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Plant Physiol. (1999) 119: 839-848
Winter Survival of Transgenic Alfalfa Overexpressing Superoxide
Dismutase1
Bryan D. McKersie*,
Stephen R. Bowley, and
Kim S. Jones
Plant Biotechnology Division, Department of Plant Agriculture,
University of Guelph, Guelph, Ontario, Canada N1G 2W1
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ABSTRACT |
To test the hypothesis that enhanced
tolerance of oxidative stress would improve winter survival, two clones
of alfalfa (Medicago sativa) were transformed with a
Mn-superoxide dismutase (Mn-SOD) targeted to the mitochondria or to the
chloroplast. Although Mn-SOD activity increased in most primary
transgenic plants, both cytosolic and chloroplastic forms of Cu/Zn-SOD
had lower activity in the chloroplast SOD transgenic plants than in the
nontransgenic plants. In a field trial at Elora, Ontario, Canada, the
survival and yield of 33 primary transgenic and control plants were
compared. After one winter most transgenic plants had higher survival
rates than control plants, with some at 100%. Similarly, some
independent transgenic plants had twice the herbage yield of the
control plants. Prescreening the transgenic plants for SOD activity,
vigor, or freezing tolerance in the greenhouse was not effective in
identifying individual transgenic plants with improved field
performance. Freezing injury to leaf blades and fibrous roots, measured
by electrolyte leakage from greenhouse-grown acclimated plants,
indicated that the most tolerant were only 1°C more freezing-tolerant
than alfalfa clone N4. There were no differences among transgenic and control plants for tetrazolium staining of field-grown plants at any
freezing temperature. Therefore, although many of the transgenic plants
had higher winter survival rates and herbage yield, there was no
apparent difference in primary freezing injury, and therefore, the
trait is not associated with a change in the primary site of freezing
injury.
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INTRODUCTION |
Winter hardiness is a complex trait that involves tolerances to
freezing, desiccation, ice encasement (severe anoxia), flooding (milder
anoxia), and disease. The combinations and severity of these stresses
that crops must tolerate vary with environment and year. There are
distinct differences among the environmental stresses that occur during
winter, but oxidative stress may be a common element. Freezing, anoxia,
and desiccation stresses have been linked with oxidative stress in
three types of correlative physiological and biochemical studies.
First, degenerative reactions associated with anoxia (Hetherington et
al., 1987 , 1988; Monk et al., 1989), desiccation (Senaratna et al.,
1987), and freezing (Kendall and McKersie, 1989) are similar to the
reactions caused by the herbicide paraquat (Chia et al., 1982; Bowler
et al., 1991) and the pollutant ozone (Van Camp et al., 1994).
Second, microsomal membranes from acclimated plants are more tolerant
of in vitro, free-radical treatment than those from nonacclimated
plants (Kendall and McKersie, 1989). Third, as plants acclimate to low
temperatures, they acquire coincidentally increased tolerance to
freezing stress, ice-encasement stress, and free-radical-generating
herbicides (Bridger et al., 1994).
We hypothesized that enhancing a plant's tolerance of oxidative stress
would improve its ability to survive the combination of stresses
associated with winter. The mechanisms to detoxify oxygen radicals are
varied and the complex interactions among the antioxidants in different
subcellular compartments, cells, and tissues are only now being
elucidated (Bowler et al., 1992; Herouart et al., 1993; Scandalios,
1993; Foyer et al., 1994; Allen, 1995). SOD is an essential component
of these defense mechanisms because it dismutates two superoxide
radicals to produce hydrogen peroxide and oxygen. Previously, a Mn-SOD
cDNA from tobacoo was introduced into alfalfa (Medicago
sativa L.); one of the primary transformants and its
F1 transgenic progeny showed increased survival and vigor
after exposure to sublethal freezing stress in the laboratory (McKersie
et al., 1993). Two of the transgenic plants also had relatively
increased tolerance of water deprivation and four had increased vigor
and winter survival in the field (McKersie et al., 1996). The previous
studies used only a few transgenic plants because of limitations of
space and facilities, and only a single alfalfa clone, called RA3, was
used because it was one of the few genotypes at that time that could be
transformed, although it had poor agronomic performance and was not
well adapted to winter conditions.
In this study we expanded our original observations by transforming two
elite alfalfa plants that are adapted to our field environment, and we
examined many more independent transgenic plants in both laboratory and
field evaluations. The results confirm that overexpression of Mn-SOD in
transgenic alfalfa plants often improves the winter survival and
subsequent herbage yield of this crop, but in some independent
transgenic plants, winter survival and subsequent
yield actually lessened. The transformation event had
a greater influence on winter survival and shoot dry-matter yield than
the subcellular site of SOD targeting. However, the improvement in
winter survival of the whole plant was not related to a change in the
primary freezing tolerance of the cells in the taproot or crown of
transgenic alfalfa.
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MATERIALS AND METHODS |
Plant Transformation
Two different clones of alfalfa (Medicago sativa),
designated N4 and S4, were selected from the University of Guelph plant breeding program to be transformed based on their field performance (Bowley et al., 1993). Petiole explants of alfalfa were cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1
Rif pMP90 containing the binary vectors pMitSOD or pChlSOD described previously (Bowler et al., 1991). The explants were cocultivated for
3 d in the dark on SH induction medium (Shetty and McKersie, 1993) containing 288 mg L 1 Pro, 53 mg L1
thioproline, 4.35 g L1
K2SO4, and 100 µM
acetosyringinone. The explants were washed in half-strength
Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the
same SH induction medium without acetosyringinone but with 500 mg
L1 claforan and 50 mg
L1 kanamycin. After several weeks somatic
embryos were transferred to BOi2Y development medium (Bingham et al.,
1975) containing no growth regulators, no antibiotics, and 50 g
L1 Suc. Somatic embryos were subsequently
germinated on half-strength Murashige-Skoog medium. Rooted seedlings
were transplanted into pots containing Turface (Plant Products,
Mississauga, Ontario, Canada) in a greenhouse at approximately
23°C/18°C (day/night) and a minimum 16-h photoperiod.
PCR Screening
Prior to transfer to the greenhouse, the putatively transgenic
plants were screened for the presence of the nos-nptII
transgene using PCR. DNA was extracted with 400 µL of homogenizing
buffer (250 mM NaCl, 25 mM
EDTA, 0.5% SDS, 200 mM Tris-HCl, pH 7.4). The
supernatant of a 13,000g centrifugation was mixed with 300 µL of isopropanol. DNA was collected at the interface, washed, and
resuspended in water. The quality and concentration of the DNA was
confirmed using a 0.8% agarose gel with ethidium bromide staining. For
the PCR reaction 25 ng of DNA was combined with 1.5 µL of 15 mM MgCl2, 1 unit of Taq
polymerase, 2.5 µL of 10× buffer, 2.5 µL of deoxyribonucleotide
triphosphate, and 2 µL of each primer made to a final volume of 25 µL with water. The primers used were
5 -AGCTGTGCTCGACGTTGTCAG-3 and 5-GGTGGGCGAAGAACTCCAGCA-3. The
PCR program included 5 min at 94°C, then 25 cycles of 94° for
15 s, 65° for 30 s, and 72° for 60 s, followed by 5 min at 72°C and holding at 4°C. PCR products were visualized on a
0.8% agarose gel with ethidium bromide.
Southern-Blot Hybridization
We treated the purified DNA from alfalfa with the restriction
enzymes EcoRV and EcoRI at a 5- to 10-fold
excess. All samples were separated using a 0.8% agarose gel. After
electrophoresis the gel was blotted overnight onto a positively charged
nylon membrane as described by Ausubel et al. (1991). After blotting the membranes were UV cross-linked. Subsequent Southern-blot analysis was based on the digoxygenin chemiluminescent system (Boehringer Mannheim) (van Miltenburg et al., 1995). We synthesized the
digoxygenin-labeled DNA hybridization probes using the Expand enzyme to
enzymatically label PCR products with digoxygenin-dUTP (PCR digoxygenin
probe synthesis kit, Boehringer Mannheim) as described by the
manufacturer. Probes were synthesized for both the kanamycin and the
mitochondrial SOD genes, using 200 pg of purified plasmid DNA as the
template. The PCR primers for synthesis of the kanamycin
probe were 1 µM each of
5-AGCTGTGCTCGACGTTGTCAC-3 and 5-GGTGGGCGAAGAACTCCAGCA-3. The
annealing temperature was 65°C and the product size was 732 bp. The
primers for the SOD gene were 5-GAGCAGACGGACCTTAGC-3 and
5-AGAAACCAAAGGGTCCTG-3, with a 55°C annealing temperature and a
511-bp product.
SOD Activity
We extracted SOD from two to three fully expanded leaf blades (or
other tissue as indicated) from a vegetative stage shoot. The sample
was frozen in liquid nitrogen, ground, and resuspended in 150 µL of
50 mM KH2PO4,
pH 7.8. We centrifuged the homogenate at 13,000g for 15 min
and determined the protein content of the supernatant (Bradford, 1976).
A constant volume (20 µL) was applied to a 13% polyacrylamide gel
with a 4% stacking gel (McKersie et al., 1993). One lane of each gel
contained 0.5 unit of bovine Cu/Zn-SOD (Sigma) as an internal standard.
The gel was stained with nitroblue tetrazolium and riboflavin (Sigma)
at 4°C and then developed on a light box for 20 min. The areas of SOD
activity were clear against a blue background.
An image of the gel was captured using a CCD (charge-coupled device)
video camera and Northern Exposure Software (Empix Imaging, Mississauga, Ontario, Canada). We used Excel (Microsoft, Redmond, WA)
to calculate the area under each SOD isozyme peak and expressed the
data as a percentage of the total activity by calculating the area of
isozyme peak per total area of all SOD peaks times 100%.
Alternatively, we expressed the data as units of SOD activity per gram
of protein, calculated as the area of individual peak per area of
internal standard times the concentration of the internal standard.
Freezing Tolerance
The transgenic plants were propagated by node cuttings and rooted
in Turface growth medium in 4- × 4- × 11-cm four-cell root trainers
(Plant Products). The plants were defoliated and grown to a height of
about 10 cm (approximately 2 weeks after defoliation). The plants were
then transferred to a 2°C growth chamber with a 12-h photoperiod, at
a light intensity of 200 µmol m2 s1 (PAR)
and acclimated for 2 or 4 weeks at these conditions. Subsequently, the
4-week-acclimated plants were frozen at  2°C overnight in the dark
and then acclimated at 2°C for another week in the growth conditions
described above. We sampled the leaf blades and fibrous roots at each
stage of acclimation. The tissues were placed in glass tubes in a
precooled aluminum block, frozen at 1°C for 1 h, inoculated
with an ice chip, and cooled at a rate of 2°C h1. One sample (tube) was removed at 6°C,
7°C, 8°C, and 9°C. We thawed the sample overnight at 2°C,
added 10 mL of water to each tube, and measured the conductivity after
1 h and after autoclaving at 120°C for 15 min to calculate the
percentage of leakage. We repeated each experiment three times on three
separate days and analyzed the data statistically as a split-plot
arrangement with stages of acclimation as the main plot.
Field Trials
The 1996 field trial was conducted at the Elora Research Station
(Elora, Ontario) following protocols authorized by the Plant Products
Division, Agriculture and Agri-Food government agency (Ottawa, Ontario,
Canada) (tests 96-UOG2-ALF03-ON0-1-01 and 96-UOG2-ALF04-ON01-01, approved April 9, 1996). The plots were established in the spring of
1996 by transplanting rooted cuttings of each transgenic and control
genotype. The soil at this location is a clayed brunisolic gray-brown
luvisol-London. Fertilizer (P and K) was applied after each harvest
according to the results of the soil test analysis. We arranged the
test in a randomized complete block design with 15 cuttings of each
control (nontransgenic) and 5 cuttings of each transgenic genotype as
the experimental units and three replications (blocks). Plants were
harvested once in the year of transplanting. We took stand counts in
the fall of 1996 and the spring of 1997 to determine survival. Plants
were defoliated on June 28, July 28, and August 28, 1997, to determine
dry-matter yields. The yields presented are the sums of the three
harvests in 1997.
The 1997 field trial was also conducted at the Elora Research Station
following protocols authorized by Plant Products Division, Agriculture
and Agri-Food Canada (tests 97-UOG1-075-ALF02-177-ONO1-01, 97-UOG1-075-ALF03-236-ONO1, and 97-UOG1-075-ALF04-224-ONO1-01; approved
May 12, 1997). Replicated plots were established on May 28, 1997, by
transplanting rooted cuttings of each transgenic and control genotype
in 1- × 2-m rectangular plots at 100 plants per plot. Each plot
consisted of a population of independent transgenic plants for each
construct. Plants were harvested twice in the year of transplanting on
July 1 and September 2, 1997. On November 19, 1997, samples were dug
from the field, rinsed, separated into taproot, crown, and leaves, and
immediately submerged in liquid nitrogen. The samples remained in
liquid nitrogen until they were ground in the laboratory and analyzed
for SOD activity on native PAGE gels as described above. Whole samples
were also dug from the field. Crowns and roots of these
field-acclimated alfalfa plants were subjected to freezing temperatures
by placing the plants in moist filter paper. The samples were frozen at
2°C overnight and then cooled at 2°C per hour to 6°C,
8°C, 10°C, 12°C, or 14°C. The frozen samples were
thawed for 1 d at 2°C. The plants were separated into crowns,
taproots, and crown buds, bisected, and assessed by viability staining
with nitroblue tetrazolium as previously described (Tanino and
McKersie, 1985). We repeated the test on December 2, 1998.
Statistical Analysis
We performed an analysis of variance using SAS for Windows, Proc
GLM (version 6.11, SAS Institute, Cary, NC). Because of missing values
in some experiments, we calculated least-square means. Significance was
determined at the 5% level of probability.
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RESULTS |
Two clones of alfalfa were transformed with A. tumefaciens containing either the pMitSOD or pChlSOD binary
vectors. Using PCR we screened the plants first for the presence of
nos-nptII transgene-conferring kanamycin resistance.
Approximately 90% of the regenerated plants scored positively in the
screen. Only PCR-positive plants were transferred to the greenhouse for
further study. Southern-blot analysis of eight transgenic plants
confirmed that there were one or two full insertions of the tDNA in the
chromosomes of each of the transgenic plants (data not shown).
We analyzed the transgenic plants from the clone N4 containing pMitSOD
or pChlSOD for SOD expression using native PAGE (Fig. 1). The nontransgenic control N4 plant
had three major SOD bands: a fast-moving chloroplastic form of
Cu/Zn-SOD, a slower-moving cytosolic form of Cu/Zn-SOD, and a
mitochondrial Mn-SOD. A small Fe-SOD peak was occasionally detected
between the Mn-SOD and cytosolic Cu/Zn-SOD, but its activity was quite
labile and not included in the calculations of total SOD activity. The
transgenic plants had an additional Mn-SOD enzyme superimposed on the
native Mn-SOD isozyme in the native gels. In the case of the pMitSOD,
the two Mn-SOD forms were not resolved by PAGE, but in the case of
the pChlSOD, two distinct Mn-SOD bands were apparent. The variance in the mobility of the mitochondrial and chloroplastic targeted forms
of Mn-SOD possibly reflects differences in the cleavage site of the
transit peptide or another posttranscriptional modification.
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| Figure 1.
Line scan of native PAGE gels for SOD activity in
leaf extracts of transgenic alfalfa plants expressing pMitSOD or
pChlSOD. Cu/Zn-SOD1 and Cu/Zn-SOD2 designate cytosolic and
chloroplastic forms of SOD, respectively. Fe-SOD activity was observed
as a small band only on some gels. The area of the Mn-SOD band was
increased in both pMitSOD and pChlSOD compared with N4. A distinct
double band was observed in Mn-SOD of pChlSOD but not pMitSOD.
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We quantified the amount of each SOD isozyme in two ways. The area of
each peak from the line scan was calculated and expressed relative to
the total area of SOD activity in each lane to determine its proportion
of total SOD activity. This method compensated for differences in the
amount of the extract applied to the gel and for differences in the
staining intensity among gels. However, the method assumed that
Cu/Zn-SOD was not affected by expression of the Mn-SOD transgene. So,
in some experiments we quantified the amount of each SOD isozyme by
expressing its area relative to the area of an internal standard
(bovine Cu/Zn-SOD) on the same gel to calculate specific activity
(units per gram of protein). The amount of bovine Cu/Zn-SOD applied was
linearly related to the area of the peak over the range used in these
experiments (data not shown).
Based on native PAGE analysis, Mn-SOD activity increased by a variable
amount among the independent transgenic plants containing either
pMitSOD or pChlSOD (Table I). In
approximately 25% of the transgenic plants from either transformation
vector Mn-SOD activity lessoned or did not change. In the majority of
the transgenic plants Mn-SOD activity increased less than 2-fold. In
only a small proportion of the transgenic plants was Mn-SOD activity
doubled relative to total SOD activity.
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Table I.
Frequency of different Mn-SOD activity classes among
independent transgenic alfalfa plants expressing pMitSOD or pChlSOD
Mn-SOD activity in leaf extracts from N4 was 19% of total SOD activity
as determined by activity staining of native polyacrylamide gels.
Values in parentheses represent the frequencies as percentages of the
total number of transgenic plants.
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We selected the highest expressing plants for more detailed analysis.
In three pMitSOD and two pChlSOD plants, Mn-SOD activity increased
in all shoot tissues sampled (Table
II), but the increase was not uniform
across tissues. The increase in Mn-SOD activity was least in apex,
nodes, and stem and greatest in the petiole and blade leaf tissues. The
response was similar in both vectors. The variation among tissues in
the transgenic plants was possibly due to differences in the
transcription of the cauliflower mosaic virus 35S promoter among the
tissues, but posttranscriptional modifications of SOD activity were
also likely to be important contributing factors.
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Table II.
Mn-SOD activity as a percentage of total SOD
activity in shoot tissues of transgenic alfalfa plants expressing
pMitSOD or pChlSOD
Activities were measured on three high-expressing pMitSOD plants and
two high-expressing pChlSOD plants.
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Bovine Cu/Zn-SOD was used as an internal standard, and the PAGE
analysis showed that Mn-SOD specific activity also increased for both
vectors (Table III). However, if the data
were expressed as a percentage of total SOD, the increase was greater
in pMitSOD than predicted. This discrepancy occurred because targeting
the Mn-SOD to the chloroplasts reduced Cu/Zn SOD activity. The plants containing pMitSOD had the same activity of both forms of Cu/Zn-SOD as
the N4 control; therefore, total SOD activity in leaf extracts increased. In contrast, targeting the Mn-SOD to the chloroplast caused an apparent feedback regulation, leading to lower
chloroplastic and cytosolic Cu/Zn-SOD activity; therefore, total
SOD activity actually lessened, even though Mn-SOD activity increased.
Although the mechanism of this apparent regulation is unknown, there
appeared to be similar regulation of the two Cu/Zn forms of the enzyme.
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Table III.
Mn- and Cu/Zn-SOD activities in independent
transgenic alfalfa plants expressing pMitSOD or pChlSOD
Values are averaged across shoot tissues from the same experiment as
described in Table II. Values in parentheses are percentages of
activity of each individual form relative to the total SOD activity.
Values within a row followed by the same letter are not significantly
different according to Duncan's multiple range test at the 5% level
of probability; n = 50 (control), 106 (pMitSOD), or 58 (pChlSOD).
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We selected four highly expressing primary transgenic plants from each
vector for growth analysis and stress-tolerance testing in the
greenhouse. The plants were propagated with cuttings. We observed no
variation in SOD activity among propagules of the same transgenic plant
(data not shown). We tested cuttings in one experiment with PCR for
nos-nptII and all remained positive (data not shown).
The transgenic plants and a nontransgenic control were grown to the
early bud stage, defoliated, and allowed to grow new shoots as a
measure of vigor. Three of the four pMitSOD plants tested had greater
shoot dry-matter production than the control (Table IV). Two pChlSOD plants had less growth
and two had more growth than the control.
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Table IV.
Vigor of primary independent transgenic alfalfa
plants expressing pMitSOD or pChlSOD
Shoot dry matter production in the greenhouse was determined at 28 d after defoliation. LSD at the 5% level of probability
for dry weight determinations = 22, n = 28.
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Freezing tolerances of leaf blades and fibrous roots were measured by
electrolyte leakage. The plants were acclimated to three different
stages and frozen to four different temperatures. Statistically significant main effects were observed for tissues, acclimation, and
temperatures (Table V). Roots were
consistently more freezing sensitive than leaves. Both leaves and roots
had less injury with acclimation. Lower temperatures caused increasing
amounts of injury. Small, statistically significant differences were
observed among the transgenic plants and compared with the control
plant. However, some transgenic plants had less injury than the
nontransgenic N4 control, whereas others had more injury. Nonetheless,
the most tolerant transgenic plant was only 1°C more freezing
tolerant than the control, indicating that any improvement in freezing tolerance per se was minimal.
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Table V.
Electrolyte leakage from leaves and roots after
freezing of independent transgenic alfalfa plants expressing
pMitSOD or pChlSOD
The experiment was factorial with a split plot design of three
acclimation stages (main plot-different weeks) × 7 plants × 4 temperatures × 3 replications (different days). Leaf and root
samples were analyzed separately. Values for the main treatment effects
are averaged across other treatments in the factorial experiment.
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Independent transgenic plants were selected from the N4 group for the
field trial if they had increased SOD activity; N4 plants with lower
SOD activity were not included. The other S4 group of plants was not
tested in the greenhouse and all available independent transgenic
plants were placed in the field. All plants had good survival in the
year of transplanting and entered the winter of 1996/1997 with close to
100% survival. The location of the field trial experienced a
relatively harsh winter in 1996/1997 because of excessively wet soil
conditions. This site was chosen because the permit for the transgenic
field trial dictated that the experimental alfalfa plants had to be
isolated by 20 m from any other alfalfa and because less than
100% survival of the nontransgenic clones was desired. Survival in
spring 1997 was less than commonly observed with these parental clones
in other tests. The average survival of the transgenic plants was
higher than that of the nontransgenic controls (Table
VI). The N4 group of transgenic plants
had higher survival and vigor in the field than the S4 group, but the
two vectors pMitSOD and pChlSOD were similar in both.
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Table VI.
Average survival and yield of independent
transgenic alfalfa plants expressing pMitSOD or pChlSOD in spaced
plantings
Two parent clones designated as N4 and S4 were transformed with the
pMitSOD or pChlSOD vectors. Values are the averages across all
independent transgenic plants within a vector with three replications
of 5 plants per independent transgenic plant and 15 plants per control.
Survival and yield of the transgenic plants were significantly greater
than the nontransgenic control plants at P = 0.0243 and P = 0.0159, respectively, according to an orthogonal contrast.
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The genetic potential of the plant and the quantity of stored
carbohydrate reserves determine the first-cut herbage yield, but winter
injury to the root, crown, or crown buds may reduce the yield. The
average yield of the transgenic plants expressing pMitSOD was higher
than that of the N4 and S4 nontransgenic controls (Table VI). The
average yield of those expressing pChlSOD was the same as N4 and
significantly greater than S4, although in the latter case there was
only one transgenic plant tested.
We observed more variation of both survival and yield within each group
of transgenic plants than between parent clones or vectors. The N4
nontransgenic control had 53% survival, and S4 had 29%. Most
transgenic plants had improved survival. Some individual transgenic
plants in both pMitSOD and pChlSOD had 100% survival, but there were
other individual plants with less survival than the nontransgenic
controls in both parental groups and with both vectors (Table
VII). Similarly, although both pMitSOD
and pChlSOD had individuals with dramatic improvements in yield,
approaching twice the yield of the N4 nontransgenic control, other
individuals had lower yields than the nontransgenic controls (Table
VIII).
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Table VII.
Frequency of plants in survival classes of
independent transgenic alfalfa plants expressing pMitSOD or pChlSOD in
spaced plantings
Values are the percentages of the independent transgenic plants in each
survival class. No. of plants and average values are shown in
Table VI.
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Table VIII.
Frequency of yield classes of independent
transgenic alfalfa plants expressing pMitSOD or pChlSOD in spaced
plantings
Values are the percentages of the independent transgenic plants in each
yield class. No. of plants and average values are shown in Table VI.
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This experiment tested only primary transgenic plants because it was
necessary to identify the elite transformation event for future genetic
and physiological evaluation. Therefore, it is possible that the
differences in winter survival among these transgenic plants were
related to the tissue culture methods used to create them. However, an
adjacent field experiment compared the effects of the three vectors
containing the yeast suc2 gene and did not identify any
with greater survival or yield than the control (D. Vadnais,
unpublished data). Therefore, improved winter survival was not a common
response observed with this transformation system. A comparison of the
winter survival of progeny of the selected primary transgenic plants
segregating for the SOD transgene is in progress.
To determine whether these field results were due to differences in SOD
expression patterns between the greenhouse and the field, a random
sample of N4 transformed and control plants was dug from the field and
analyzed using native PAGE (Table IX). Results were similar to previous results from the greenhouse-grown plants. Mn-SOD (expressed as a percentage of total SOD) increased significantly in both pMit-SOD and pChl-SOD vectors (P = 0.03). On
average, 53% of total SOD activity was due to cytosolic Cu/Zn-SOD, and
23% was due to chloroplastic Cu/Zn SOD. Chloroplastic Cu/Zn-SOD had
lower activity in both pMit-SOD and pChl-SOD plants (data not shown).
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Table IX.
Mn-SOD activity as a percentage of total SOD
activity in tissues of transgenic alfalfa plants expressing pMitSOD
or pChlSOD
Activities were measured on plants taken from the field on November 19, 1997. LSD at the 5% level of probability = 14;
n = 3. Average Mn-SOD activity = 1398 units
g1 protein.
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The crowns and roots of field-acclimated plants were sampled from the
1997 field trial in November and again in December, and then they were
subjected to freezing temperatures. Viability was determined by vital
staining with tetrazolium. In the samples removed from the field on
December 2, 1998, the cortex of the taproots lost viability below
8°C (no red staining as shown in Fig.
2). At 10°C the pith of the crowns
was affected and the first signs of injury appeared in the axis and
innermost scales of the crown buds. At 14°C the bud axis and inner
scales were dead. The vascular cylinder of the taproots was almost
white, although the endodermis still stained red. The vascular system and cortex of the crown also remained viable. When the same test was
performed 2 weeks before, the extent of injury in all tissues was much
greater. In the crowns the vascular system was the least vulnerable,
and in the crown buds the very tip of the primordia survived 14°C
but the vascular tissues were no longer viable. We found no differences
in the sites or pattern of freezing injury between the nontransgenic
and any of the transgenic plants (data not shown). At 10°C and
14°C we observed the same extent of freezing injury in all of the
transgenic plants and in the control (nontransgenic) plants.
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| Figure 2.
Viability staining of the roots and crown of
alfalfa. Plants were dug from field plots on December 2, 1997, frozen
to 6°C, 10°C, and 14°C, and stained with nitroblue
tetrazolium to detect viability. Cells stained red have active
respiration and are considered alive. Those stained white have been
injured. There is essentially no difference in the injury seen between
control and SOD-transgenic plants at any freezing temperature
examined.
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DISCUSSION |
Transgenic alfalfa plants constitutively expressing a Mn-SOD cDNA
had greater Mn-SOD activity, significantly greater survival in the
field after one winter, and greater total shoot dry-matter production
(yield) in the field than the nontransgenic control plants. This effect
has now been observed in three alfalfa clones (N4 and S4 in this study
and RA3 in a previous study [McKersie et al., 1993, 1996]) with two
SOD vectors having different subcellular targeting sequences. However,
this study shows that there was a large amount of variation among
transgenic plants regenerated from a single vector in a single clone.
This variation occurred in all of the parameters measured, including
Mn-SOD activity, yield in the greenhouse and in the field, and winter
survival. The variation among plants that had been transformed with the same vector was greater than that due to subcellular targeting of SOD
or due to the alfalfa clone into which the tDNA was inserted. We now
see that our earlier experiments with RA3 were fortuitous in showing a
positive effect because it is clear from these data that there was a
possibility of selecting individual transgenic plants with less
hardiness, the same hardiness, or improved hardiness from the same set
of transgenic plants.
These data indicate that we must be cautious in interpreting
experiments that measure stress-tolerance effects in transgenic plants
using only a few independent transgenic plants from each vector, such
as in our previous study (McKersie et al., 1996), because conflicting
results are likely. For example, in the case of SOD overexpression,
Tepperman and Dunsmuir (1990), Pitcher et al. (1991), and Payton et al.
(1997) found no improvement in tolerance to oxidative and related
stresses, whereas Bowler et al. (1991), Gupta et al. (1993a, 1993b),
Perl et al. (1993), McKersie et al. (1993, 1996), and Van Camp et al.
(1994, 1996) found significant improvements. Several studies (Pitcher
et al., 1991; Gupta et al., 1993a, 1993b) evaluated only two transgenic
plants. The present data suggest that a reliable prediction of a
general trend should require more than 20 independent transgenic
alfalfa plants.
The alfalfa plants that we produced varied considerably in SOD
activity, so we attempted to select plants in the greenhouse to reduce
the number of primary transgenic plants evaluated in the field and to
speed the selection of plants for cross-pollination and seed
production. Prescreening the transgenic plants for SOD activity, vigor,
or freezing tolerance in the greenhouse was not effective at
identifying individual transgenic plants with improved field
performance. One may have anticipated this because survival of the
crowns and roots determine winter survival of alfalfa, and we measured
SOD activity in shoots in the greenhouse. Although the greenhouse is a
very different environment than the field, we assumed that the
cauliflower mosaic virus 35S promoter would be constitutive and that
therefore individuals with high expression in one environment and one
tissue would have correspondingly high expression in another. Either
this assumption was not valid or other factors interacted with SOD
activity to modify performance.
Shoot growth in the greenhouse was also not a reliable prediction of
growth in the field. Comparing the yields of transgenic plants common
to Tables IV and VIII by regression analysis gave r2 = 0.32. A greenhouse screen for vigor
may have eliminated a few poorly performing plants, but it could not
identify the highly performing plants.
Freezing tolerance increased by only 1°C in the best transgenic plant
when the plants acclimated to controlled environments and when we
measured viability by electrolyte leakage. When the crowns and taproots
of field-acclimated plants were frozen, there was no apparent
difference in freezing tolerance between transgenic and control plants,
if we measured viability by tetrazolium staining. Therefore, these
studies do not indicate that overexpression of SOD changed the primary
site of freezing injury in alfalfa. Yet, we did observe differences in
winter survival among the plants. Previously, we had observed
differences when viability was measured as the amount of
shoot regrowth from crowns after a freezing stress (McKersie et
al., 1993). Additional SOD activity may have improved the cellular
repair mechanisms, thereby allowing the transgenic plant to better
recover from freezing injury. Perhaps cellular viability was not a good
indicator of whole-plant viability in this instance. In addition,
freezing may not have been the most appropriate laboratory stress test,
and better outcomes may have resulted from ice-encasement, flooding, or
long-duration freezing tests. Unfortunately, despite the current
regulatory requirements for permits to test transgenic plants in the
field, field testing remains the most suitable measure for winter
survival and yield potential.
| |
FOOTNOTES |
1
Financial support for this research was provided
by the Natural Sciences and Engineering Research Council of Canada and
the Ontario Ministry of Agriculture Food and Rural Affairs.
*
Corresponding author; e-mail: mckersie{at}plant.uoguelph.ca; fax
1-519-763-8933.
Received June 29, 1998;
accepted December 3, 1998.
| |
ABBREVIATIONS |
Abbreviation:
SOD, superoxide dismutase.
| |
ACKNOWLEDGMENTS |
The authors thank Dirk Inzé, Universiteit Gent, Belgium,
who kindly provided the binary vectors pMitSOD and pChlSOD. Molian Deng
transformed the N4 alfalfa plants, and Ranjith Pathirana transformed
the S4 plants. Lori Wright confirmed the transformations using PCR and
conducted the Southern-blot hybridizations. Heather Anderson, Andrea
Fiebig, and Carol Hannam analyzed SOD activity using PAGE. Ning Chen
maintained the plants in the greenhouse and conducted the freezing
experiments. Donna Hancock, Jennifer Thatcher, and Julia Murnaghan
conducted the field trials.
| |
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Top
Abstract
Introduction