplasmic transcriptional factor virG through another phosphorylation event. Activated Vir G initiates the transcription of individual vir operon. These gene products produced in this process transfers the DNA fragment into the plant using the T-DNA present in the Tumour inducing (Ti) plasmid. T- strand is integrated with plant genome via non-homologous recombination by plant encode proteins.   Most laboratory experiments use a binary system consisting of two plasmids. The oncogenes have been removed from the T-DNA sequence and replaced with DNA of interest.  Different strains of A. tumefaciens display different virulence.

Once the plant cell has been introduced the new DNA in a stable manner, the next step is to regenerate a plant from the transformed cells. The isolation of transgenic plants is limited by lack of generation occurring in the transformed cell population. Variability in the frequency and scope of regeneration among angiosperm species is high.  Regeneration is done by two ways: organogenesis and somatic embryogenesis. Organogenesis is the formation of organized shoots and roots. Somatic embryogenesis is the formation of embryo like structure from somatic tissues.  These two process are mutually exclusive process.

Materials

Supplies and Equipment

  1. GA7 tissue culture boxes with lids.
  2. Laminar flow hood.
  3. Forceps.
  4. Scalpels.
  5. Sterile, disposable Petri dishes.
  6. Sterile filter paper.
  7. Cork borers (0.7 mm).
  8. Cork borer sharpener.
  9. Bunsen burners.
  10. Inoculation loops.
  11. Environmental shaker incubators (25°C).
  12. Environmental growth chambers.
  13. P20, P200, P1000, and P5000, Pipetman (or equivalent) micropipettors and appropriate tips.
  14. 1.5-mL Microfuge tubes.
  15. 15- and 50-mL capped centrifuge tubes (Falcon or equivalent).
  16. 15% (v/v) Household bleach.
  17. 70% Ethanol.
  18. Laboratory sealing film (Parafilm or equivalent).
  19. Heated water bath at 55°C.
  20. Disposable 10-mL sterile syringes.
  21. Acrodisc 0.2-μ m syringe filter sterilization units (or equivalent).

Reagents, Solutions, and Media

  1. 1/2X MSO, pH 5.8: half-strength Murashige and Skoog (MS) medium solidified with 0.8% Phytagar.
  2. Agrobacterium strains: any one of several common disarmed (non-gall-forming) laboratory strains (e.g., EHA 101, 105, C58, and LBA4404) containing an engineered binary transformation vector.
  3. YEP medium, pH 7.2: 5.0 g/L of Bacto-yeast extract, 10.0 g/L of Bacto-peptone, 10 g/L of NaCl, 15 g/L of Bacto-agar.
  4. Filter-sterilized MS20IM Agrobacterium induction medium, pH 5.25: MS salts and vitamins supplemented with 2 % (w/v) sucrose, 100 μ M acetosyringone, 1 mM betaine phosphate or proline, and 2.5 mM 2-(4-morpholino)ethanesulfonic acid (MES).
  5. Cocultivation medium, pH 5.8: MS medium supplemented with 4.5 μ M benzylaminopurine (BA), 0.5 μ M naphthalene acetic acid (NAA) and solidified with 0.8% (w/v) Phytagar.
  6. MSBN1.1 shoot regeneration medium, pH 5.8: identical to the cocultivation medium shown above with the exception that selective agents are used as appropriate.
  7. MSHF rooting medium, pH 5.8: MS medium solidified with 0.8% Phytagar and supplemented with selective agents when appropriate.

Methods

Growth and Propagation of Tobacco (Nicotiana tabacum)

Most plants offer a number of tissues that will regenerate under the proper conditions. However, efficiencies may vary greatly. Plants or plant tissues grown under axenic conditions in culture offer the most consistent results with respect to regeneration, as some of the environmental conditioning that varies with season in plants grown outside of the laboratory has been  eliminated. Material from cultures also leads to fewer downstream contamination problems. Shown below is a procedure for growing tobacco plants under axenic conditions. It should be noted that the methods have been optimized for the cultivar “Xanthi”; however, others, such as “SR1” also have been successfully transformed using this procedure.

  1. Sterilize the surface of tobacco seeds by placing them in 15-mL conical centrifuge tubes and filling them with 10 mL of a 15% bleach solution plus one drop of Tween-20.
  2. Shake the tubes continuously for 15 min on a gyratory shaker at 110 rpm.
  3. Allow the seeds to settle, pipet off the Clorox solution, and rinse three times with sterile distilled water. Rinsing is accomplished by filling the centrifuge tube with 10 mL of sterile distilled water, then allowing the seeds to settle and pipetting off the rinse water. Remove all but 1 mL of water during the final rinse.
  4. Dispense the last milliliter of water with seeds using a pipet onto 100 × 20 mm Petri dishes containing 25 mL of agar solidified 1/2X MSO.
  5. Incubate plates at 26°C under soft fluorescent lights with a 16-h photoperiod.
  6. After 10–14 d, transfer germinating green seedlings to Magenta boxes containing 50 mL of autoclaved MSHF.
  7. Plants may be multiplied by removing expanded leaves from rooted plants, cutting the remaining stem between nodes, and inserting the resulting stem pieces into Magenta boxes containing MSHF. Individual plants may be maintained indefinitely without multiplication by simply propagating the shoot tip in a similar manner. Repeat subcultures to fresh medium once every 4 wk.

Growth of Agrobacterium and Preparation of Inoculum

Compared to other laboratory strains of bacteria such as Escherichia coli, Agrobacterium grows relatively slowly. To grow overnight cultures of sufficient densities consistently and conveniently, it is important to inoculate them with cells actively growing on solid medium.

  1. Prepare a 50-mL culture tube containing 10 mL of YEP media containing the appropriate selective antibiotics.
  2. Inoculate the tube with one loopful of active bacteria ( tumefaciens containing a binary vector with the gene[s] of interest) taken from a selection plate kept at 4°C.
  3. Grow 20–24 h at 25°C with agitation of 100–150 rpm. If an environmental shaker is unavailable, room temperature should be sufficient.
  4. Determine the optical density of the cultures spectrophotometrically at 420 nm. Calculate the amount of culture needed to provide an optical density of 0.5 when diluted to 20 mL.
  5. Centrifuge the appropriate amount of culture in a 50-mL Falcon tube for 15 min at 2500g.
  6. Pour off the supernatant
  7. Resuspend the pellet in 20 mL of MS20IM medium
  8. Induce the Agrobacterium for transformation by shaking on a rotary shaker (100– 150 rpm) for 5 h at 20–25°C (room temperature).

Preparation and Infection of Leaf Disks

The overall objective in preparing plant material is to maximize the number of wounded, cut surfaces for Agrobacterium attachment while maintaining enough healthy tissue that will later support efficient regeneration.

  1. Remove expanded leaves from rooted plants growing axenically in culture and float them in 100-mm Petri dishes containing sterile MS20IM.
  2. Cut disks from the leaves in dishes under MS20IM using a flame-sterilized 0.7-cm cork borer. Prepare leaf disks in batches of approx 50/plate.
  3. Set aside approx 16 leaf disks to serve as controls for the transformation/regeneration procedure by transferring them directly to 100 × 15 mm Petri dishes containing cocultivation medium overlaid with sterile filter paper (8 disks/plate) after gently blotting away excess MS20IM using sterile filter paper.
  4. Decant the MS20IM from the plates containing the remaining leaf disks using a sterile pipet and replace it with induced A. tumefaciens suspension. Incubate at room temperature (approx 25°C) for 10–20 min with occasional swirling.

Cocultivation

Agrobacterium attachment to plant tissue is completed during the earlier stages of cocultivation. The physical transfer of genetic material occurs later.

  • Remove each disk individually, gently blot off excess culture using sterile filter paper, and transfer to 100 × 15 mm Petri dishes containing cocultivation media overlaid with sterile filter paper. Place about 16 disks/plate.
  • For large scale experiments we routinely cut about 800 disks and inoculate Petri dishes with approx 24 disks/plate.
  • Seal all Petri dishes with laboratory sealing film (Parafilm or equivalent)
  • Incubate cultures at 20°C in the dark for 3 days.

Selection and Regeneration of Transgenic Tobacco Shoots

Several important events occur during selection and regeneration. Antibiotic(s) that do not affect plant cells are used to eliminate or arrest the growth of A. tumefaciens. Conditions are also optimized for the adventitious, organogenic regeneration of new plant tissues. Additional selective agents are incorporated into the regeneration medium to enrich the population of new growth with transgenic tissues. It aids the purposes of genetic selection. Genetic selection is the process of selecting preferentially for those cells that have been transformed by the incoming transgenes. A selective advantage can be conferred on the transformed cells through the introduction of genes encoding antibiotic resistance or resistance to some metabolic inhibitor such as a herbicide. The untransformed cells die in the presence of the antibiotic or herbicide, whereas the transformed cells grow and multiply. If no form of genetic selection were used, then one would be faced with the option of screening every shoot that regenerated in a transformation experiment. If the transformation frequency is high and we can use this method of screening. However, for other species with lower transformation frequencies, this would become a laborious if not impossible task. Therefore, genetic selection is an essential component of any plant transformation protocol and has been accomplished by using various marker genes.

  1. Subculture the disks to selective medium. All those infected with tumefaciens and half of the control disks (no infection with A. tumefaciens) should be transferred to MSBN1.1 regeneration medium containing the appropriate selective agents in 100 × 15 mm Petri dishes. The control disks under these conditions will provide an indication of nontransgenic regeneration (“escapes”) under selection. Transfer the remaining control disks to regeneration medium (MSBN1.1) containing only the selective agent used to eliminate Agrobacterium (this is a control to evaluate overall regeneration frequency). In all cases, plate at a density of approx 8 disks/plate.
  2. Maintain cultures at 20°C in low light (approx 45 μE/m2s). Check regularly for contamination. If contamination is discovered, unaffected disks within the plate may be subcultured to fresh MSBN1.1.
  3. All disks should be subcultured to fresh selection plates every 2–3 wk. The disks will expand and develop callus over time. Try to ensure that the expanded disks establish good contact with the media. Shoots will appear in 3–4 wk.

Rooting of Transgenic Shoots to Recover Complete Plantlets

The next step is to recover complete plants from any regenerated shoots through root organogenesis. In addition, the first meaningful screen to test for transformation is often the rooting procedure, as root organogenesis is usually more sensitive to the incorporated selective agents than shoot regeneration. Shoots recovered from selective regeneration procedures that do not root under selection are rarely transgenic and should be discarded.

  1. Carefully remove regenerated shoots by cutting them at their base using a sterile scalpel and forceps and place them in GA7 boxes (about four shoots per vessel) containing 50 mL of MSHF supplemented with selective agents. Roots should become visible within approx 10 d.
  2. Subculture only the shoots that have rooted by cutting off the shoot with the top four internodes and introducing these individually into a GA7 box containing 50 mL of MSHF supplemented with the appropriate selective agents. These individual shoots may be considered as putative transformants.
  3. Rooted shoots can be maintained and/or propagated to establish individual lines at monthly intervals. Alternatively, the plants may be acclimatized and transferred to the greenhouse to produce seeds. It takes about 3 months to set seed, depending on conditions.

Analysis of Transgenic Plants

Recovered plants are typically analyzed on a number of different levels to determine that they are in fact transgenic. Once plants grow large enough to provide enough tissue for analyses without compromising health, they may be assayed for transgene expression and molecularly for the presence of the appropriate sequences. The assay for gene expression is conducted using methods consistent with the transgene coding sequence and desired results. If such a procedure is impossible or inconvenient, polymerase chain reactions (PCRs) may also be performed. Plants that give a positive result must then be analyzed using a DNA blotting procedure (Southern) to confirm the presence of transgenes and their abundance.