Presentation by Dr. Abu Sefyan I. Saad (ARC, Sudan) at Wheat for Food Security in Africa conference, Oct 9, 2012, Addis Ababa, Ethiopia.

1. Expression of Fusarium trehalose synthase
genes, TPS1 and TPS2 enhances salinity
stress tolerance into wheat crops
Abu Sefyan I. Saad1, 2, Xu Li1, Chun-Sheng Gao1, He-Ping Li1,3, Yu-Cai Liao1,4*
1Molecular Biotechnology Laboratory of Triticeae Crops, Huazhong Agricultural University, Wuhan 430070, Hubei, P.R.
China，emails: sefian_ib@yahoo.com; lixu@webmail.hzau.edu.cn; chunshenggao@webmail.hzau.edu.cn
2Agricultural Research Corporation (ARC), PO Box 126, Wad Medani, Sudan.
3College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, P.R. China; emails:
hepingli@mail.hzau.edu.cn
4College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, P.R. China; emails:
yucailiao@mail.hzau.edu.cn
*Corresponding author. Emails address: yucailiao@mail.hzau.edu.cn; ycliao06@yahoo.com.cn (Y.-C. Liao)

2. outlines
 Introduction.
 Materials and Methods
I. Plasmids
II. Plant transformation and tissue culture
III.Molecular characterizations of transgenic TPS1 and
TPS2 wheat plants
IV. Salinity stress tolerance assay
 Results and Discussions
 Conclusion

3. Introduction
Trehalose is a non-reducing disaccharide sugar, widely
distributed in anhydrobiotic micro-organisms (bacteria, fungi,
etc.), and plants
These anhydrobiotic organisms can survive in drought
conditions over a long period of time and recover within hours
when in contact with water (Hottiger, et al. 1994; Elbein, et al.,
2003).
In plant, trehalose is produced from glucose by trehalose-6-
phosphate synthase (TPS) and trehalose-6-phosphate
phosphatase (TPP) pathway similar to that reported in several
species of bacteria (Avonce, et al. 2006).
Trehalose serves as sugar storage, metabolic regulator and
protects against abiotic stress by the stabilization of proteins
structures and biomembranes Elbein, et al.,( 2003).

4. Introduction
The overexpression of trehalose biosynthetic
genes in some plants such like tobacco, potato,
rice, Arabidopsis, tomato, alfalfa, and maize has
led to an improvement in abiotic stress tolerance
(Holmstrom, et al., 1996; Yeo, et al., 2000; Garg, et
al., 2002; Avonce, et al., 2004; Cortina and
Culianez-Macia, 2005; Suarez, et al., 2009; Jiang,
et al. 2010).
In wheatTPS from E. coli improve drought (A)
and Salinity stress tolerances (B) (Abebe et al.
2003).
Therefore, the stress tolerance of these
transgenic plants was successfully improved.

5. Introduction
The aims of this study are to improve drought and salinity
tolerance in wheat
through genetic transformation by inserting two stress
response genes (trehalose-6-phosphate synthase, TPS1,
and trehalose-6-phosphate phosphatase, TPS2), which were
isolated from Fusarium graminearum.

6. 2. MATERIALS AND METHODS

7. pAHC25-Ubi-TPS1 or TPS2
Materials and Methods
PAHC25
Plasmids
TPS1
TPS2
A B
1.159 KB
Fig.1 Structure of two a recombinant plasmids pUbiTPS1 and
pUbiTPS2 each containing bar gene and ubiquitin promoter from
maize used for transformation, A, the plasmid vector pAHC25, with the
bar gene, ubiquitin promoter and TPS1 gene; B, the plasmid vector
pAHC25, with the bar gene, ubiquitin promoter and TPS2 gene.

8. Materials and Methods
Plant transformation and tissue culture
Wheat transgenic approaches
Agrobacterium Gene gun Others
Electroporation
Pollen tube
PEG
…..etc

9. Materials and Methods
BIOLISTIC
Bombardment parameters
Pressure: 1100 psi
Distance: 9 cm
Vacuum: 28 inches
9

10. Materials and Methods
The basic process of biolistic transformation of wheat
1 mm 5 mm 5 mm 1cm
A B C D
E 1cm
1 cm F
5 mm
G 1 cm f
10

11. Materials and Methods
PCR and Southern blot analysis
M H2o CK M1 M2 M H2o CK P1 P4 P6
A B D
14Kb
400bp
300bp
5 kb
H2O CK P1 X1 M
C
405bp
330bp
Fig. 2 Molecular characterizations of transgenic TPS1 and TPS2 wheat plants
(A), PCR analyses from TPS1 transgenic line and non transgenic line. TPS1
gene specific fragment 405 bp by primers TPS1P1/ TPS1P2. (B), PCR analyses
from TPS2 transgenic line and non transgenic line, TPS2 gene specific
fragment 330 bp by primers TPS2P1/ TPS2P2. (C), PCR analyses of the cross
between two transgenic, TPS1 (M1) X TPS2 (P1) lines, resulted in X1 two
genes specific fragments by four primers specific for both genes. (D),
southern Blotting for genes, TPS1 transgenic lines and non transgenic line
(Y158) and three TPS2 transgenic lines alone with non transgenic (Y12).

12. Materials and Methods
Salinity stress tolerance assay:
In all experiments, the T2 generation seeds of
TPS1and TPS2 transgenic lines were used for
assays. One independent transgenic line M1
and three independent transgenic lines P1, P4,
and P6 along with non transgenic line Y158 or
Y12 as a control, were used to represented
TPS1 and TPS2 transgenic wheat plants,
respectively.

13. RESULTS AND DISCUSSIONS

14. A C
**
D
*
E
*
B
+7day
Fig. 3 Salinity stress tolerance for TPS1 transgenic line and non-transgenic
(Y158). (A), the appearance of T2 lines of TPS1 M1 and Y158 non transgenic
seedlings under salt stress 200 mM NaCl . (B), the appearance of T2 lines of
TPS1 M1 and Y158 non transgenic seedlings, 7 days recovery from 350 mM
NaCl concentration. (C), roots length after 10 days recovery. (D), root dry
weight after 10 days recovery. (E), Plant fresh weight (biomass) after 10 days
recovery.
*, ** indicate significant differences at P <0.05, <0.01 respectively. Three
replications

15. A
C
***
*** ***
*
D
*** ***
**
E
*** ***
***
B
+7 day
Fig. 4 Salinity stress tolerance for TPS2 transgenic line and non-transgenic
(Y12).
A)The appearance of T2 lines of TPS2 P1, P4 and P6 and Y12 non transgenic
seedlings during 200 mM NaCl concentration.
B)The appearance of T2 lines of TPS2 three transgenic lines and Y12 non
transgenic seedlings, 7 days recovery from 350 mM NaCl concentration.
C) Roots length after 14 days recovery.
D) Root dry weight after 14 days recovery.
E) Plant fresh weight (biomass) after 14 days recovery.
**, *** indicates significant differences at P <0.05, <0.01 respectively.

16. Table 2 Comparison of root length (R.L ,cm), root dry weight (R.D ,mg) and plant fresh
weight (F.W ,g) after 14 days recovery of transgenic TPS2 & TPS1 plants and non-
transgenic Y12 or Y158 grown in normal conditions, survival rate of transgenic lines in
350 mM NaCl.
TPS2 TPS1 CK1(Y158) Ck1(Y12) Ck(Y158) Ck(Y12)
R.L(cm) 11.0±0.79ns 10.6±0.19 ns 11.1±0.6 13.9±0.26
R.D(mg) 26.0±1.6 ns 18.2± 0.93 ns 24.4±2.7 32.8±1.5
F.W(g) 0.7±0.04 ns 0.6±0.01ns 1.0±0.1 1.0±0.22
SR% 45.7±6.4 45.2±15.4 0 0

17. Conclusion:
 Over-expression of either TPS1 or TPS2
gene resulted in an enhanced salinity
tolerance in wheat without growth effects.
 Trehalose genes played a role in root growth
under salt stress.
 This also presents trehalose potential as a
candidate for engineering salt tolerance in
wheat crops.

18. Acknowledgement
This work was supported by the Ministry of
Agriculture of China and for a Doctoral
fellowship from the Chinese Exchange
program