In planta transformation

EMBO COURSE

Practical Course on Genetic and Molecular Analysis of Arabidopsis

Module 5

In-planta Agrobacterium mediated transformation of adult plants by vacuum infiltration and floral dip

Nicole Bechtold and Georges Pelletier

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1. Introduction ...... 3 1.1 Arabidopsis transformation ...... 3 1.2 Brief summary of T-DNA transfer ...... 3 2. In planta transformation methods...... 4 2.1. Growth conditions of the plant material before infiltration...... 4 2.2. Agrobacterium culture and preparation...... 4 2.3. Vacuum infiltration...... 5 2.4. Floral dipping ...... 5 3. Screening of transformants...... 6 3.1. In the greenhouse ...... 6 3.2. In vitro...... 6 4. Control of Arabidopsis pests...... 7 5. Materials...... 7 5.1. Greenhouse materials ...... 7 5.2. Laboratory materials...... 8 5.2.1 Equipment...... 8 5.2.2 Medium...... 8 5.2.2.1. Agrobacterium culture medium...... 8 5.2.2.2. Infiltration medium ...... 8 5.2.2.3. Floral dipping medium ...... 9 5.2.2.4. Sterilisation solution ...... 9 5.2.2.5. In vitro culture medium...... 9 5.2.3. Plant materials ...... 10 5.2.4. Agrobacterium strains and vectors...... 10 References ...... 10

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1. Introduction

1.1 Arabidopsis transformation

Plant genetic transformation was initiated and developed in the eighties thanks to the convergence of constant progress in (i) the protocols for regeneration from tissue culture; (ii) molecular techniques leading to well expressed marker genes after transfer in plant cells; and in (iii) the diversification of DNA delivery methods. Arabidopsis thaliana can be transformed through direct DNA uptake in protoplasts ( Damm et al, 1989) or after cocultivation of leaf or root explants with Agrobacterium (Valvekens et al, 1988)um It is also possible to transform this species by directly applying Agrobacterium to the plant and recovering transformants in the progeny. The first "in-planta" method was described by Feldmann and Marks in 1987 and consisted of the imbibition of seeds with Agrobacterium. Another whole plant transformation procedure is that of Hong-Gil Nam (1990) in which young inflorescences are cut off and the wounded surfaces are inoculated with Agrobacterium. These procedures offer two main advantages. Tissue culture and the resulting somaclonal variations are avoided and only a short time is required in order to obtain entire transformed individuals. However, the mean frequency of transformants in the progeny of such inoculated plants is relatively low and very variable. The infiltration method proposed later by Bechtold et al (1993) was based on the assumption that the stage at which the T-DNA transfer takes place with these methods is late in the development of the plant, either at the end of or at the zygote stage. This assumption was deduced from the observation that transformants are hemizygous for T-DNA insertions. The following protocol makes use of adult plants which are infiltrated with Agrobacterium at the reproduction stage. Each treated plant gives, on average, 10 transformants which can be selected from the progeny after four to six weeks (0.4% of the seeds). A new method based on the same principle was developed by Clough and Bent in 1998. They simplified the infiltration medium and substituted a surfactant for vacuum infiltration allowing penetration of the agrobacteria in the plant tissues, (Silwet L-77). The frequency of transformants obtained by this method is nearly the same as for infiltration: about 0.5% of the progeny of the treated plants. As with infiltration this frequency varies with experiments. The floral dip protocol is described afterthe vacuum infiltration protocol (Chap 2.4).

1.2 Brief summary of T-DNA transfer

In Agrobacterium, the transferred DNA (T-DNA) is a defined part of the Tumor inducing plasmid (pTi for A. tumefaciens) and does not encode any genes important for the transfer process. These genes, the vir genes, are located elsewhere on the pTi or can be located on another plasmid and act in trans (binary vectors). The only cis-acting elements required during the transfer are two imperfect direct repeat sequences of 25 bp, the left border (LB) and the right border (RB). These sequences delimit the region of DNA to be excised from the pTi and can border any DNA sequence to be introduced into plant cells (Hooykaas and Schilperoort, 1992). The first step of the T-DNA transfer (Zupan and Zambryski, 1997; Tinland, 1996) is bacterium-plant cell recognition and the attachment of the bacterium to the plant cell. The genes required are located on the bacterial . The vir genes are then induced by the 5 - 3 In planta transformation presence of phenolic compounds (acetosyringones) produced by wounded plant cells. The constitutive VirA protein will be induced and activates the VirG protein which stimulates the expression of the other normally repressed vir genes. The VirD2 and VirD1 proteins excise the single-stranded T-DNA from the Ti plasmid. Upon this excision, VirD2 stays covalently attached to the 5'-end of the released DNA (RB); this molecule is then exported to the plant cell through a channel probably composed by VirB proteins. VirE2 proteins are associated with the single-stranded T-DNA and from part of the T-complex structure. VirD2 and VirE2 probably mediate the entry of the complex into the nucleus through the nuclear-pores. The exact mechanism of the integration of the T-DNA into the plant remains unknown. One model proposed by B. Tinland is that the 3'-end (LB or adjacent sequence) of the single- stranded T-DNA pairs with a short (5 bp) complementary sequence in the plant DNA, thus determining the point of intergration. The 5'-end (RB) will be ligated to the plant DNA near this region and after endonucleolytic digestion of the displaced plant DNA, the upper strand is extended by repair replication to copy the T-DNA. As a consequence, the sequence before the LB is poorly conserved whereas the sequence before the RB is frequently conserved up to the nucleotide that was attached to the VirD2 protein.

2. In planta transformation methods

2.1. Growth conditions of the plant material before infiltration

1. Prepare some plastic trays ( 20x30 cm) with compost and wet them moderately.

2. Sow 50 carefully separated seeds on the surface of the compost.

3. Place the trays at 4°C for 64h for stratification (optional).

3. Place the trays in the greenhouse (sixteen-hour day photoperiod, 15°C night and 20 to 25 °C day temperature with additional artificial light (105 mE/m2/s) and subirrigate with a layer of tap water under the trays, until germination. Afterwards water moderately for 4 to 6 weeks. Plants must be as vigorous as possible. A better development is observed when they are grown during the rosette stage under relatively short days (13 h) . It is also preferable to avoid etiolation by providing sufficient lighting. The optimal stage for infiltration is when the plants have the first siliques formed and the secondary floral stems are appearing.

2.2. Agrobacterium culture and preparation

1. Prepare a preculture by inoculating 10 ml of LB medium containing the appropriate antibiotics with 100 ml of a fresh culture or a glycerol stock, or with a colony taken from a dish. It is convenient to maintain (for 1 month) a sample of the most recent culture at 4°C as this allows the inoculation of the next culture without the necessity of a preculture.

2. Place at 28°C with good aeration for one night.

3. Inoculate a 2 litre erlenmeyer flask containing 1 liter of LB medium and the appropriate antibiotics with 10 ml of preculture.

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4. Grow at 28°C with good aeration until the OD(600nm) reaches at least 0.8. For the MP5-1 strain the culture needs 15 hours to reach the desired OD, but it may take longer for other strains.

5. Spin the culture at 8000 g for 8 minutes. Gently resuspend the bacteria with 1/3 of the initial volume of infiltration medium for vacuum infiltration and in one volume of 5% sucrose + 200 ml/l Silwet L-77 for floral dipping. It is convenient to centrifuge the culture in a GSA rotor with 250 ml tubes. To resuspend the bacteria pellet, it is just necessary to add a small volume of infiltration medium and to shake the tube.

2.3. Vacuum infiltration

1. Carefully remove the 4-6 week-old plants from the soil with the roots intact. It is also possible to leave the plants in the tray and to infiltrate only the leaves and the stems. In this case, the number of transformants is generally lower but may be sufficient if only a few are needed.

2. Briefly rinse the roots in water to eliminate any adhering soil particles.

3. Put 25 to 50 plants in an aluminium tray (22x16 cm) and stack a second (perforated) tray inside the first one to hold the plants in place. Put 300ml of fresh bacterial suspension into the trays. The same suspension can be used several times. The plants must be entirely immersed in the suspension. It is possible to use other trays depending on the size of the plants and the volume of the vacuum chamber. Avoid infiltrating more than 50 plants at once or the transformation frequency will decrease dramatically.

4. Place the trays in a 10-liter vacuum chamber and apply 104 Pa (0.1 atm) of vacuum pressure for 20 minutes. Gently break the vacuum and remove the trays. Twenty minutes are needed to obtain a complete infiltration of the plants with the suspension. The principal cause of plant death after the treatment is when the drop in pressure is too rapid (due to the power of the pump), or when the vacuum is broken too suddenly. Nevertheless the pump must allow 300 ml of liquid to bubble in the vacuum chamber.

5. During the infiltration fill a plastic tray (28x38 cm) with compost, treat and water.

6. Immediatly replant the infiltrated plants (T0) in the trays; 30 plants per tray. Cover with a perforated plastic wrap or a seed tray incubator and place some water underneath. Replanting must be done immediately after the treatment. Don't let the plants dessicate after the treatment and before replanting (don't treat too many plants at once). Be careful to put only the roots in the soil. If leaves or the base of the rosette is in the soil, it usually causes the deterioration of the plant. Replanting must be performed using latex gloves on a table covered with a plastic wrap. This permits the destruction of all things which have been in contact with Agrobacterium. Avoid high illumination and water condensation while the plants are recovering in greenhouse. Don't completely cover the plants.

7. Remove the cover 3-4 days later. Water the plants moderately until maturity (4-6 weeks) and then let the plant dry progressively.

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Following the treatment, the leaves dry rapidly but the floral stems hold up and continue to flower. If the plants do not continue flowering it may be due to the vacuum conditions or to a high temperature in the greenhouse after infiltration.

8. Harvest the seeds from 50 plants in bulk. Let the siliques dry at 27°C for 2 days, then thresh and clean the seeds. To prevent contamination when an in vitro selection of transformants is done, avoid harvesting soil particles with the seeds.

2.4. Floral dipping

1. Put the bacteria suspension in an appropriate recipient, depending the length of the floral stems (beaker, trays…). The plants should not be watered before transformation to avoid soil falling in the suspension

2. Dip the whole plants or just the floral stems (if they are long enough) in the suspension for 2 minutes.

3. Put the plants in a greenhouse or growth chamber and cover them with a seed tray incubator for 2 days. Cultivate the plants as described in 2.3.7 and 2.3.8.

3. Screening of transformants

3.1. In the greenhouse

1. Sow each bulk of seeds in a 55x36 tray containing Perlite and a top layer of fine sand, previously wet with water containing an appropriate herbicide. The nature of the herbicide should vary depending on the selection marker used; preferably phosphinothricin (Final 7.5 mg/l) or glyphosate (Roundup, 18 mg/l). Perlite is useful to lighten the trays. The size of sand particles has to be small enough to allow a constant imbibition of the seeds sown on the surface. The selection with herbicide is more efficient (to avoid escaped plants) with a minimal medium of tap water and sand, instead of compost. Take care to homogenize the distribution of seeds sown on the trays to allow the even development of the transformants. It is advisable to supply the plants with a nutrient solution when the transformants reach the 2-leaf stage.

2. Synchronize germination at 4°C for 64 hours.

3. Place the trays in the greenhouse. Sub-irrigate permanently with water containing the herbicide, as described previously, for 4 weeks. Transformants (T1) (normal green cotyledons and first leaves formed) can be observed after two weeks. Untransformed plantlets are blocked just after germination (no expansion of cotyledons which rapidly turn yellow). A continuous presence of the herbicide is necessary because the germination of all the seeds can take 4 weeks. To prevent the sand drying, a constant watering is required.

4. Water and treat prepared pots (5.5 cm diameter) containing compost.

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5. Transfer resistant plantlets into individual pots when they are sufficiently developed (4-5 leaf-stage) and cover to facilitate rooting.

6. Water moderately and alternatively with tap water and a nutrient solution until the flowering stage. At this time, care must be taken to individualize plants, to prevent cross-pollination and/or seed contaminations. Progressively reduce watering while plants finish producing flowers. Plants must be staked and may be individualized with perforated transparencies rolled up around the pot.

7. When siliques are dry, harvest and clean the T2 seeds from each T1 plant. Generally, enough seeds are obtained for most experiments without further propagation. The in vitro segregation of the T-DNA selectable markers and Southern blotting experiments allow the estimation of the number of loci and the number of copies of T-DNA. One can expect to obtain more than 50% of transformants with a T-DNA insertion at a single mendelian locus. 70% of the T-DNA insertions are in tandem.

3.2. In vitro

1. Divide the seeds into 1.5 ml microtubes; 100 ml of seeds per tube.

2. Add 1 ml of the sterilisation solution and close the tubes and mix.

3. Lay the tubes down in a laminar flow cabinet for 8 minutes to disperse the seeds into the solution. Don't sterilize more than 10 tubes at once to prevent too long contact of the seeds with the sterilization solution (no more than 8 minutes).

4. Remove the solution with a pipette and rinse twice with 1 ml of pure 95% ethanol. Remove as much of the ethanol as possible. Let the seeds dry in the flow cabinet for one night.

5. Sow no more than 500 seeds in sterile conditions on a 10 cm Petri dish containing the selective medium. Close the dishes with only 2 pieces of adhesive tape to prevent high levels of humidity.

6. Place the dishes at 4°C for 64 h.

7. Transfer the trays into a growth chamber (16 hours day length; 20°C).

8. Transformants (green rooted plants) may be scored 10 days later for kanamycin selection.

9. Plant the resistant plantlets into individual pots when they are sufficiently developed (4-5 leaf-stage). Transfer to a growth chamber and cover to facilitate rooting.

10. Continue the process as in 3.1.6 and 3.1.7.

4. Control of Arabidopsis pests.

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Several insect and fungal pests adversely affect Arabidopsis plants. Below are some of the pests that affect Arabidopsis when grown in INRA Versailles greenhouses, and a description of the pesticides used to combat them. Be careful if you have to use other pesticides because Arabidopsis is very sensitive to many of them and often they cause necrosis on the leaves or stop the floral induction or kill the plants. A regular control of these pests is necessary and if possible an annual cleaning and disinfection by fumigation with dichlorvos (125 mg/m2) of the empty greenhouse or the growth chamber should be done. Not leaving old plants around or stagnant water and a regular inspection of your plants are the best way to have healthy and well developed Arabidopsis plants.

- Thrips can infect the flowers and reduce the fertily, and can transmit virus. Spraying abamecin (9 mg/l) or formetanate (500 mg/l) (homogeneously wet the plants) can be used to control them.

- Aphids accumulate on the stems and leaves. These can be controlled with regular nicotine ( 150 mg/m2) fumigation in the greenhouse.

- Sciarid flies lay eggs in the soil and their larvae eat the vegetative leaves. Small black flies are seen in large numbers around the plants. Spray abamecin (9mg/l) or cyromazine (300 mg/l) on the trays after sowing or on the plants after floral induction.

- Gray rot is a fungal infection that affects the plants when the humidity is kept high. This can be controlled by spraying vinchlozoline (750 mg/l).

5. Materials

5.1. Greenhouse materials

22x16 cm aluminium alimentary trays (Bourgeat, 38490 Les Abrets, France), net pots diameter = 5.5 cm (TEKU, D2842 Lohne/Oldb, Germany),45x33x3.5 cm incubator for seed trays (BHR, 71370 St Germain du Plain, France), 28x38 cm carrying tray (KIB NL5140 AD Waalwijk, Netherlands), 40-well multipot trays (KIB, Netherlands), perforated plastic wrap (1000 holes/m2) subirrigation potting mix (WOGEGAL, 37700 St Pierre-des-Corps, France), 0.5 mm sieved sand, perlite, Hypnol (nicotine ) plant louse treatment (CP Jardin 59570 Bavay, France), Vertimec (abamecin) sciarid flies and thrips treatment (Merck-Sharp), Dicarzol 200 (formetanate) thrips treatment (Hoechst), Trigard 75 WP (cyromazine) sciarid flies treatment (Merck-Sharp), Ronilan (vinchlozoline) gray rot treatment (BASF), Dedevap (dichlorvos) empty greenhouse disinfection (Bayer), FINAL™ (phosphinothricin) transformed plant selection (Hoechst), Roundup (glyphosate) transformed plant selection (Monsanto).

5.2. Laboratory materials

5.2.1 Equipment

Rotary shaker (PROLABO), vacuum oil pump (ALCATEL/CIT) , dessicator (Nalgene, 10 l volume).

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5.2.2 Medium

5.2.2.1. Agrobacterium culture medium

LB (Luria-Bertani) medium (g/l)

Bacto-tryptone 10 Bacto-Yeast extract 5 NaCl 10 pH=7 pH ajusted with NaOH 1M. The medium is sterilized by autoclaving at 115°C for 20 min.

5.2.2.2. Infiltration medium

Macroelements (mg/l)

NH4NO3 1650 KNO3 1900 CaCl2,2H2O 440 MgSO4,7H2O 370 KH2PO4 170

Microelements (mg/l)

H3BO3 6.3 MnSO4,4H2O 22.3 ZnSO4,7H2O 8.6 KI 0.83 Na2MoO4, 2H2O 0.25 CuSO4,5H2O 0.025 CoCl2,6H2O 0.025

BA 0.010 Sucrose 50000 pH 5.8

The microelements and 6-benzylaminopurine (BA) are made up as concentrated stock solutions (at 1000x and 1 mg/l respectively) and stored at 4°C. The pH is adjusted with KOH and the medium is sterilized by autoclaving at 115°C.

5.2.2.3. Floral dipping medium

H2O + Sucrose 5% + 200 ml/l Silwet L-77 (OSI Specialities S.A. 7 rue du Pré-Bouvier CH- 1217 MEYRIN SWITZERLAND).

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5.2.2.4. Sterilisation solution

Dissolve 1 tablet of Bayrochlore (contain sodium dichlorocyanate and releases 1.5g of active chlorine. Bayrol GMBH, D-800 München 70) in 40 ml of distilled water and add some drops of 1 % tween. Take 5 ml in 45 ml Ethanol 95%.

5.2.2.5. In vitro culture medium

Macroelements (mg/l)

KNO3 506

KH2PO4 340 MgSO4(7H2O) 493 Ca(NO3)2(4H2O) 472

Microelements (mg/l)

H3BO3 4,3 MnCl2(4H2O) 2,8 CuSO4(5H2O) 0,13 Na2MoO4(2H2O) 0,05 NaCl 0,58 ZnSO4(7H2O) 0,29 CoCl2(6H2O) 0,0025

Morel and Wetmore vitamins (mg/l)

Myo-inositol 100 Calcium panthothenate 1 Niacine 1 Pyridoxine 1 Thiamine HCl 1 Biotin 0,01

MES 700 Sucrose 10000 Agar BIOMAR 7000 pH=5,8

5 ml/l of a filter sterilised ammoniacal iron citrate stock solution (1%) and for kanamycin selection 1 ml/l of a filter sterilised kanamycin stock solution (100 mg/ml) must be added after autoclaving at 115°C. Macroelements, microelements, vitamins and MES are made up as concentrated stock solutions and stored at room temperature or at 4°C for the microelements and the vitamins The stock solutions are made as followed: 200x KNO3 (1M), 400x KH2PO4 1M, 500x MgSO4(7H2O) 1M, 500x Ca(NO3)2(4H2O) 1M, 1000x microelements, 500x Vitamins, 100x

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MES (14 %). Agar is added in each bottle before autoclaving. The pH is ajusted with KOH. The medium is sterilized by autoclaving at 115°C.

5.2.3. Plant materials

Arabidopsis thaliana (L.) Heyn., ecotype Wassilevskija (WS) was used for all the experiments. Ecotype Columbia (Col0), Nossen (No0) and Landsberg erecta were also used with good efficiency.

5.2.4. Agrobacterium strains and vectors

We used the Agrobacterium strain MP5-1 for most of the experiments. This strain carries the binary vector pGKB5 (Bouchez et al., 1993), which was constructed for T-DNA insertional mutagenesis. This plasmid is very stable in Agrobacterium under non-selective conditions and confers resistance to kanamycin. It was introduced into the helper strain C58C1(pMP90) (Bouchez et al, 1993), which contains a disarmed C58 Ti plasmid, to produce strain MP5-1. The T-DNA contains a promoterless GUS reporter gene fused to the right border, and kanamycin and Basta resistance genes as plant selection markers. Other binary vectors and helper strains could also be used (strains ABI, ASE, GV3101; vectors pBin19, pOCA18, pCGN, pDE1001). However, strain C58C1(pMP90) gave the best results in our conditions. Commonly used binary vectors confer resistance to kanamycin, and selection of transformants has then to be done in vitro under sterile conditions.

References

Bechtold N, Ellis J, Pelletier G (1993)In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris, Life Sciences 316, 1194-1199. Bent A.F., Kunkel B.N., Dahlbeck D., Brown K.L., Schmidt R., Giraudat J., Leung J., and Staskawicz B.J. (1994) RPS2 of Arabidopsis thaliana : a leucine rich repeat class of plant disease resistance genes. Science 265, 1856-1860. Bouchez D, Camilleri C, Caboche M (1993) A binary vector based on Basta resistance for in planta transformation of Arabidopsis thaliana. C R Acad Sci Paris, Life Sciences 316, 1188-1193. Chang S S, Park S K, Nam H G (1990) Transformation of Arabidopsis by Agrobacterium inoculation on wounds. The Plant Journal 5(4), 551-558. Clough S. and Bent A. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16(6), 735-743. Damm B., Schmidt R., and Willmitzer L. (1989) Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts. Mol. Gen. Genet. 213, 15-20. Feldmann K A, Marks M D (1987) Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana : a non-tissue culture approach. Mol Gen Genet 208, 1-9. Hooykaas P. and Schilperoort R.(1992) Agrobacterium and plant genetic engineering. Plant Mol Biol., 35, 205-218. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204, 383-396.

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Tinland B. (1996) The integration of T-DNA into the plant genome. Trends in plant science, 1(6), 178-184. Valvekens D., Van montagu M., and Van Lijsebettens M. (1988) Agrobacterium tumafaciens mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA. 85, 5536-5540. Zupan J. and Zambryski P. (1997) The Agrobacterium DNA transfer complex. Critical Reviews in Plant Sciences, 16, 279-295.

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