Performance of Four Mannitol-Accumulating Transgenic Wheat Lines Under Moisture Stress

and Non-Stress Conditions

MATERIALS AND METHODS The four mannitol-accumulating transgenic lines, the non-transformed parent line ‘Bobwhite’, and five conventional check varieties (Reeder, RT01-12, Butte 86, Kauz, and MTRWA116) were planted on April 29, 2005 at the ARDEC research station north of Ft. Collins, CO.

In two of the lines (pTA5-1 and pTA5-2), the transgene construct contained the chloroplast transite peptide sequence to transport the mtlD product to the chloroplast. In lines pTA2-1 and pTA2-2 (without the transit peptide sequence), mtlD is expected to accumulate in the cytosol.

In order to establish the stands, both the irrigated and rain-fed trials were furrow irrigated until June 17, when a diversion ditch was dug between the trials, cutting off the irrigation to the rain-fed plots.

To compensate for stand variability between plots, grain weight, 200-kernel weight, harvest index, head number, and number of kernels was calculated from a one-meter sample taken from one row in each plot

Soil moisture was determined with a Delta T Instruments HH2 Moisture Meter used with a six-sensor, one meter long soil probe.

Statistical analysis was conducted with SAS/STAT v. 9.1 (Cary, NC). Analysis of variance was followed by mean separation with Duncan`s New Multiple Range Test (p<0.05), and simple phenotypic (Pearson) correlation.

Experimental Design:

Complete Randomized Block

Three Replications

Plot Measurement: 5’ X 5’

INTRODUCTIONMoisture stress is one of the greatest factors affecting yield in agronomic crops worldwide. Traditional breeding

methods have met with only limited success at increasing the drought-tolerance of field crops. Some of the most current attempts at improving the performance of crops in water-limited environments have used biotechnology

to transfer novel genes conveying drought-tolerance into crop species. A research team from Oklahoma State University has transformed the spring wheat variety ‘Bobwhite’ with the mannitol-dehydrogenase gene (mtlD)

from E. coli that causes the wheat to produce the sugar alcohol mannitol. Mannitol is found in several plant species, and is thought to aid in stress tolerance by scavenging reactive oxygen species (ROS) produced during

stress.

OBJECTIVES To evaluate the performance of four transgenic lines containing the mtlD gene in the ‘Bobwhite’ background under well watered and moisture stress conditions

To compare the performance of two lines with transgenes containing the chloroplast transite peptide sequence to two lines without the sequence.

To gain insights on the drought tolerance mechanism of the mtlD gene.

CONCLUSIONS

Grain Weight – 1 Meter

200-Kernel

Wt.

Harvest Index

# of Heads – 1

meter

Head Length

# of Kernels – 1 meter

Grain Weight– 1 Meter

0.802190.0052*

0.903980.0003*

0.765870.0098*

-0.468100.1724

0.98267<.0001*

200-Kernel

Wt.

-0.090840.8029

0.648340.0426*

0.460940.1800

-0.023590.9484

0.684290.0291*

Harvest Index

0.93223<.0001 *

-0.035360.9228

0.781680.0076*

-0.423810.2223

0.926640.0001*

# of Heads – 1

meter

0.884860.0007 *

-0.152240.6746

0.896600.0004 *

-0.711180.0211*

0.809950.0045*

Head Length

-0.533690.1121

0.597410.0682

-0.412400.2363

-0.662710.0368 *

-0.565720.0883

# of Kernels – 1 meter

0.97122<.0001 *

-0.311500.3809

0.915860.0002 *

0.878500.0008 *

-0.637660.0473 *

Pearson Correlation CoefficientsCorrelations in Fully Irrigated Trial Shown Above Diagonal,

Correlations in Partially Irrigated Trial Shown Below

(Correlations significant at the 0.05 level are denoted by an asterisk *)

Source of Variance

Grain Weight – 1 Meter

200-Kernel

Wt.

Harvest Index

# of Heads – 1

meter

Head Length

# of Kernels – 1 meter

Treatment

**** **** *** ns ns ****

Rep (Treatme

nt)ns * ns ns ns ns

Entry **** **** **** **** **** ****

Treatment* Entry

* ** ns Ns ns ns

CV (%) 26.3 6.8 15.7 17.2 4.9 21.5

Summarized ANOVA Table (all treatments)

*, ****: Significant at the 0.05 and 0.0001 levels of probability, respectively.

ns: not significant at the 0.05 probability level.

Grain Weight - 1 meter

Reeder RT01-12 Bobwhite pTA2-1 pTA2-2 pTA5-1 pTA5-2

Gra

ms

0

20

40

60

80

100

120

140

160

Number of Heads - 1 meter

Reeder RT01-12 Bobwhite pTA2-1 pTA2-2 pTA5-1 pTA5-2

# of

Hea

ds

0

20

40

60

80

100

120

140

160

180

Fully IrrigatedPartially Irrigated

Head Length

Reeder RT01-12 Bobwhite pTA2-1 pTA2-2 pTA5-1 pTA5-2

Cen

tim

eter

s

0

2

4

6

8

10

12

14

16

200-Kernel Weight

Reeder RT01-12 Bobwhite pTA2-1 pTA2-2 pTA5-1 pTA5-2

Gra

ms

0

1

2

3

4

5

6

7

Number of Kernels - 1 meter

Reeder RT01-12 Bobwhite pTA2-1 pTA2-2 pTA5-1 pTA5-2

# of

Ker

nel

s

0

1000

2000

3000

4000

5000

Harvest Index

Reeder RT01-12 Bobwhite pTA2-1 pTA2-2 pTA5-1 pTA5-2

Per

cent

0.0

0.1

0.2

0.3

0.4

0.5

Means of Selected Traits

a

ab

a

a

cc

c

c

cc

cc

bc

c

ab

abc

a

cd d

cd cdbcd

a ab

cd

ab

ab abc

a

ab

a

cdd

b

bc cd

a

dcd

bcd

ab

d

a

bcd

f

decde

e f

bc de

a

b

d

cd

bcd

bc

cd

e

bcdab c

d

ab

abc abc

f

bcd

a

d

abcd

abc abc

a

a

c

bc

bbc bc

a a

b

b b

b

b

(Means within treatments followed by same letter are not statistically significant at the 0.05 level)

Chart 1a Chart 1b Chart 1c

Chart 1d Chart 1e Chart 1f

Chart 3a

Chart 3b

Soil Moisture 06/28/05 (Heading)

Soil Depth

200mm 300mm 400mm 600mm 1000mm

% M

oist

ure

0

5

10

15

20

25

30

35

Fully IrrigatedPartially Irrigated

Soil Moisture 07/28/05 (Grain Filling)

Soil Depth

200mm 300mm 400mm 600mm 1000mm

% M

oist

ure

0

5

10

15

20

25

30

35

Fully IrrigatedPartially Irrigated

Relative Water Content - Partially Irrigated

Jointing Anthesis Anth+10 Days

RW

C (%

)

70

75

80

85

90

95

ReederRT01-12BobwhitepTA2-1pTA2-2pTA5-1pTA5-2

Relative Water Content - Fully Irrigated

Jointing Anthesis Anth+10 Days

RW

C (%

)

76

78

80

82

84

86

88

90

92

94

96

ReederRT01-12BobwhitepTA2-1pTA2-2pTA5-1pTA5-2

RESULTS AND DISCUSSION Due to heavy rains early in the season (refer to the raınfall chart), both trials were well watered until anthesis (refer to soil moisture chart). Hot and dry weather during grain filling resulted in a divergence in soil moisture profiles (refer to other soil moisture chart) and a severe stress on the partially irrigated trial. Mean grain weight per plot in the stressed trial was reduced 43% from the well watered trial.

In the analysis of variance, the effect of entries was highly significant for all variables, the treatment effect was significant for all variables except number and length of heads, and the treatment x entry effect was siginificant only for grain weight and 200-kernel weight (refer to anova table).

Several significant correlations were found both in the fully irrigated and partially irrigated trials (refer to correlation table). Correlations found in the fully irrigated trial, but not in the partially irrigated trial include grain weight and 200-kernel weight, harvest index and 200-kernel weight, and 200-kernel weight and number of kernels. One correlation between head length and number of kernels was found in the partially irrigated trial, but not the fully irrigated trial.

Poor emergence in the transgenic lines and the `Bobwhite` control resulted in low plant stands relative to the cultivars. Partially for this reason, the cultivars outperformed the transgenic lines for most variables (refer to mean graphs).

For most traits in both treatments, the transgenic lines did not differ from the `Bobwhite`control (refer to mean graphs). One or more transgenic lines exceeded the control for harvest index, kernel number, and head number.

There was no clear pattern of difference between lines with and without the chloroplast trasit peptide.

Partially Irrigated Trial

Diversion Ditch

Fully Irrigated

Trial

Rainfall During the Growing Season

May Jun Jul Aug Sep

Inch

es o

f R

ain

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Data this year showed no clear differences among the transgenic lines or between the transgenics and the parent line ‘Bobwhite’

This year’s evaluation may have been complicated due to late planting, small plot size, and/or poor stand establishment

Late season rain may have also lessened the amount of moisture stress

Trials will be repeated next year

REFERENCES

Abebe, Tilahun, Arron C. Guenzi, Bjorn Martin and John

C. Cushman. “Tolerance of Mannitol-Accumulating

Transgenic Wheat to Water Stress and Salinity.” Plant

Physiology 131: 1748-1755.

William Nobles1, Patrick Byrne1, Arron Guenzi2, Bjorn Martin2, Sathya Elavarthi2, Brett Carver2 and Mohamed Mergoum3, (1)Colorado State University, Soil and Crop Sciences Dept., Campus Delivery 1170, Fort Collins, CO 80523-1170, (2)Oklahoma State University, Oklahoma State University, 368 Ag Hall, PASS, Stillwater, OK 74078, (3)Plant

Sciences Department, NDSU, Loftsgard Hall, P.O. Box 5051, Fargo, ND 58105