in planta answers (II)

Jimmy Botella J.Botella at botany.uq.edu.au
Fri Jun 7 09:13:41 EST 1996


Here it goes the second half:

SECOND HALF OF IN PLANTA ANSWERS



I don't know what's the problem but perhaps with this protocol we use in 
Versailles to create a collection of transformants you  will find the 
problem

Nicole

----------------
Nicole Bechtold
Station de Genetique et d'Amelioration des plantes
INRA
route de St Cyr
78026 VERSAILLES Cedex
Tel: 33 1 30 83 30 15
Fax: 33 1 30 83 33 19
Email: bechtold at versailles.inra.fr


In-planta Agrobacterium mediated transformation of adult Arabidopsis 
thaliana plants by vacuum infiltration.

Nicole Bechtold and Georges Pelletier.

1. Introduction

Plant genetic transformation was initiated and developed in the eighties 
thanks to the convergence of constant progress in (i) the protocol of 
regeneration from tissue culture; (ii) molecular techniques leading to 
well expressed marker genes after transfer in plant cells; and (iii) the 
diversification of DNA delivery methods.
Arabidopsis thaliana can be transformed through direct DNA uptake in 
protoplasts ( see chapter ?) or after cocultivation of leaf or root 
explants with Agrobacteria ( see chapter ? and ?).
It is also possible to transform this species by directly applying 
Agrobacteria to the plant and recovering transformants in the progeny.
The first "in-planta" method was described by Feldmann and Marks in 1987 
(1) and consisted of the imbibition of seeds with Agrobacteria. Another 
whole plant transformation procedure is that of Hong-Gil Nam (2) 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. (3) 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 gametogenesis 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.

2. Materials

2.1. Greenhouse materials

1. 22x16 cm aluminium alimentary trays (Bourgeat, 38490 Les Abrets, 
France)

2. Net pots diameter = 5.5 cm, 28x38 cm transport-tray tray (TEKU, D2842 
Lohne/Oldb, Germany)

3. 45x33x3.5 cm incubator for seed trays (BHR, 71370 St Germain du Plain, 
France)

4. 40-well multipot trays (KIB, Netherlands)

5. Perforated plastic wrap (1000 holes/m2)

6. Subirrigation potting mix (WOGEGAL, 37700 St Pierre-des-Corps, France)

7. 0.5 mm sieved sand

8. Perlite

9. Birlane CE40 compost disinfection treatment (chlorfenvinphos, 
Agrishell)

10. Hypnol (nicotine) plant louse treatment (CP Jardin 59570 Bavay, 
France)

11. FINAL* (phosphinothricin) transformed plant selection (Hoechst).

2.2. Laboratory materials

1. Rotary shaker (PROLABO)

2. Vacuum oil pump (ALCATEL/CIT) 

3. Dessicator (Nalgene, 10 l volume).

4. 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 20 
mn at 115!C.

5. Infiltration medium and in vitro culture medium

Composants	Infiltration medium (mg/l) (see Note 1)	In vitro culture 
medium (mg/l) (see Note 2)
Macroelements		
NH4NO3	1650	0
KNO3	1900	506
CaCl2,2H2O	440	0
Ca(NO3)2(4H2O)	0	472
MgSO4,7H2O	370	493
KH2PO4	170	340
Microelements		
H3BO3	6,3	4,3
MnSO4,4H2O	22,3	0
MnCl2(4H2O)	0	2,8
ZnSO4,7H2O	8,6	0,29
KI	0,83	0
Na2MoO4, 2H2O	0,25	0,05
CuSO4,5H2O	0,025	0,13
CoCl2,6H2O	0,025	0,0025
NaCl	0	0,58
Morel and Wetmore vitamins		
Myo-inositol	0	100
Calcium panthothenate	0	1
Niacine	0	1
Pyridoxine	0	1
Thiamine HCl	0	1
Biotine	0	0,01
		
BA	0,010	0
Sucrose	50000	10000
MES*	0	700
Agar BIOMAR	0	7000
Ammonium iron (III) citrate		50
pH	5,8	5,8

(*MES=4-morpholineethanesulfonic acid)

6. Sterilization solution

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

2.3 Plant materials

Arabidopsis thaliana (L.) Heyn., ecotype Wassilevskija (WS) was used to 
perfect the infiltration protocol. Ecotypes: Columbia (Col0), Nossen 
(No0) and Landsberg erecta may also be used with good efficiency.

2.4. Agrobacterium strains and vectors

We used the Agrobacterium strain MP5-1 for most of the experiments (4). 
This strain carries the binary vector pGKB5, 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) (5), 
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.

3. Methods

3.1. Growth conditions of the plant material before infiltration

1. Prepare some plastic trays (20x30 cm) with compost. Wet and treat them 
against Sciridae larvae by spraying with a commercial preparation of 
clorfenvinphos.

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

3. Place the trays at 4!C for 64h for stratification.

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) (see note 3) and subirrigate with a layer of tap water 
under the trays, until germination. Afterwards water moderately for 4 to 
6 weeks.

3.2. Agrobacterium culture and preparation

1. Prepare a preculture by inoculating 10 ml of LB medium containing the 
appropriate antibiotics with 100 ml of a last culture or a glycerol, or 
with a colony taken from a Petri dish (see Note 4).

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

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

4.Grow at 28!C with good aeration until the OD(600nm) reaches at least 
0.8 (see Note 5).

5. Spin the culture at 8000 g for 7 minutes. Gently resuspend the 
bacteria with 1/3 of the initial volume of infiltration medium (see Note 
6).

3.3. Infiltration

1. Carefully remove the 4-6 week-old plants from the soil with the roots 
intact (see Note 7).

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 (see Note 8).

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 (see Note 9).

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 (see Note 10).

7. Remove the cover 3-4 days later. Water the plants moderately until 
maturity (4-6 weeks) and then let the plant dry progressively (see Note 
11).

8. Harvest the seeds from 30 plants in bulk. Let the siliques dry at 27!C 
for 2 days, then thresh and clean the seeds (see Note 12).

3.4. Screening of transformants

3.4.1 In 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 (Basta) 5 to 10 
mg/l) or glyphosate (Roundup, 50 mg/l) (see Note 13).

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), in the Basta selection, (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) (see Note 14).

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

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 (see Note 15).

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 (see Note 16). 

3.4.2 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, 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 (see Note 17).

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 dishes into a growth chamber (16 hours day length; 20!C).

8. Tranformants (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 3.4.1.6 and 3.4.1.7.

4. Notes

1. 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 for 20 min.

2. 5 ml of a filter sterilized ammoniacal iron citrate stock solution 
(1%) and for kanamycin selection 1 ml of a filter sterilized kanamycin 
stock solution (100 mg/ml) must be added after autoclaving at 115!C for 
20 min.
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 (KNO3 1M, KH2PO4 1M, MgSO4(7H2O) 1M, 
Ca(NO3)2(4H2O) 1M, microelements 1000x, Vitamins 500x, 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 for 20 min.

3 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.

4. It is convenient to maintain (for 1 month) a sample of the last 
culture at 4!C as this allows the inoculation of the next culture without 
necessity of a preculture preparation.

5. For the MP5-1 strain the culture needs 15 hours to reach the desired 
OD, but it may take longer for other strains.

6. 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.
A better transformation frequency is obtained with a suspension which is 
3 times more concentrated than the original suspension.

7. It is also possible to leave the plants in the tray and to infiltrate 
only the leaves and the stems (6). In this case, the number of 
transformants is generally lower but may be sufficient if only a few are 
needed.

8. Plants must be entirely immersed into the suspension. It is possible 
to use other trays depending on the size of plants and of the volume of 
the vacuum chamber. Avoid infiltrating more than 50 plants at once 
otherwise the transformation frequency decreases dramatically.

9. 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 where 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 be sufficiently powerful to degas 300 ml of liquid in the 
vacuum chamber.

10. 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 
chirurgical gloves on a table covered with a plastic wrap.This permits 
the elimination of all things which have been in contact with the 
Agrobacterium as they may be destroyed afterwards. Avoid high 
illumination and water condensation while the plants are recovering in 
greenhouse. Don't completely cover the plants. 

11. Following the treatment, the leaves dry rapidly but the floral stems 
become erect 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.

12. To prevent contamination when an in vitro selection of transformants 
is done, avoid harvesting soil particles with the seeds.

13. Perlite is usefull 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 (at 
avoiding the selection of sensitives plants) with a minimal medium of tap 
water and sand, instead of compost. Take care to homogenize the seed 
sowing 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.

14. A continuous presence of the herbicide is necessary because the 
germination of all the seeds can take 4 weeks. To prevent the drying of 
the sand a constant watering is required. 

15. Plants must be staked and may be individualized with perforated 
transparencies rolled up around the pot.

16. 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.

17. Don't sterilize more than 10 tubes at once to prevent a too long 
contact of the seeds with the sterilization solution (no more than 8 
minutes).

References

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. Bent A F, Kunkel B N, Dahlbeck D, Brown K L, Schmidt R, Giraudat J, 
Leung J, Staskawicz B J (1994) RPS2 of Arabidopsis thaliana: a 
leucine-rich repeat class of plant disease resistance genes. Science 265, 
1856-1860.
 

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                         '''''
                         (O O)
            +------oOO----(_)--------------+
            |         Jimmy Botella        |
            |   University of Queensland   |
            |  J.Botella at botany.uq.edu.au  |
            +---------------------oOO------+
                         |_||_|
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                         oOOOOo



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