ORIGINAL PAPER

The transformation of pea (Pisum sativum L.): applicablemethods of Agrobacterium tumefaciens-mediated gene transfer

Petra Krejcı Æ Petra Matuskova Æ Pavel Hanacek ÆVilem Reinohl Æ Stanislav Prochazka

Received: 23 January 2006 / Accepted: 11 September 2006 / Published online: 12 January 2007� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2007

Abstract Three methods of transformation of pea

(Pisum sativum ssp. sativum L. var. medullare) were

tested. The most efficient Agrobacterium tumefaciens-

mediated T-DNA transfer was obtained using embry-

onic segments from mature pea seeds as initial

explants. The transformation procedure was based on

the transfer of the T-DNA region with the reporter

gene uidA and selection gene bar. The expression of

b-glucuronidase (GUS) in the regenerated shoots was

tested using the histochemical method and the shoots

were selected on a medium containing phosphinothri-

cin (PPT). The shoots of putative transformants were

rooted and transferred to non-sterile conditions.

Transient expression of the uidA gene in the tissues

after co-cultivation and in the course of short-term

shoot cultivation (confirmed by histochemical analysis

of GUS and by RT-PCR of mRNA) was achieved;

however, we have not yet succeeded in proving stable

incorporation of the transgene in the analysed plants.

Keywords Agrobacterium-mediated transformation �b-glucuronidase � Pea � Phosphinothricin

Introduction

The number of genetically modified organisms (GMO)

where the required genes were transferred using vari-

ous methods changing the original genome, has re-

cently increased. In this way many agriculturally,

economically and pharmaceutically important plant

genera and species were modified.

Legumes are a group of plants, which present a

number of problems with which we have to cope in the

course of in vitro regeneration and transformation

procedures. Until lately they were considered to be

‘‘recalcitrant’’ to these procedures (Atkins and Smith

1997; De Kathen and Jacobsen 1995; Grant et al.

1995). In spite of this fact, however, methods of

transformation and regeneration of soybean, bean,

lentil, pea, broad bean, chickpea, peanut, mungbean

and a number of other legumes and fodder plants of

the family Fabaceae have been developed (see Atkins

and Smith 1997 for some of the methods that were

involved).

Since the ninteenth century pea (Pisum sativum L.)

has served as a model plant in genetics, plant physiol-

ogy and lately also many workplaces around the world

have used it for gene manipulation, too. The aim was to

obtain plants with a higher quality of proteins or other

nutritive substances, plants resistant to herbicides,

diseases and pests.

Several methods of pea transformation have been

developed so far that tested various types of initial

explants, e.g. epicotyl segments (Puonti-Kaerlas et al.

1990; De Kathen and Jacobsen 1990), apical meristems

(Hussey et al. 1989), protoplasts (Hobbs et al. 1990;

Puonti-Kaerlas et al. 1992), cotyledonary node seg-

ments (Davies et al. 1993) or lateral cotyledonary

Communicated by J. Sadowski.

P. Krejcı (&) � P. Matuskova � P. Hanacek �V. Reinohl � S. ProchazkaDepartment of Plant Biology,Mendel University of Agriculture and Forestry in Brno,Zemedelska 1, 613 00 Brno, Czech Republice-mail: xkrejci8@mendelu.cz

123

Acta Physiol Plant (2007) 29:157–163

DOI 10.1007/s11738-006-0020-3

meristems (Jordan and Hobbs 1993; Bean et al. 1997),

segments of the embryonic axis (Schroeder et al. 1993;

Polowick et al. 2000) and in vivo approaches (Chowr-

ira et al. 1995, Svabova et al. 2005) among others.

Some of these procedures report various drawbacks,

such as poor regenerability, complicated and long-term

regeneration of pea plants via a callus phase, the

unstable incorporation of transgenes into plant tissues

and associated loss of transgene activity in subsequent

generations, reduced fertility, phenotypic abnormali-

ties, altered ploidy etc. (Bean et al. 1997). Despite this,

a number of studies have reported the obtaining of

fertile transgenic plants (Puonti-Kaerlas et al. 1990;

Davies et al. 1993; Schroeder et al. 1993; Bean et al.

1997; Polowick et al. 2000).

We tested and modified three methods of pea

transformation using a hypervirulent strain of Agro-

bacterium tumefaciens in our pursuit to create a simple

and easily recurrent procedure of pea transformation.

Material and methods

Plant material

In all experiments untreated mature dry seeds of the

following varieties of garden pea (Pisum sativum ssp.

sativum L. var. medullare) were used: Vladan, Ctirad,

Cezar, Havel, (BS SEMO Smrzice, CR), Zazrak z

Kelvedonu (retail network) and Puget as a control

(John Innes Institute, Norwich, UK).

Agrobacterium tumefaciens

The hypervirulent Agrobacterium tumefaciens strain

EHA 105 with incorporated pGT89 plasmid was ob-

tained from John Innes Institute in Norwich, UK. The

plasmid T-DNA contains the 2 · 35S::uidA gene

modified with pea CHS-1b intron that renders the gene

non-expressible in procaryotic cells, and NOS::bar

gene coding the phosphinothricin acetyltransferase

(PAT) conferring resistance to the herbicide phosphi-

nothricin (PPT) and a gene located outside of the T-

DNA region coding for bacterial resistance to kana-

mycin (nptII).

Preparation of the Agrobacterium tumefaciens

suspension

One of the Agrobacterium colonies was transferred

into the liquid LB medium of pH 7.2, with antibiotics

(50 mg 1–1 of kanamycin and 10 mg 1–1 of rifampicin)

and left overnight on the shaker at a temperature of

28�C and 200 rpm. The suspension was then centri-

fuged at 3,000 g for 20 min and the pellet re-suspended

with the same volume of MS medium without antibi-

otics and left on the shaker for at least 1 h.

Media for co-cultivation and cultivation

For the preparation of the media mixtures of major

elements, minor elements and vitamins of Murashige

Skoog, Gamborg B5 and Luria Broth media (Duchefa)

were used.

Preparation of the plant material

To sterilise the seeds for in vitro culture 70% ethanol

for 30 s and then a 15% solution of the commercial

bleach Savo (min. 5% NaClO) for 15 min were ap-

plied; the seeds were then rinsed 3· with sterile dis-

tilled water.

Transformation procedures

1. Transformation of stem segments and regeneration

of the transformants from the callus

The sterilised seeds were germinated for 7 days on

sterile expanded perlite EP AGRO (Agroperlit, pro-

ducer Perlit, Senov, CR) at 22�C and 16 h of light/8 h

of darkness. The stem parts were then excised and cut

into 5–7 mm long segments. These explants were

soaked for 10 min in the prepared Agrobacterium

suspension, dried on sterile filter paper and transferred

onto the B5 co-cultivation solid medium without anti-

biotics for 48 h. After co-cultivation the segments were

rinsed in a liquid MS medium with 500 mg 1–1 of

augmentin, dried on sterile filter paper and transferred

onto the solid B5 medium containing 300 mg 1–1 of

augmentin, 0.5 mg 1–1 BAP and 2,4-D, pH 5.7 for

3 weeks until a callus was formed. The callus was then

transferred several times onto the fresh initiation B5

medium containing antibiotics and after 1–2 months

onto the regeneration B5 medium (5 mg 1–1 BAP and

kinetin). After the first sub-culture the callus was

sampled to test the GUS activity using histochemical

method I (Futterer 1995).

2. Transformation of axillary buds

The modified method of Bean et al. (1997) was used

for the experiments and seeds of the variety Puget used

in the original work served as a control.

The sterilised seeds were imbibed in sterile distilled

water for 24 h on a shaker at 150 rpm and then they

were germinated for 48 h in moistened sterile Agrop-

158 Acta Physiol Plant (2007) 29:157–163

123

erlite. After germination the seeds were inoculated

according to the original method and after co-cultiva-

tion they were transferred onto the solid B5 medium

with 300 mg 1–1 of augmentin, 4.5 mg 1–1 BAP and

0.1 mg 1–1 IBA, pH 5.7. For the first 3 weeks the co-

cultivated explants were kept without selection pres-

sure to help full development of the regenerating

shoots from the wounded axillary buds. These regen-

erated shoots were excised and transferred onto a

selection medium containing 2.5 mg 1–1 of PPT. After

another 3 weeks 5 mg 1–1of PPT was added to the

medium.

The histochemical method was used for continuous

analyses of the GUS activity in co-cultivated explants

(samples were taken from the neighbourhood of the

inoculated buds immediately after co-cultivation) as

well as in the regenerated shoots. The estimated

transformants were transferred onto the rooting med-

ium, acclimatised and then transferred to ex vitro

conditions. On some shoots we tested the method of

grafting on a rootstock.

3. Transformation of embryonic segments

The method is based on the work of Schroeder et al.

(1993). The dry mature seeds were sterilised and im-

bibed for 24 h in sterile distilled water on a shaker at

150 rpm. After removal of the testa and one cotyledon

the exposed embryo was cut longitudinally into halves

and the greater part of the radicle was removed. These

embryonic segments were immersed in a liquid 1/2 MS

medium, the Agrobacterium suspension was added and

the segments were co-cultivated for 1 h on a shaker at

150 rpm. After co-cultivation the segments were dried

on sterile filter paper and placed onto the solid co-

cultivation medium P1 (MS with B5 vitamins) without

antibiotics for another 48–72 h. The explants were then

rinsed in a liquid 1/2 MS medium with 500 mg 1–1 of

augmentin and transferred to the regeneration medium

P1 with 300 mg 1–1 of augmentin, 2 mg 1–1 BAP and

NAA. When the shoots were more than 10 mm long,

they were gradually cut off from the original explants

and then cultivated to a stage when they were capable

of rooting or grafting. Samples were taken during

regeneration to test the presence of GUS using histo-

chemical method II. At the stage of short shoots

selection was applied by adding a 2.5–5 mg 1–1 con-

centration of PPT to the selection medium (P2 with

4.5 mg 1–1 BAP and 0.02 mg 1–1 NAA).

Histochemical analysis of GUS

Two protocols with different compositions of the

reaction mixture were used.

I. (Futterer 1995): The reaction mixture contained

100 mM Na2HPO4/KH2PO4, pH 7, 10 mM potassium

ferri- and ferrokyanide, 0.1% Triton X-100, 0.3% X-

Gluc (5-bromo-4-chloro-3-indolyl-b-D-glucuronic

acid). The prepared samples were immersed in the

mixture and vacuum infiltrated at 200 mbar; then they

were incubated overnight at 37�C.

II. (Stromp 1991): The reaction mixture contained

0.1M NaPO4 buffer, pH 7, catalysts of oxidation—0.5M

potassium ferri- and ferrokyanide and 10 mM EDTA,

pH 7, 0.1% Triton X-100 and the substrate 1 mM X-

Gluc. The prepared samples were immersed in the

mixture and incubated overnight at 37�C.

Two step RT-PCR analysis

RNA was isolated from embryonic segments after co-

cultivation by using RNeasy Plant Mini Kit (Qiagen).

The total RNA was treated using RNase-Free DNAse

Set (Qiagen). cDNA was synthesized using Enhanced

Avian RT-PCR Kit (Sigma). The PCR of cDNA of

uidA was performed using primers ‘‘Gusint1’’ (5¢-GAT

CGC GAA AAC TGT GGA AT) and ‘‘Gusint2’’ (5¢-TCT GCC AGT TCA GTT CGT TG), located up- and

downstream of the intron. The uidA RT-PCR resulted

in 355 bp amplificate for cDNA and 483 bp amplificate

for bacterial DNA. RNA from transformed tobacco

leaves was used as a positive control. PCR reactions

ran for 35 cycles: at 94 �C (30 s), 56.5 �C (30 s) and 72

�C (30 s).

Results

Method no. 1

Three varieties of pea (Vladan, Zazrak z Kelvedonu

and Puget) were tested in ten experiments. Calli were

formed over the entire surface of almost 97% explants.

The best callus formation was observed in variety

Vladan (Table 1).

The GUS activity was tested 1 month after co-cul-

tivation on 15 samples of the callus of each variety per

experiment (altogether 150 samples per variety) from

Table 1 Results of callus formation (method no. 1)

Variety Number ofevaluatedexplants

Callus formationfrom explants

Percentage ofcallus formation

Vladan 400 395 99Zazrak 400 387 97Puget 200 187 95

Acta Physiol Plant (2007) 29:157–163 159

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randomly selected parts of calli using histochemical

method I. Positive reactions (blue sectors) were ob-

served in all varieties (Fig. 1). The best results were in

variety Vladan (45% of analysed samples with positive

reactions) and less GUS staining was observed in

variety Puget (Fig. 2). We also tried to regenerate

shoots from the formed calli, but after six months

without any positive results with regeneration we

abandoned this method.

Method no. 2

In this method 6 pea varieties were tested (three

varieties from method no. 1 and three other varie-

ties—Ctirad, Cezar and Havel). In two series of

experiments approximately 2,720 seeds (i.e. about

5,440 lateral cotyledonary meristems) were treated.

About 93% of all inoculated meristems started to form

shoots 1 week after co-cultivation (altogether about

5,030 buds). About 11,500 shoots regenerated from

inoculated axillary buds were excised; about 6,800

regenerated shoots were longer than 10 mm. The most

promising regeneration of transformed axillary buds

after co-cultivation in the first series was achieved in

the variety Puget (average number of excised shoots

was 3.4 per meristem), followed by Zazrak z Kelve-

donu (2.9 excised shoots per meristem)—Table 2. The

best regeneration in the second series was achieved in

the variety Ctirad (3.3 excised shoots per meri-

stem)—Table 3.

In our experiments, only about 1.5% of shoots sur-

vived the selection pressure. The GUS activity was

tested after co-cultivation and it was confirmed that in

about 30% of cases the transgene was transferred to

the tissue. Twenty-one days after co-cultivation the

percentage of positive reactions dropped down to 1%

(Fig. 3—results of the first series of experiments). The

best results with transgene transfer in the first stages

after co-cultivation were achieved in the variety Zaz-

rak z Kelvedonu (about 42% of selected samples

showed a positive GUS reaction). Further analyses of

cultivated plants originating from GUS positive ex-

plants did not confirm the presence of the transgene.

Method no. 3

Six pea varieties were tested in this method. In three

series of experiments approximately 2,420 embryonic

segments were cultivated. A callus started to form from

the basal part of the halved embryos, while 3–4 shoots

per one explant regenerated from the apical part

within 7 days after co-cultivation. About 1,760 regen-

erated shoots more than 10 mm long were cut off the

explants.

With regard to long-lasting problems with internal

infection we used only the varieties Vladan, Ctirad,

Cezar and Havel for further tests in the second series

and the varieties Vladan, Cezar und Havel in the third

series of experiments.

One thousand segments were selected in the third

series. About 3,500 shoots regenerated on explants and

about 760 shoots longer than 10 mm were cut off the

explants. These shoots were then cultivated on a

selection medium with PPT.

The best regeneration was achieved in the variety

Havel (the coefficient of multiplication = average

number of regenerated shoots per one explant, was 3.9)

and Cezar (the coefficient of multiplication was 3.6)

The percentage of shoots surviving selection on

medium with PPT was very low (about 2.5%). The best

resistance on the selection medium was achieved in the

variety Cezar (5% survived shoots after 3 weeks on

selection medium); the differences between varieties

were statistically significant—Table 4.

Fig. 1 Regenerated callus from nodal segments with GUSpositive assay (blue sectors)

Fig. 2 Positive GUS assays in callus formation after co-cultiva-tion

160 Acta Physiol Plant (2007) 29:157–163

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The GUS activity was tested on segments immedi-

ately after co-cultivation (Fig. 4) and within 14 days

after co-cultivation. The 60–70% reduction of the

percentage of positive reactions in all varieties was

evident (Fig. 5).

The transgene expression in the explants after co-

cultivation was proven by RT-PCR. The used plasmid

pGT89 contained the intron modified uidA gene non-

expressible in procaryotic cells and though the result of

RT-PCR on RNA template (Fig. 6) provided sufficient

confirmation of uidA expression. Further tests were

performed on shoots, which survived selection; how-

ever, no incorporation of the transgene into the shoot

tissues, rooted plants and cultivated mature plants was

proven (data not shown).

Discussion

In the presented experiments three methods of pea

transformation were tested. In the first method stem

segments were used as initial explants for transforma-

tion. In most cases green and viable callus was formed.

After a number of sub-cultures on fresh medium we

made an attempt to achieve shoot regeneration on a

regeneration medium with a changed content of

growth regulators (BAP, kinetin). We obtained no

regenerated shoots from callus for six months. Simi-

larly Puonti-Kaerlas et al. (1989, 1990); De Kathen

et al. (1990); Lulsdorf et al. (1991); Hussey and Gunn

(1984) and others introduced the regeneration proce-

dure through the stage of the callus, but only De Ka-

then et al. (1990) and Puonti-Kaerlas et al. (1990)

obtained viable plants.

Using the further two transformation methods we

succeeded in obtaining a large number of regenerated

shoots in a relatively short time, either from lateral

cotyledonary meristems wounded in the course of

inoculation, or from apical parts of the embryonic seg-

ments. Other authors achieved similar results with pea

(Davies et al. 1993; Schroeder et al. 1993; Jordan and

Hobbs 1993; Bean et al. 1997 and Polowick et al. 2000).

When cutting the embryos into thin longitudinal

segments we modified the original method (Schroeder

et al. 1993). We tried to cut the embryo into 3–5 seg-

ments and compared the viability and regeneration

capacity with halved embryos (embryo cut longitudi-

nally into two explants only). In most cases the very

thin segments did not survive co-cultivation with

Agrobacterium, in contrast to the halved embryos,

which tolerated co-cultivation very well and whose

regeneration was excellent. The majority of authors

used immature embryos (Schroeder et al. 1993; Polo-

wick et al. 2000), while in our experiments embryos of

mature seeds were used; this might be one reason why

only transient transgene expression appeared. Many

studies mentioned the effect of the age of the explant

on transgenosis and regeneration, whereas the re-

sponse of the younger explants to transformation was

better, while the older explants responded better to

regeneration (De Kathen et al. 1995; Jaiwal 2001).

Table 2 Results of the first series of experiments (method no. 2)

Variety Number ofregeneratedexplants

Number ofexcised shoots

Average numberof excised shootsper meristem

Vladan 835 1,753 2.1Zazrak 969 2,779 2.9Puget 538 1,827 3.4

Table 3 Results of the second series of experiments (method no.2)

Variety Number ofregeneratedexplants

Number ofexcised shoots

Average numberof excised shootsper meristem

Vladan 525 1,238 2.4Zazrak 92 98 1.1Puget 162 370 2.3Cezar 584 1,705 2.9Havel 287 377 1.3Ctirad 388 1,282 3.3

Fig. 3 Positive GUS assays in lateral meristems after co-cultivation and 21 days later

Table 4 Results of shoot selection on selection medium withPPT (method no. 3)

Variety Number ofsegments

Number ofexcisedshoots

Number ofselectedshoots

Percentage ofselectedshoots

Cezar 400 367 18 5.0Vladan 400 330 5 1.5Havel 200 163 2 1.0

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In our experiments with the transformation of

embryonic segments, the transient expression of

transgenes in tissues was a limiting factor to obtain

fertile transgenic plants. GUS activity tests proved that

the expression immediately after co-cultivation was

higher than in the later stages of cultivation. This

is probably due to transient expression when the

transgene is imported only into the cytoplasm of the

plant cell, but stable incorporation into the nuclear

DNA does not occur. A number of authors presented

similar results with pea, soybean and other legumes

(Hobbs et al. 1990; }Ozcan 1995; Trick and Finer 1998;

Jaiwal et al. 2001; De Clercq et al. 2002). Sonication

was applied to the segments in the presence of Agro-

bacterium; compared to the untreated culture the

percentage of GUS-positive sectors in the co-cultivated

segments indeed increased (data not shown). Trick

et al. (1998), Santarem et al. (1998) and Meurer et al.

(1998) reported similar results in soybean.

Based on our experiments the most effective meth-

od seems to be the method of transformation of seg-

ments of mature pea embryos. We achieved transient

expression of the uidA gene in the tissues after co-

cultivation and in the course of short-term shoot cul-

tivation (confirmed by histochemical analysis of GUS

activity and by RT-PCR), however we have not yet

succeeded in proving the stable incorporation of the

transgene into the analysed plants. The described

method will be further used with the aim to obtain

stable transformants.

Acknowledgments Supported by the Ministry of Education ofthe Czech Republic, Project No. MSM 432100001 and by theMinistry of Agriculture of the Czech Republic, Project no.QF3072.

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