ORIGINAL ARTICLE

Combination of ammonium nitrate, cerium chlorideand potassium chloride salts improves Agrobacteriumtumefaciens-mediated transformation of Nicotiana tabacum

Priti Maheshwari • Igor Kovalchuk

Received: 28 October 2011 / Accepted: 12 June 2012 / Published online: 1 July 2012

� Korean Society for Plant Biotechnology and Springer 2012

Abstract The frequency of plant transformation can be

improved by addition of various chemical into transfor-

mation media. In the past, we showed that exposure of

tobacco, wheat and triticale explants to ammonium nitrate,

cerium and lantanium chloride and potassium chloride

resulted in an increase in the frequency of transformation.

Here, we tested whether a combination of increased con-

centrations of the aforementioned salts yielded a higher

transformation frequency. We found that exposure to

61.8 mM ammonium nitrate caused a 5.0-fold increase in

transformation frequency, whereas exposure to 1.0 lM

cerium chloride or 47.0 mM potassium chloride resulted in

1.2- and 2-fold increases, respectively. Exposure to

61.8 mM ammonium nitrate and 1.0 lM cerium chloride

led to a 4.8-fold increase in transformation frequency,

whereas exposure to 61.8 mM ammonium nitrate and

47.0 mM potassium chloride let to a 5.2-fold increase.

Finally, exposure to 61.8 mM ammonium nitrate, 1.0 lM

cerium chloride and 47.0 mM potassium chloride produced

a 5.1-fold increase. The analysis of the intactness of

T-DNA borders showed that plants exposed to ammonium

nitrate and a combination of ammonium nitrate with other

salts had the more intact right borders and the less intact

left borders. The best results were observed when all three

salts (ammonium nitrate, potassium chloride and cerium

chloride) were used. Thus, we concluded that the addition

of cerium chloride and potassium chloride does not sub-

stantially improve the transformation rate beyond the

improvement observed upon treatment with 61.8 mM

ammonium nitrate, but may slightly improve the intactness

of T-DNA borders.

Keywords Plant transformation efficiency � T-DNA

integration � Agrobacterium tumefaciens � Nicotiana

tabacum � Ammonium nitrate � Cerium chloride �Potassium chloride

Introduction

Plant transgenesis allows production of plants with novel

traits and is a tool to study gene function. Plant transfor-

mation mostly relies on the use of Agrobacterium tum-

efaciens. In dicots, Agrobacterium-mediated transformation

is typically an efficient process.

The frequency of transformation can be improved

through variety of approaches with main ones relying on

the increase of the regeneration capacity of explants and

the increase of the frequency of transgene integration. The

temperature during the phase of plant co-cultivation with

Agrobacterium (Li et al. 2003) as well as the temperature

during the regeneration step (Immonen 1996) influences

the frequency of transformation.

The addition of various chemicals to transformation and

regeneration media can also improve transformation effi-

ciency. Nitrogen compounds (Immonen 1996), cupric sul-

phate and EDTA salts (Sahrawat et al. 2003; Kothari et al.

2004), spermidine (Khanna and Daggard 2003), silver ions

salts (Dias and Martins 1999; Sahrawat et al. 2003), cal-

cium salts (Malabadi and Staden 2006; Perl et al. 1992) and

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11816-012-0243-2) contains supplementarymaterial, which is available to authorized users.

P. Maheshwari � I. Kovalchuk (&)

Department of Biological Sciences, University of Lethbridge,

Lethbridge, Canada

e-mail: igor.kovalchuk@uleth.ca

P. Maheshwari

e-mail: priti.maheshwari@uleth.ca

123

Plant Biotechnol Rep (2013) 7:147–154

DOI 10.1007/s11816-012-0243-2

silver thiosulfate (Perl et al. 1992) have been demonstrated

to have an effect on regeneration.

In Agrobacterium-mediated transformation, the inte-

gration of T-DNA into the plant genome is controlled by

host factors involved in strand break repair and chromatin

organization factors (Citovsky et al. 2007). Strand breaks

in the host genomes are repaired by two major mecha-

nisms: the predominant non-homologous end-joining

(NHEJ) and the rare homologous recombination (HR)

repair. The former is an error-prone mechanism that fre-

quently causes deletions and insertions of various sizes; the

latter is a relatively precise mechanism (Shrivastav et al.

2008). It is hypothesized that the main reason why many

integration events consist of truncated transgenes is that

transgene integration occurs predominantly via NHEJ

(Tzfira et al. 2003). A shift towards HR in strand break

repair may increase the frequency of intact T-DNA inte-

grations and may allow for the more frequent occurrence of

gene targeting—precise transgene integrations at defined

genome positions (Shaked et al. 2005).

We have recently demonstrated that the addition of

various salts to growth media improves both the efficiency

of explant regeneration and the frequency of transgene

integration (Boyko et al. 2009, 2011). Specifically, we

found that exposure of tobacco leaf explants and wheat

scultella to NH4NO3 caused a concentration-dependent

increase in both the regeneration capacity of explants and

the frequency of transgenesis (Boyko et al. 2009; Greer

et al. 2009). Next, while testing a variety of salts for their

capacity to increase the frequency of homologous recom-

bination, we found that the supplementation of tissue cul-

ture media with CeCl3, LaCl3 or KCl also increased the

frequency of transformation (Boyko et al. 2011). We also

showed that exposure of explants to these salts improved

the quality of transformation events: integrated transgenes

had the better preserved right borders (Boyko et al. 2011).

This is a significant improvement as a number of trans-

formation events cannot be used because substantial por-

tions of T-DNA are truncated upon integration. We

hypothesize that high concentrations of ammonium nitrate

change the chromatin structure, which leads to the more

efficient integration via the homologous recombination-

dependent pathway (Boyko et al. 2009; Ziemienowicz et al.

2011).

To further improve transformation efficiency, we ana-

lyzed whether the simultaneous use of several salts,

including ammonium nitrate, potassium chloride and cer-

ium chloride, would result in a higher transformation rate

than exposure to a single salt. We found that exposure to

61.8 mM NH4NO3 caused a *5-fold increase in the

transformation frequency and addition of CeCl3, or and

KCl does not improve transformation frequency any

further.

Materials and methods

Preparation of Nicotiana tabacum plants

for Agrobacterium-mediated transformation

Seeds of N. tabacum cultivar Big Havana were surface-

sterilized and plated on Murashige and Skoog (1962) (MS)

medium supplemented with various amounts of ammonium

nitrate, CeCl3 and KCl (Table 1) as described before

(Boyko et al. 2009). In all cases, plants were grown under

high-light conditions (32.8 lEm-2 s-1) at 22 �C under a

16-h light regime and at 18 �C under an 8-h dark regime

with a constant humidity of 65 %. Explants were prepared

for transformation as described before (Boyko et al. 2009).

In brief, 1-week-old tobacco plants were transferred from

MS medium to a sterile 250-mL glass flask containing

sterile liquid control MS or modified MS medium

(Table 1). Then, the plants were grown for 3 weeks in

flasks with continuous shaking at 50–75 rpm. Four-week-

old plants were removed from the flasks, and leaves were

used for transformation with Agrobacterium. After trans-

formation, the regenerated plants were grown in soil at

22/18 �C, 16/8 h light/dark regime. The transformation

experiments were repeated twice, with a 3- to 4-month

interval between repetitions.

Agrobacterium strains and constructs

The Agrobacterium GV3101 strain containing the

pPM6000 helper plasmid was used for transformation. The

binary vector consisted of a T-DNA cassette containing

the active luciferase (LUC) gene driven by the N-gene

promoter from tobacco and the hph gene conferring resis-

tance to hygromycin. Agrobacterium was grown overnight

at 28 �C with the appropriate antibiotics (rifampicin

25 mg/mL and gentamicin 25 mg/mL) to the optical den-

sity of 0.6 measured at 600 nm, and prepared for trans-

formation as previously described (Kovalchuk et al. 2000).

Leaf transformation was performed as described (Boyko

et al. 2009). Leaves of 3–4 cm in size were blotted dry on

sterile filter paper, then completely submerged upside-

down in Petri dishes lined with Whatman paper soaked

with Agrobacterium. Several parallel incisions (avoiding

touching the side veins and cutting the leaf margins) of

5–7 mm were done with surgical blade. After soaking for

10 min, leaves were again blotted dry, placed upside-down

in Petri dishes with solid MS medium, and incubated for

3 days in the dark at 22 �C. Next, the leaves were rinsed

with sterile distilled water, blotted dry and transferred to

another Petri dish containing solid MS medium supple-

mented with IAA (0.8 mg/L), kinetin (2 mg/L) to induce

callus formation. Ticarcillin (100 mg/L) and potassium

clavulanate (3 mg/L) were added for the control of

148 Plant Biotechnol Rep (2013) 7:147–154

123

Agrobacterium growth. No selection was applied, and after

4 weeks of cultivation on callus-inducing medium, shoots

were excised and transferred to the root-inducing standard

solid MS medium containing 1-naphthaleneacetic acid

(NAA) (0.5 mg/L), ticarcillin (100 mg/L) and potassium

clavulanate (3 mg/L). After *2 weeks of root induction,

the plantlets were transplanted to soil.

Visualization of the luciferase reporter gene expression

When established, plantlets (T0) were checked for the

expression of luciferase. The seeds of these plants were col-

lected and 2-week-old T1 progeny plants additionally tested

for luciferase expression. The number of T0 plants expressing

luciferase was divided by the total number of regenerated

plants to obtain the transformation frequency. The expression

of the luciferase gene was visualized using a CCD camera

(Gloor Instruments, Basel, Switzerland). The solution con-

taining 0.5 mM beetle luciferin (Promega) and 0.05 %

Tween-80 was sprayed on plants and plants were incubated in

the dark for 30–45 min. Next, plants were photographed in the

dark and in the light using a CCD camera.

DNA extraction

Genomic DNA was extracted from the transgenic and

control plants using the Gene Elute Plant Genomic DNA

Miniprep Kit (Sigma) according to the manufacturer’s

protocol. For Southern blot analysis, genomic DNA was

extracted from T0 transgenic plants expressing luciferase.

For PCR analysis, genomic DNA was extracted from single

copy (segregation analysis was performed by checking

luciferase expression) T1 transgenic plants.

PCR amplification of T-DNA borders

The intactness of the left and right borders of the T-DNA

was analyzed using several sets of T-DNA-specific primers

as previously described (Boyko et al. 2009). In brief, each

PCR amplification was performed with a set of 4 primers, 1

outward (from integrated T-DNA) and 3 inward. PCR of

the intact left border results in amplification of 3 fragments

of 332, 315 and 153 nt in length. PCR of the intact right

border results in 3 fragments of 323, 296 and 251 nt in

length. When no fragments were observed, internal primers

were used to confirm the presence of the transgene.

Reaction conditions and primers sequences have been

published before (Boyko et al. 2009). PCR was repeated

twice. The analysis was performed on 10–12 plants, with

three replicates of 3–4 plants each. In all cases, single copy

transgenic lines were used for PCR analysis. Copy number

has been checked by segregation analysis based on lucif-

erase expression (data are not shown).Ta

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Plant Biotechnol Rep (2013) 7:147–154 149

123

Southern blot analysis

T-DNA copy number was analyzed by Southern blot

hybridizations as previously described (Boyko et al. 2009).

The intact T-DNA should give a fragment of 6.3 kb or

longer (from the NdeI site to the right border) (Figure S1).

Statistical treatment of data

In all cases, the mean and standard error or the standard

deviation was calculated. The statistical significance of the

experiment was confirmed either by the two-tailed, paired

Student’s t test with a = 0.05 or a = 0.1 (comparing data

from two treatments) as well as by a single-factor ANOVA

(comparing data from three or more treatments). The sta-

tistical analysis was performed using the JMP 5.0 software

(SAS Institute).

Results

Ammonium nitrate improves the Agrobacterium-

mediated transient transformation of N. tabacum

The analysis of the number of regenerated shoots showed

that none of the medium combinations had any positive or

negative effect; there was also no difference in the average

number of regenerated plantlets per leaf incision (data not

shown).

We found that incubation in the medium containing 39

ammonium nitrate was the most efficient way to increase

the transformation frequency as estimated through lucif-

erase expression (Table 2). The number of transgenic

plants regenerated in 39 ammonium nitrate was 4.97 times

higher than that in 19 ammonium nitrate (P \ 0.01).

Exposure to 1 lM CeCl3 and 47 mM KCl increased the

transformation rate by 20 % (P [ 0.1) and 100 %

(P \ 0.05), respectively, whereas exposure to 94 mM KCl

decreased it by *70 % (P \ 0.05). A combination of 39

NH4NO3 with either 1 lM CeCl3 or 2.59 KCl resulted in

4.81- and 5.24-fold increases in the transformation rate,

respectively (P \ 0.01 in both cases). A combination of

39 NH4NO3 with 59 KCl resulted in a *70 % decrease in

the transformation rate (P [ 0.1). Finally, a combination of

39 NH4NO3 with 1 lM CeCl3 and 2.59 KCl led to a 5.09-

fold increase in the transformation rate (P \ 0.001). The

statistical analysis showed that the increase in the trans-

formation rate observed upon exposure to 39 NH4NO3 and

exposure to 39 NH4NO3 ? 1 lM CeCl3, 39 NH4NO3 ?

2.59 KCl or 39 NH4NO3 ? 1 lM CeCl3 ? 2.59 KCl

was similar (P [ 0.1 in all cases).

Exposure to ammonium nitrate results in the more

intact integrations at the right T-DNA border

and the less intact integrations at the left border

The processing of T-DNA involves VirD2-dependent

cleavage of the right border between nucleotides 3 and 4

(Kim and Veena 2007). The intact right border sequence

should then start at nucleotide position 4. We performed a

PCR test to amplify the left and right borders using a

combination of one outward and three inward primers. If

annealing sequences of all three inward primers was pre-

served, the amplification of the left border would result in

three 153-, 315- and 332-nt-long fragments; this would be

an indication that either there was no deletion or the

deletion at the left border was smaller than 9 nt (Figs. S2,

S3). At the same time, the presence of 0, 1 and 2 fragments

would suggest truncations longer than 188, 36–188 or

9–36 nt, respectively. Similarly, if the annealing sequence

of all three inward primers was preserved, the amplification

of the right border would result in three (251-, 296- and

323-nt long) amplicons, indicating that either there was no

deletion or the deletion at the left border was smaller than

13 nt (Figs. S2, S4). The presence of 1 or 2 amplicons

indicates truncations of 40–85 or 13–40 nt, respectively.

Table 2 T0 Nicotiana tabacum plants regenerated from media supplemented with NH4NO3, KCl and CeCl3

19 NH4NO3 39 NH4NO3 19 Ce3? 2.59 KCl 59 KCl 39 NH4NO3

19 Ce3?39 NH4NO3

2.59 KCl

39 NH4NO3

59 KCl

39 NH4NO3

19 CeCl32.59 KCl

Replication 1

Total plants 81 90 96 94 107 95 47 89 52

LUC? 3 20 4 6 1 19 8 1 10

% 3.7 22.2* 4.1 6.4* 0.9* 20.0* 17.0* 1.1* 19.2*

Replication 2

Total plants 290 210 245 190 116 222 137 198 105

LUC? 10 28 11 15 3 32 28 2 18

% 3.4 13.3* 4.5 7.9* 2.5 14.4* 20.4* 1.0* 17.1*

* Significant difference between the percentages of transgenic plants for treatment and control groups (19 NH4NO3)

150 Plant Biotechnol Rep (2013) 7:147–154

123

The complete absence of amplicons indicates truncations

larger than 85 nt.

The analysis showed that in 40 % of the cases, in control

plants, the size of deletions at the right border was longer

than 13 nt. In contrast, plants regenerated from media

supplemented with higher concentration of ammonium

nitrate (39), potassium chloride (2.59), CeCl3 (1 lM) or a

combination of these three salts had a smaller percentage of

such deletions (Table 3). The analysis of the left border

showed that exposure to the above-mentioned salts resulted

in a higher frequency of deletions. Whereas in 70 % of

cases, control plants had deletions smaller than 9 nt, plants

regenerated from 39 NH4NO3, 2.59 KCl or 1 lM CeCl3had only 10 % deletions of such size, with the rest of them

being much larger (Table 1). Plants regenerated from

media supplemented with all three salts had the left border

preserved in 50 % of the cases. It should also be noted that,

at the left border, truncations longer than 188 nt occurred

more frequently in control plants.

Southern blot analysis of selected transgenic lines

To further confirm that the regenerated tobacco plants

expressing luciferase were indeed transgenic, we per-

formed Southern blot analysis of randomly taken trans-

genic lines regenerated from different media. Since we

used a restriction enzyme (NdeI) that cleaves T-DNA only

at a single position outside the region of probe hybridiza-

tion (Fig. S1), the number of fragments observed by

Southern blot is expected to correspond to the number of

loci carrying the transgene(s).

The analysis showed that, out of 3 lines analyzed, the

control group had 2 plants carrying a single transgene locus

and 1 plant with 2 loci (Fig. 1). In the 39 NH4NO3 group,

all 3 plants had only one locus containing transgene(s). In

the 2.59 KCl, 1.0 lM CeCl3 and 39 NH4NO3 ? 2.59

KCl ? 1.0 lM CeCl3 groups, there were 2 plants with a

single transgene locus and 1 plant with 2 loci.

Discussion

In this work, we extended our study of the role of ammo-

nium nitrate, KCl and CeCl3 in the improvement of

transformation frequency. In the past, we showed that these

chemicals increased the recombination and transformation

frequencies. Here, while testing the influence of combina-

tions of these chemicals on transformation efficiency, we

found that exposure to 61.8 mM ammonium nitrate alone

or in combination with either KCl or CeCl3 or both com-

pounds simultaneously results in a high increase in

recombination frequency. The study demonstrated that

exposure to ammonium nitrate improves the intactness of

the right border, while decreasing the intactness of the left

border. In plants regenerated upon exposure to all three

chemicals, the left border was better preserved than in

plants exposed to ammonium nitrate alone.

All the tested chemicals, namely NH4NO3, KCl and

CeCl3, were shown to have an influence on recombination

frequency (Boyko et al. 2006, 2009, 2010, 2011). It is

possible that exposure to these chemicals improves trans-

formation efficiency by exerting an effect on the recom-

bination machinery. Exposure to chloride salts may change

the ionic and osmotic balance that could positively influ-

ence the transformation efficiency (Patade et al. 2012). Our

previous analysis showed that chlorine ions and not

sodium, potassium or magnesium ions had a positive effect

on HR (Boyko et al. 2010). Chlorine ions may cause sin-

gle- and double-strand DNA breaks via production of free

radicals (Hasegawa et al. 2000). The increased number of

strand breaks may result in an increased frequency of

transgene integrations, as was reported for tobacco (Puchta

1999). It is also hypothesized that exposure to ammonium

nitrate can exert a positive influence on metabolism (Cline

et al. 2006), hormone signaling (Takei et al. 2001), cell

division and expression of HR-related proteins (Chen et al.

1997). Higher level of cytokinins, triggered by application

of ammonium nitrate, may assist Agrobacteria in the

Table 3 Intactness of the right

and left borders of T-DNA,

shown as the percentage of lines

with deletions out of all lines

tested at the right and left

borders

* Significant difference

between the percentages for

treatment and control groups

Size ranges

of deletions

Control (%) 39 NH4NO3 (%) 2.59 KCl (%) 39 NH4NO3 ? 1 lM

CeCl3 ? 2.59 KCl (%)

1 lM

CeCl3 (%)

Right border (nt)

\13 60 72.7* 90* 80* 70

13–40 10 9.1 0 0 0

40–85 10 9.1 10 10 10

[85 20 9.1 0* 10 20

Left border (nt)

\9 70 10* 10* 50* 10*

9–36 10 70* 70* 20 40*

36–188 0 20* 20* 20* 30*

[188 20 0* 0* 10 20

Plant Biotechnol Rep (2013) 7:147–154 151

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transformation process (Hwang et al. 2010). In animals,

ammonium nitrate has been shown to stimulate RAD51

activity; therefore, it seems possible that this salt may have

a similar effect in plants (Sigurdsson et al. 2001; Liu et al.

2004; Shim et al. 2006). Thus, ammonium nitrate among

several other salts was shown to aid recombination

machinery in formation of long protein/ssDNA filaments

needed for invading and pairing the homologous sequences

(Liu et al. 2004).

Recently, we also showed that ammonium nitrate and

chlorine ions can stimulate the single-strand binding

activity of RecA (Ziemienowicz et al. 2011). It can be

hypothesized that these salts have a similar effect on

RAD51 activity, although this remains to be studied.

Similarly, CeCl3 was shown to have a positive effect on

nitrogen metabolism and thus could also have an influence

on the efficiency of integration (Weiping et al. 2003). It

cannot be excluded that CeCl3 can have a more direct

effect either on the activity of the proteins involved in HR

or on the chromatin structure, favoring more frequent

integrations.

How is it possible that the improvement of cell metab-

olism and cell division does not improve the regeneration

of calli but rather the efficiency of transgene integration?

Cells undergoing active cell division are likely to be more

frequently targeted by Agrobacteria. Indeed, an efficient

Agrobacterium-mediated transformation of petunia is only

possible in cells that pass through the S-phase (Villemont

Fig. 1 Southern blot analysis of transgenic plants. The Southern blot

analysis was done with genomic DNA of potential transgenic plants

expressing luciferase. Genomic DNA was digested with NdeI that

cuts close to the left border; the probe used for the detection of

fragments was from the luciferase gene (Figure S2). Thus, the number

of detected fragments is equal to the number of loci carrying the

transgenes. - negative control, a wild-type plant; ? positive control,

10 pg of plasmid. Lanes 1–3 showing 3 independent plants. a Plants

from the control group; b plants from the 39 NH4NO3 group; c plants

from the 39 NH4NO3 ? 2.59 KCl ? CeCl3 group; d Plants from the

2.59 KCl group (lanes 1–3) and the CeCl3 group (lanes 4–6)

152 Plant Biotechnol Rep (2013) 7:147–154

123

et al. 1997). Moreover, the transformation of tobacco

protoplasts during the S–M phase resulted in a higher

transformation frequency as compared to protoplasts

transformed while being in the G1 phase (Meyer et al.

1985; Okada et al. 1986).

It is interesting that almost no additive effect on trans-

formation frequency was found when media containing 39

ammonium nitrate was supplemented with 2.59 KCl and/

or 1.0 lM CeCl3. We can only hypothesize that exposure

to 61.8 mM NH4NO3 results in saturation of the capacity of

tobacco cells for transformation. It is difficult to prove

whether such a phenomenon exists, but it is possible that

there are only a certain number of totipotent cells that are

receptive for transgene integration. In this case, exposure to

61.8 mM NH4NO3 may increase the number of trans-

formed cells to the maximum limit. Indeed, earlier we

showed that exposure to *100 mM NH4NO3 resulted in a

decrease in transformation frequency (Boyko et al. 2009).

The microhomology-dependent model of transgene

integration suggests that T-DNA integrates through

annealing of the T-DNA 30 end to an area of microho-

mology in the genome, followed by similar annealing at the

50 end and synthesis of the second DNA strand. This model

may explain how both T-DNA borders get truncated.

Exposure to 61.8 mM NH4NO3, 47 mM KCl, 1.0 lM

CeCl3 or a combination of three salts resulted in an

increase in the intactness of the right T-DNA border.

Similarly, our previous studies showed a positive correla-

tion between an increase in the amount of NH4NO3, KCl or

CeCl3 in the medium and the preservation of the T-DNA

right border (Boyko et al. 2009, 2011). In contrast, the left

border got severely truncated in all plants regenerated from

media supplemented with the aforementioned salts. It

should be noted that exposure to media supplemented with

all three salts resulted in the lowest frequency of left border

truncations.

It is possible that changes in the mechanism of protec-

tion of T-DNA borders reflect changes in the mechanism of

transgene integration. According to a combined integration

model proposed by Tzfira et al. (2004), the invading

T-DNA strand undergoes a certain degree of degradation at

the left border—the unprotected 30 end. The 50 end appears

to be protected by the attached VirD2 protein. More

nucleotides can be lost at the unprotected left border upon

conversion to a dsDNA form. If integration steps occur

with the help of VirD2, the search for microhomology at

both ends would result only in a substantial degradation at

the 50 end. However, our data show that, in plants regen-

erated from control media, both T-DNA borders are trun-

cated with a similar frequency.

The improvement of protection of the right border may

suggest that integration of a transgene in plants exposed to

all the above-mentioned salts occurs through a different

mechanism, the one that includes the protection of the 50

end with VirD2. Also, since in the past we showed that

exposure to these salts increased the frequency of recom-

bination (Boyko et al. 2009, 2010, 2011), it can be

hypothesized that the HR machinery may play a more

crucial role in transgene integration.

It is quite possible that the aforementioned salts (pri-

marily ammonium nitrate) have a direct effect on transgene

integration. Indeed, in animals, ammonium sulfate has a

direct positive effect on the activity of human Rad51 by

inducing changes to the protein shape and by promoting the

formation of long filaments allowing for efficient recom-

bination to occur (Liu et al. 2004; Shim et al. 2006;

Sigurdsson et al. 2001). It remains to be shown whether a

similar mechanism operates in plants.

Acknowledgments We would like to thank Valentina Titova for

proofreading the text. NSERC Strategic and AARI are acknowledged

for financial support.

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