summary in planta transformation

U6063168 at HERMES.UCS.UNIMELB.EDU.AU U6063168 at HERMES.UCS.UNIMELB.EDU.AU
Thu Nov 30 00:55:53 EST 1995


Dear Netters,

Here is a summary of the responses to the questions I asked about "in planta"
transformation

Carolyn Schultz

Clarke Lab
University of Melbourne
Australia
email U6063168 at ucsvc.ucs.unimelb.edu.au


The question again, for those who missed it!

I would like to know what there is to know about "in plant" transformation
using vacuum infiltration, as I heard along the grape vine that this method
gives superior tranformation efficiencies.

I have the method that was posted to the bulletin board by Andrew Bent Jan 94.
Does anyone have any adjustments/refinements to this method.

A BRIEF SUMMARY OF THE RESPONSES

TWO DETAILED PROTOCOLS ARE INCLUDED AT THE END

modification 1 (Greg Heck, Fernandez Lab)

The biggest difference between our protocol and the Bent one is that we
really subject our plants to vacuum.  The Agro solution really boils.  I
haven't found a great correlation with time but we time for 2 minutes after
the solution really starts to boil.  We also do selection on plates in the
presence of sucrose (1%).  The protocol from Bent I have does not call for
sucrose ion the selection plates.  Somehow we seem to get much crisper Kan
selections this way.  It worked without sucrose, but with sucrose the
transformants are shockingly obvious.

modification 2 (N. Bechtold, D. Bouchez, see below for detailed protocol)

plants (approx 25) are removed from soil for vacuum infiltration in a tray 
of agrobacterium suspension (500ml).  Infiltration iS for 20 minutes at 104 Pa
(0.1atm).   Plants are then transfered back to soil

modification 3 (K. Meyer, Pam Greens Lab, see below for detailed protocol)

- method adapted from Nicole Bechtold, Andrew Bent and Takashi Araki.  No
claims that any of the steps necessary or ideal!!

No clipping of bolts.
Infiltration at 400mm Hg (about 17 inches) for five minutes.


1. What  efficiencies are people generally getting?

>   i).  up to 10 transformants per plant (N. Bechtold, D. Bouchez)
>   ii). 0.05%-0.2% based on Kan Resist. seed  (Shinhan Shiu)
>   iii) up to 5%, i.e. a couple dozen transformants per pot of 9-11
	 plants (G. Heck, Fernandez Lab)
>   iv)  up to 1% (K Meyer, Pam Greens Lab)


2. what Agro strains are best etc?

>   most labs just had not tested different strains, so here are the ones that
>   are working.

>   i)   MP5-1, carries binary vector pGKB5. (see attached protocol for more
>        details (N. Bechtold, D. Bouchez) 

>   ii)  GV1301 (G. Heck, Fernandez Lab and Shinhan Shiu)
>   iii) C58 Agro strain (C58 pGV3850), harboring pGA482 derived binary vector
>        (K. Meyer, Pam Greens Lab). 


3. I would also like to know if anyone is using this technique in other plant
species especially tobacco (or is the method not suitable for bigger plants?)

> i)  we tried to transform rapeseed but we didn't succeed, perhaps just
>     because the progeny of these plants is lower than for arabidopsis
      (Nicole Bechtold (1) and David Bouchez (2))

>  the general consensus here, is that size is a definite problem with
>  using this method for other plant species.


OTHER USEFUL TIDBITS FROM GREG HECK (FERNANDEZ LAB)

Most of the transformations appear
independent, based on limited Southern ananlysis and the frequency of
distinctive embryo lethal mutations.  The number of insertion loci mostly
fall in the range  of 1-2.
		
DETAILED PROTOCOLS

Protocol #1
 
Nicole Bechtold and David Bouchez
                                
Preprint published in :
"Gene transfer to plants" (Springer Laboratory)
Springer Verlag, 1994, I. Potrykus, Ed.


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



Nicole Bechtold (1) and David Bouchez (2)

(1) Station de Genetique et d'Amelioration des Plantes 
	Fax: (33.1) 30.83.33.19
(2) Laboratoire de Biologie Cellulaire
	Fax: (33.1) 30.83.30.99
Institut National de la Recherche Agronomique
78026 Versailles Cedex FRANCE



3.1 Materials and chemicals
	[Equipment and instruments]
	[Infiltration medium]
	[Plant material]
	[Agrobacterium strains and vectors]
3.2 Experimental procedure
	3.2.1 Preparation of plant material
	3.2.2 Agrobacterium culture
	3.2.3 Vacuum infiltration of Arabidopsis plants
	3.2.4 Screening of transformants
3.3 Safety considerations
3.4 Conclusions


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

Nicole Bechtold and David Bouchez

In this chapter, we describe a new protocol for obtaining transgenic 
Arabidopsis thaliana plants by a so-called in planta transformation 
method. Such methods, with no in vitro culture or regeneration step, 
have already been described. Briefly, germinating seeds (Feldmann and 
Marks 1987) or wounded plants (Chang et al. 1990) are inoculated with 
an appropriate Agrobacterium strain ; plants are then grown to 
maturity and their seeds collected. Transformants are selected at low 
frequency among the progeny of inoculated plants. The main advantages 
of these methods are the simplicity of the procedure and the low 
frequency of somaclonal variants in the transgenic lines, which is 
particularly useful for T-DNA mutagenesis strategies (Feldmann 1991).
The mechanism(s) involved in these transformation procedures are 
largely unknown. However, it appears, based on the fact that all the 
transgenic individuals obtained are independant and hemizygous 
(Feldmann 1991, Bouchez et al. 1993, Bechtold et al. 1993), that the 
target cells for Agrobacterium-transformation are likely to be the 
zygote itself or the gametes. 
Therefore, one can expect higher transformation efficiency if plants 
are inoculated as late as possible. A method largely used by plant 
pathologists to inoculate adult plants is vacuum infiltration with a 
bacterial suspension. Considering this, we set up a protocol to 
inoculate adult plants with an Agrobacterium suspension using vacuum 
infiltration (Bechtold et al. 1993). The plants are then transplanted 
to soil and seeds are collected from these inoculated plants. This 
method has been used succesfully in several laboratories to obtain 
transgenic Arabidopsis plants with various constructs. In our 
laboratory, we can routinely achieve a transformation frequency of up 
to 10 transformants from one inoculated plant.


3.1 Materials and chemicals

[Equipment and instruments]

	Greenhouse materials:
Aluminium alimentary trays 22x16 cm (Bourgeat, 38490 Les Abrets, 
France), net pots diameter = 5.5 cm (TEKU, D2842 Lohne/Oldb, Germany), 
incubator for seed trays 45x33x3.5 cm (BHR, 71370 St Germain du Plain, 
France), carrying tray 28x38 cm (KIB NL5140 AD Waalwijk, Netherlands), 
plastic alimentary trays 13x10 cm (Alphaform, 92100 Boulogne 
Billancourt, France), 40-well multipot trays (KIB, Netherlands), 
perforated plastic wrap 1000 holes/m2, subirrigation potting mix 
(WOGEGAL, 37700 St Pierre-des-Corps, France), sieved sand 0.5 mm, 
perlite, compost disinfection treatment Birlane CE40 (chlorfenvinphos, 
Agrishell), plant louse treatment Hypnol (nicotine ) (CP Jardin 59570 
Bavay, France), transformed plant selection BASTA(TM) 
(phosphinothricin) Hoechst.

	Greenhouse conditions:
Sixteen-hour day photoperiod, 15 deg C night/ 20 to 25 deg C day 
temperature, artificial light (105mE/m2/s).

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

[Infiltration medium]

Macroelements (mg/l)

NH4NO3            16500
KNO3              19000
CaCl2,2H2O         4400
MgSO4,7H2O         3700
KH2PO4             1700

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

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


	[Plant material]
Arabidopsis thaliana (L.) Heyn., ecotype Wassilevskija (WS) was used 
for all the experiments. Ecotype Columbia was also used with good 
efficiency.

	[Agrobacterium strains and vectors]
We used the Agrobacterium strain MP5-1 for most of the experiments 
(Bouchez et al. 1994). 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) (Koncz and Schell  1986), 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 genes resistance as plant selection markers.
Other binary vectors and helper strains could also be used. However, 
strain C58C1(pMP90) gave the best results in our hands. Commonly used 
binary vectors confer resistance to kanamycin, and selection of 
transformants has then to be done in vitro under sterile conditions.

3.2 Experimental procedure

	3.2.1 Preparation of plant material

1. Sow 50 Arabidopsis seeds (ca 1 mg), on the surface of an aluminium 
tray (22 X 16 cm) containing wet sowing compost. It is advisable to 
treat the compost against Sciridae larvae, for example by using a 
commercial preparation of clorfenvinphos. We routinely use Arabidopsis 
ecotypes Wassilevskija and Columbia. Other ecotypes should be tested 
before large-scale experiments.

2. Synchronize germination by incubation at 4 deg C for 64 h.

3. Place the trays in the greenhouse

4. Water moderately by sub-irrigation with tap water. After 4 to 6 
weeks, plants are ready for infiltration.

	3.2.2 Agrobacterium culture

1. Inoculate 1 liter bottles containing 500 ml of liquid LB medium 
containing appropriate antibiotics with 1 ml of an overnight culture 
of Agrobacterium. Best results are obtained with the helper strain 
C58C1(pMP90) (Koncz and Schell  1986). Grow this strain in LB 
supplemented with 50 mg/l rifampicin and 25 mg/l gentamycin, plus 50 
mg/l kanamycin for the selection of the binary vector.

2. Grow at 28 deg C with a good aeration (rotary shaker, 200 rpm) to 
an OD (600 nm) = 0.8 (approximately 14 hours).

3. Spin the culture at 2500 g for 15 minutes. Gently resuspend the 
bacteria with 1/3 of the initial volume of infiltration medium.


	3.2.3 Vacuum infiltration of Arabidopsis plants

1. Plants are taken at 4-6 weeks of age, when the primary 
inflorescence is 10-15 cm high, the first siliques are formed and 
secondary inflorescences appearing. Carefully remove the plants from 
soil.

2. Briefly rinse the roots with water to remove adhering soil 
particles.

3. Put 25 plants in a plastic tray (13x10 cm) containing 500 ml 
bacterial suspension. To prevent the plants from floating, it is 
necessary to maintain them submerged with another perforated plastic 
tray fitted into the first one. The same suspension can be used 
several times.

4. Place the trays in a 10-liter vacuum chamber. Apply 104 Pa (0.1 
atm) for 20 minutes. Gently break the vacuum and remove the trays.

5. Immediately transfer the infiltrated plants (T0) to soil, in 28 X 
38 cm carrying trays containing humidified potting mix. Cover the 
trays with a perforated plastic wrap or an incubator for seed trays to 
maintain a high humidity, in order to allow the plants to recover.

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

7. Harvest the seeds in bulk from 50 plants. Let the siliques dry at 
27 deg C for 2 days, then thresh and clean the seeds.

	3.2.4 Screening of transformants

1. For each bulk, prepare three 55 X 36 cm sowing trays containing 
Perlite and a top layer of fine sand. Wet the trays with water 
containing the herbicide phosphinothricin (Basta, 5 to 10 mg/l).

2. Sow the seeds on the surface of the trays and synchronize 
germination at 4 deg C for 64 hours. Resistant plantlets can be 
isolated at very high densities (up to 100-150 seeds per cm2) if 
needed.

3. Place the trays in the greenhouse. Sub-irrigate with water 
containing Basta as before. Transformants (T1) are scored two weeks 
later.

4. When resistant plantlets have 4-5 leaves, transfer them into 
individual pots (5.5 cm diameter) containing potting mix and cover to 
facilitate rooting.

5. Water moderately with water, and once or twice with a nutrient 
solution during flowering. At this stage, care must be taken to 
individualize plants, to prevent cross-pollination and/or seed 
contamination. Progressively reduce watering when plants finish 
producing flowers.

6. When siliques are dry, harvest and clean T2 seeds from each T1 
plant. Generally, enough seeds are obtained for many experiments 
without any further propagation. The segregation of the T-DNA 
marker(s) is scored by sowing 1 100 seeds on selective medium. 
Screening of mutants can also be performed at this stage.

3.3 Safety considerations

Large-scale transformation experiments generate a lot of material 
contaminated with Agrobacterium. Therefore, all the contaminated 
glassware and plasticware, as well as soil, sand, etc, were sterilized 
after use at 120 deg C, 4 h in a Pasteur oven.
All the greenhouse effluents were collected and decontaminated 
with sodium hypochlorite.

3.4 Conclusions

This transformation method was originally devised for the production 
of a saturated collection of T-DNA insertion lines in Arabidopsis. 
However, this method is now routinely used in our laboratory and 
others to obtain transgenic Arabidopsis plants for any purpose. Its 
main advantage is the simplicity of the procedure : all the steps are 
performed in horticultural, non-sterile conditions. No in vitro 
culture and regeneration steps are needed. Selection of transformants 
with Basta allows large populations of progeny to be screened in the 
greenhouse. Primary transformants generally produce enough seeds for 
many experiments without further propagation.
Furthermore, one can expect lower levels of somaclonal variation, 
compared to in vitro methods. However, for unknown reasons, only a 
fair proportion of the mutations observed seem not to be linked to a 
functional T-DNA insert. Thus, the method appears to have a mutagenic 
effect, as already described (Feldmann 1991).
Experiments are in progress in our laboratory to apply this method to 
other plant species. However, many aspects of Arabidopsis biology 
(size of the plant, cycle length, reproductive biology, etc) may play 
a role in the success of this method. This perhaps might prevent this 
transformation procedure from being applied to many other plant 
species.

3.5 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 SS, Park SK, Kim BC, Kang BJ, Kim DU, Nam H G (1994) Stable 
genetic transformation of Arabidopsis thaliana by Agrobacterium 
inoculation in planta. The Plant Journal. 5(4):551-558
3      Feldmann K A (1991) T-DNA insertion mutagenesis in Arabidopsis 
: mutational spectrum. Plant J 1: 71-82.
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      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.
6      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.

Protocol #2
from Knut Meyer

VACUUM INFILTRATION TRANSFORMATION OF ARABIDOPSIS

October 21, 1995
Source:	Pam Green's lab

This protocol is adapted from protocols by Nicole Bechtold, Andrew Bent and
Takashi Araki. No claims are made that any of the steps are necessary or
ideal these experiments have not been done. However, this protocol gives us
very good results, with 95 % of all infiltrated plants giving rise to
transformants, and a transformant in up to 1 in 25 seeds.

1.Sow seeds of ecotype Columbia in lightweight plastic pots prepared in the
following way: mound Arabidopsis soil mixture into 3 to 4 inch pots,
saturate soil with Arabidopsis fertilizer, add more soil so that it is
rounded about 0.5 above the edge, dust with fine vermiculite, cover soil
with a square of window mesh and secure mesh with a rubber band. Any spots
where the mesh isn't in contact with the soil can be pushed down and secured
with toothpicks.

2.Grow plants under conditions of 16 hours light / 8 hours dark at 20=B0 C,
fertilizing with Arabidopsis fertilizer once a week from below, adding maybe
0.5 to each flat. Thin plants to about one plant per square inch. After 4-6
weeks, depending on your conditions, plants will be ready to infiltrate when
they are at this stage: the primary inflorescence is 10-15 cm tall and the
secondary inflorescences are appearing at the rosette. No clipping of bolts
is necessary before infiltration.

3.ln the meantime, transform your construct into Agrobacterium tumefaciens
strain C58Cl (pMP90) (Koncz and Schell 1986). When plants are ready to
transform, inoculate a 500 mL culture of YEP medium containing 50 mg/L
rifampicin, 25 mg/L gentamycin and the appropriate antibiotic for your
construct with a 1 mL overnight starter culture.

4.	Grow culture overnight at 28=B0 C with shaking, until culture OD600 is
greater than 2.0. Spin down the culture and resuspend it in 1 L of
infiltration medium.

	lnfiltration medium (1 liter)		2.2 g MS salts
						1 X B5 vitamins
						50 g sucrose
						0.5 g MES
						pH to 5.7 with KOH
						0.044 =B5M benzylaminopurine
						200 =B5L Silwet L-77 (OSl 
							Specialties)

5.	Place resuspended culture in a Rubbermaid container inside a large bell
jar. lnvert pots containing plants to be infiltrated into the solution so
that the entire plant is covered, including rosette, but not too much of the
soil is submerged. One good way to do this is to place the corners of the
pots on rubber stoppers sitting in the culture. Make sure no large bubbles
are trapped under the plant.

6.	Draw a vacuum of 400 mm Hg (about 17 inches). Once this level has been
obtained, close the suction and let the plants stay under vacuum for five
minutes. Quickly release the vacuum. Briefly drain the pots, place them on
their sides in a tray, cover the tray with plastic wrap to maintain
humidity, and place the flats back in a growth chamber. The next day,
uncover the pots and set them upright. Keep plants infiltrated with
different constructs separated in different trays from this stage on.

7.	Allow plants to grow under the same conditions as before. Stake plants
individually as the bolts grow. When plants are finished flowering,
gradually reduce water and then stop watering to let them dry out. Harvest
seeds from each plant individually.

8.	Prepare large selection plates: 	4.3 g/L MS salts
						1 X B5 vitamins (optional)
						1 % sucrose
						0.5 g/L MES
						pH to 5.7 with KOH
						0.8% phytagar
=09
	Autoclave. Add antibiotics (30 =B5g/mL works well for kanamycin). 
Pour into 150 X 15 mm plates.

9.Dry out plates well in the sterile hood before plating. Twenty minutes to
half an hour with the lids open is usually sufficient.

10.	For each plant sterilize up to 100 =B5L of seeds (approximately 2500
seeds) and plate out individually. Sterilize seeds (7 minutes rocking in 50
% bleach / 0.02 % Triton X-100, 3 rinses in sterile distilled water).
Resuspend seeds in 8 mL sterile 0.1% agarose and pour onto large selection
plates as if plating phage. Tilt plate so seeds are evenly distributed, and
let sit 10-15 minutes. After a while the liquid should soak into the medium
if it's too slow open the plate in the hood and let dry out until the excess
liquid is gone. Parafilm plates and place in a growth room.

11. 	After 5 to 7 days transformants will be visible as dark green plants.
Transfer these onto "hard selection" plates (100 x 15 mm plates with same
recipe as selection plates but with 1.5 % phytagar) to eliminate any
pseudo-resistants. Replace in growth room.

12. 	After 6-10 days plants will have at least one set of true leaves.
Transfer plants to soil, cover them with plastic, and move to a growth
chamber with normal conditions. Keep covered for several days.  I usually
move just one transformant to soil from any one plant that was infiltrated,
to ensure independent transformants.











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