Plant Cell Tissue and Organ Culture

Enhanced tolerance to salinity following cellular acclimation to increasing NaCl levels in Medicago truncatula Adel M. Elmaghrabi1 Sergio Ochatt2 Hilary J. Rogers1* Dennis Francis1

1 A.M. Elmaghrabi, H. J. Rogers, D. Francis: School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff CF10 3TL, U.K 2 S. Ochatt : INRA,UMR 1347 Agroécologie, Pôle GEAPSI, Laboratoire de Physiologie Cellulaire, Morphogenèse et Validation (PCMV), B.P. 86510 21065 Dijon Cedex France

Email of corresponding author: rogershj@cf.ac.uk

ExplantsMANA EID MS4 MPIC

Leaves 96 ± 4.7a 85.3 ± 9.9a 81.5± 9.8a 60.8 ± 9.5a

Cotyledons 77.7 ± 6.7a 71.8 ± 5.5a 69.8 ± 5.1a 51.8 ± 5.1a

Hypocotyls 10.8 ± 1.2b 18.7 ± 3.8b 25.5 ± 8.5b 17.5 ± 3.8b

Supplementary Table 1. Optimisation of media and explant source (a) Optimising best medium and most responsive explant in terms of mean (± S.E.) percentage frequency of callus growth (n = 12). (b) Compositions of the four different media: all modified from MS (Murashige and Skoog, 1962) medium, used to optimise the ideal conditions for in vitro growth of callus derived from Medicago truncatula callus. Superscript letters indicate significant differences within each column (P < 0.05)

(a)

MANA Benzyl amino purine (BA)Napthyl acetic acid (NAA)

0.52.0

EID BA2,4-Dichlorophenoxy acetic acid

(2,4-D) Casein hydrolysate

0.21.0

1,000

MS4 BA2,4-D

0.24.0

MPIC KinetinPicloram

0.50.2

(b)

Key: MANA= medium MS+NAA (as in Ochatt et al., 2000), EID= Embryo induction development MS4= MS + 4 mg/L 2,4-D, MPIC= medium MS+Picloram (as in Ochatt et al., 2000).

Months NaCl (mM)

A B C D E F

0 50 100 150 250 350

1 0.094e 0.090d - - - -

2 0.101e 0.096d 0.046d - - -

3 0.120d 0.100d 0.052d 0.038d - -

4 0.250c 0.220c 0.140c 0.094c 0.046b 0.028a

5 0.540b 0.430b 0.340a 0.190b 0.065a 0.022a

6 0.700a 0.770a 0.330b 0.270a 0.025c 0.014b

AGR 0.100 0.113 0.058 0.058 - -

Supplementary Table 2. Growth of calli Monthly FW (g) during, and absolute growth rate (AGR, g m-1) following (a) 6 months at each NaCl concentration; (b) in 17 month old calli, and (c) in 23 month old calli. Within each column, means given the same super-scripted letter are not significantly different within each treatment compared with the 0 mM NaCl control. Different letters denote significant differences compared with the control (P ≤ 0.05)

(a)

Months 0 50 100 150 250 350

1 0.141d 0.117d 0.02c 0.0088d -0.0014a -0.013c

2 0.198c 0.191b 0.09c 0.0129c -0.0029a -0.0086b

3 0.255a 0.214a 0.116b 0.0538b -0.025c -0.0125c

4 0.211b 0.174c 0.198a 0.189a -0.006b -0.0012a

AGR 0.0175 0.014 0.045 0.045 - -

Months 0 50 100 150 250 350

1 0.25a 0.14d 0.50c 1.04a 0.030a -0.011b

2 0.16b 0.23a 0.40c 0.89b 0.020a 0.004a

3 0.24a 0.20c 0.81a 1.10a 0.0020c -0.023c

4 0.26a 0.22b 0.76b 1.10a 0.005b -0.022c

AGR 0.003 0.020 0.065 0.015 - -

(b)

(c)

Supplementary Table 3 PCR primers used for real-time PCR

Target gene Primer pair

Oligonucleotide sequence ProductSize (bp)

MtSERK1 MtSERK1-FMtSERK1-R

GTTGTGGGGGATTTTGGATTAGTCGAGCAAGGTCAAAAGC

200

Pyrroline-5-carboxylate synthease

Mt P5cs1-FMt P5cs1-R

CGTAGGTCTTGCCAACAACA AGAGAAGCCCATTCCCACTT

206

SOS SOS-FSOS-R

ATATCCATCTCGCGTTGAGGCCCTTTGCTCTACCAACCAA

195

WEE1 WEE1-FWEE1-R

TCCGATTGAGGAAGGAGATGAATGAATGACCAGGCAGGAG

197

MtCC52B Mtccs52B-FMtccs52B-R

GGCCACCAATTGAACTCTGT CCCCAGCACCAGTCACTATT

198

Mt 18S Mt 18S-FMt 18S-R

TGACGGAGAATTAGGGTTCGCCTCCAATGGATCCTCGTTA

194

Supplementary Figure 1. Representative images of calli showing various levels of organogenesis and embryogenesis response (a-c) profuse embryogenesis (arrows; SE) in healthy callus following the entire tissue culture protocol at 100 mM NaCl and transfer to ECR medium for one month at 100 mM NaCl (d) shoot morphogenesis (SM) and (e) root morphogenesis (RM) following 5 months of pre-treatment on MANA medium (Bar scale =10 mm) (f) a necrotic callus following the entire acclimation protocol at 350 mM NaCl. (Bar scale =10 mm) (g) Representative calli by the end of acclimation and subcultured for one month to ECR medium with 0, 50, 100 or 150 mM NaCl. Arrows point to somatic embryo genesis (Bar scale = 5 mm) (h) transfer of callus cultured at 100 mM NaCl from EDM to ECR medium for two months to induce roots (arrow, r; Bar scale = 1.84 mm).

SM

d

0

150100

50g

r

h

f

RM

e

SE

SE a

0.2 mm

Embryogenesis

c

1mm

SE

SESE

1mm

b

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 50 100 150 250 350

NaCl treatments (mM)

aa

bb

cc

FW

(m

g)

Supplementary Figure 2. Preliminary acclimation experiment. Mean (± S.E.) callus fresh weights (mg) following culture on MANA medium for 5 months and then sub-culture to the various NaCl treatments continuously for 3 months. Different letters above each bar compared with 0 mM indicate significant differences between treatments (P ≤ 0.05) n=12; y=0.218 -0.0007x (P = 0.002).

0 50 100 150 250 350

Osmolarity of calli 409 546 725.5 761.25 1393.5 1196.33Osmolarity of medium

338 409.4 561.6 635.4 710.6 853.2

0

200

400

600

800

1000

1200

1400

1600

cd

b b

a

a

fe

dc

ba

Osm

ola

rity

(m

mo

les

kg-1

)

NaCl (mM)

Supplementary Figure 3. Osmolarity of calli and culture medium Mean (± S.E.) osmolarity (mmoles kg-1) in the callus tissues and in the culture medium and mean percentage (± S.E.) water content of callus tissues by the end of 6 months culture on the various NaCl concentrations. Different letters between bars of same colour indicate significant differences between treatments (P ≤ 0.05) (n=4).

Supplementary Figure 4. Water content of calli. Mean percentage (± S.E.) water content in the callus following 6 months at the various NaCl concentrations. Different letters indicate significant differences between treatments compared with 0 mM NaCl control (P 0.05, n=3)

ab a

bbc

cc

0 50 100 150 250 35046

50

60

Wat

er c

on

ten

t (%

)

[NaCl] (mM)

150mM NaCl

250mM NaCl

350mMNaCl

100

100

100

100

0mM NaCl

File: Mtr leaf-brut Date: 17-05-2010 Time: 15:26:15 Particles: 15500 Acq.-Time: 440 s

0 200 400 600 800 10000

320

640

960

1280

1600

FL4

coun

ts

0 200 400 600 800 10000

320

640

960

1280

1600

FL4

coun

ts

PK1PK2

partec PAS

Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%PK1 <None> 11124 11124 - 71.77 98.11 6.05 - - PK2 <None> 1525 1525 - 9.84 193.67 7.86 - -

Speed: 1.0Enable Parameter Gain Log L-L U-L

FL4 360.0 lin 75.0 999.9

100

Supplementary Figure 5. Levels of endoreduplication in calli. Frequency of FCM peaks of cells from calli following 6 months of culture at the different NaCl concentrations. The in-built computation programme of the cytometer identifies peaks as G1 (2C), S-phase (>2 and < 4 C) G2 cells (4C), where 1C is the nuclear DNA amount in a gamete (n = 3000). Note a third peak indicating 8C (i.e. an onset of endoreduplication) in the 50 mM NaCl treatment, and skewing of “S-phase” cells in the 100 and 150 mM NaCl treatments (indicating an abnormally high population of nuclei undergoing DNA synthesis) and absence of the second (4C) peak in the 350 mM NaCl treatment, indicating an arrest of cell divisions (probably reflecting an onset of senescence of tissues).

100mM NaCl

50mM NaCl

100

0

cou

nts

5