2
.
5
. Statistical methods Segregation ratios were analysed using a X
2
analysis. A two-sample t-test Minitab was applied in
the analysis of TI activity measurements and com- parison of populations of T
3
seeds.
3. Results
3
.
1
. Isolation of promoter The TI
1
gene, corresponding to one of two genes encoding the major pea seed inhibitors, was
isolated as a genomic clone containing approxi- mately 1 kb of promoter sequence. This promoter
contained a number of elements that may influ- ence its activity Fig. 1C. Relative to the tran-
scription start, a putative TATA box − 40, a RY-like element CCTGCATG: − 467 to − 473,
together with a number of G-box elements, were evident. Three G-box elements, TAC ACGT
ATG, TAC ACGT GTA and ATC ACGT GAC were at positions − 135 to − 144, − 226 to − 235
and − 571 to − 580, respectively. In the case of the most distal element, an overlapping ‘C-repeat’-
like element was observed T GACCG A: − 568 to − 577, together with an upstream CE1-like
element TAC CACC AC: − 586 to − 594. The significance of many G-box elements is unclear but
their context and interaction with other elements can determine a range of promoter responses [23 –
25]. The TI
1
gene promoter was used in the con- struction of transgenes designed to express either
antisense TI mRNA at the appropriate stage of seed development or the marker enzyme, ß-glu-
curonidase, for the assessment of the behaviour of the promoter.
3
.
2
. Reco6ery of transformed plants The transformation efficiency of grain legumes
is consistently low; for pea, in particular, percent- age success rates of 1 – 3 have been cited, based
on recoveries of primary transformed plants [15,17,26]. Two genotypes have been employed for
the work described in this paper — BC117 and cv. Puget. Transformation efficiencies, based on
recovery of individual explants producing resistant shoots, were calculated to be 0.13 and 0.8, re-
spectively, for BC117 and cv. Puget. The latter value is within error of the values reported for cv.
Puget [17]. Transformation efficiencies were much lower, however, when based on recovery of indi-
vidual explants for which T
2
transformed plants were obtained. Transmission of transgenes was
observed for only 40 of explants. Multiple shoots were often recovered from individual ex-
plants; since these can result from independent transformation events, all shoots were grafted T
1
plants; lines and grown for seed production. Fur- ther details of the lines generated with the two
constructs are given below.
3
.
2
.
1
. Transgenic lines generated using RSGIT In all, 19 T
1
plants derived from explants of cv. Puget, both bar and GUS genes were detected by
Southern analysis data not shown. GUS activity could not be detected in any T
2
seeds tested from eight lines up to 55 seeds per line, whereas all
seeds tested from four lines up to 15 seeds per line were positive for GUS activity. Seeds from a
further seven lines showed segregation of GUS activity between 8 and 15 seeds tested with ratios
ranging from 1:1 to 9:1 and probabilities in the range 0.05 – 0.9 for a single locus 3:1 ratio. Where
all seeds tested positive for GUS activity, a mini- mum of two loci containing transgenes could be
inferred.
All the GUS positive seeds produced plants which were resistant to the herbicide and both bar
and GUS genes were detected on Southern blot analyses. All the GUS negative seeds produced
plants sensitive to the herbicide and neither bar nor GUS genes could be detected. Where there
was no evidence for GUS activity in any T
2
seeds from any one line, or for either bar or GUS
transgenes in the T
2
plants, non-transmission of the transgenes through meiosis could be assumed;
this was the case for 42 of the T
1
plants. Lines 6:C and 12:A, showing segregation of transgenes
among T
2
seeds and plants at a frequency of 9:4 Fig. 2 and 9:1, respectively, were used for further
analyses P = 0.6 and 0.3, respectively, for a 3:1 ratio.
3
.
2
.
2
. Transgenic lines generated using E
6
ASTIJIT Sixteen T
1
plants, derived from explants of BC1 17 and cv. Puget, were analysed. Fourteen lines
showed some resistance to the herbicide in the leaf
Fig. 2. Southern blot analysis of EcoRI-digested genomic DNA from thirteen tracks 1 – 13 T
2
plants from the trans- genic line 6:C, hybridised with a GUS gene probe. A 2.8 kb
fragment, predicted from Fig. 1A, was evident in plants that were resistant to the herbicide, giving a segregation ratio of
9:4 for both the transgenes. The sizes of DNA markers are indicated kb.
ratios, respectively. Lines 1:B and 1:C were used for further analyses.
3
.
3
. Analysis of lines transformed with RSG
1
T
3
.
3
.
1
. GUS acti6ity in de6eloping embryos Embryos were harvested at 33, 42 and 53 days
after flowering DAF from line 6:C:4, which was identified as a T
2
plant homozygous for the GUS transgene, following analysis of mature seed see
below. GUS activity was detected at low levels in embryos at 33 DAF but increased dramatically
between 42 and 53 DAF, both on a per unit fresh weight and a per unit protein basis Fig. 3. These
data show that GUS expression in embryos, under the control of the TI
1
promoter, follows that of TI activity, previously shown to increase dramatically
during the late stages of embryogenesis in geno- types grown under the same conditions [10,22].
3
.
3
.
2
. Response of the TI
1
promoter to drought stress
Populations of T
3
seeds from individual T
2
seeds from lines 6:C and 12:A were assayed for GUS
activity to determine which populations were derived from T
2
seeds that were homozygous for the presence or absence of the transgene or were
hemizygous. Measurements were made on 30 seeds from each population. Plants from populations
that were homozygous for the presence or absence of the transgene were subjected to either drought
or control growth conditions. GUS activity mea- surements were made on extracts from roots and
leaves harvested after the 42-day-treatment. Very low background GUS activity B 1 RLUmg
protein × 10
− 3
was recorded for non-transgenic plants. A low level of GUS activity was detected in
roots and leaves from control transgenic plants Fig. 4. The activity in roots from drought-treated
plants was greatly elevated compared with control levels on a unit dry weight basis and on a unit
protein basis Fig. 4, line 6:C. In analyses of line 12:A, an even greater fold increase in GUS activity
was observed for drought-treated roots Fig. 4. Western analysis Fig. 5a – d showed a large in-
crease in the amount of GUS protein , Fig. 5c and d in the drought-treated roots compared with
those of the control plants, in agreement with activity measurements [a non-specific low molecu-
lar weight protein that reacted with the antibody appeared to be constitutively present in all the
Fig. 3. GUS activity measured as relative light units RLU; mean of 3 determinations per mg fresh weight and per
mg protein
in developing T
3
embryos from the transgenic line 6:C T
2
plant 6:C:4 of Fig. 2. Embryos were harvested at three developmental stages.
painting test 1:1 total: partial resistance, whereas two were sensitive to the herbicide. The bar gene
was detected by Southern analyses not shown in all plants including those sensitive to the herbicide.
All T
2
plants from 13 lines were sensitive to the herbicide and the bar gene could not be detected
by Southern analysis; 10 – 20 seeds per line were sown and analysed. In this instance, therefore, the
transgenes were not transmitted through meiosis in 80 of primary transformants. Three lines,
derived from the genotype BC117, showed herbi- cide resistancesensitivity among T
2
plants at the following frequencies: 10:0 line 1:B, 17:3 line
1:C and 37:2 line 1:D with probabilities P of 0.4, 0.3 and 0.8 for 15:1, 3:1 and 15:1 segregation
extracts when roots of plants analysed in Fig. 5a – d were extracted directly in sample buffer and
in all the analyses of plants derived from line 12:A not shown]. Northern analysis of RNA from
these plants showed increased levels of GUS mRNA in roots of drought-treated plants Fig.
5e – h, estimated to be 10-fold by scanning autora- diographs. Control non-transgenic plants showed
no hybridisation to the GUS probe Fig. 5e and f. TI mRNA was detected in the roots of the two
sets transgenic and non-transgenic of drought- treated plants Fig. 5j and l but not in either set
of well-watered plants Fig. 5i and k.
3
.
4
. Analysis of seeds from lines transformed with E
6
ASTIJIT TI activity TIA measurements were made on
T
3
seeds originating from five T
2
plants; seeds from the T
2
plant 1:C:6, which segregated for absence of the bar gene, were included as controls.
Analyses of TIA measurements on these seeds showed there was a significant difference at the
0.1 level in three of the populations when com- pared with the non-transgenic control population
Table 1 with reductions in TIA of up to 45. There was no significant difference between the
Fig. 4. GUS activity measured as relative light units RLU; mean of 4 determinations in leaves and roots of T
3
plants, derived from the two transgenic lines 6:C T
2
plant 6:C:4 of Fig. 2 and 12:A, subjected to control or drought conditions. Activities are presented per mg dry weight for 6:C and 12:A and per mg protein for 6:C.
Fig. 5. Western blot analysis a – d of protein 20 mg from roots of non-transgenic a and b and transgenic c and d T
3
plants, derived from T
2
plants 6:C:1 and 6:C:4, respectively Fig. 2, subjected to control a and c or drought b and d conditions. The blot was developed using a commercial antibody to GUS [M
r
approximately 68 000 in c and d ] which reacts non-specifically with a low molecular weight protein in all extracts. Northern blot analysis of total RNA e – l from roots of non-transgenic e,
f, i, j and transgenic g, h, k, l plants subjected to control e, g, i, k or drought f, h, j, l conditions. One blot e – h; 3 mg RNA per sample was hybridised with a GUS probe and the second blot i – l; 9 mg RNA per sample with the insert from pT1 4 – 41.
The positions of two ribosomal RNA bands approximately 3600 and 1800 bases, determined by subsequent hybridisation to a ribosomal DNA probe, are indicated . TI mRNA, previously estimated to be approximately 650 bases, is indicated .
Table 1 Trypsin inhibitor activity TIA in transgenic seeds from
plants transformed with an antisense TI gene
a
T
3
population P t-test
Mean TIA 1:C:6 control
2.9 9 0.7 n.a.
0.001 1.9 9 0.4
1:C:8 0.45
1:C:14 2.6 9 1.0
0.001 1.7 9 0.6
1:B:1 0.001
1:B:9 1.6 9 0.5
a
Mean TIA for populations of seeds from four transgenic T
2
plants in comparison to seeds from a control T
2
plant that segregated for absence of the transgene. The T
3
seeds analysed from the four transgenic T
2
plants all gave rise to transgenic plants. Mean TIA was based on three determina-
tions for n individual seeds from the T
3
populations; n = 15 for C and n = 10 for B populations.
second TI
1
gene were not isolated in these exper- iments [10]. The behaviour of the TI
1
gene pro- moter was examined, therefore, as a marker gene
construct, in transgenic peas under normal and drought stress conditions.
Analysis of GUS activity in developing embryos showed that the TI
1
promoter was active at the appropriate stage of development with a pattern
of expression Fig. 3 that agreed with that previ- ously documented for pea seed-expressed TI genes
[10]. Analysis of transgenic plants subjected to the same drought conditions employed in previous
studies showed, however, that the TI
1
promoter responded to drought conditions with concomitant
increases in GUS RNA, protein and enzyme activ- ity in roots Figs. 4 and 5. Furthermore, a low
level of GUS expression was evident in roots of control well-watered transgenic plants, despite
the apparent inactivity of the homologous native promoter in roots from these plants Fig. 5. Thus,
the TI
1
promoter-transgene is behaving in a simi- lar, but not identical, way to that of native ho-
mologous TI
1
genes. It is possible that sequences upstream of the 1 kb promoter fragment utilised
are necessary for conferring total seed specificity; analyses of other legume seed promoters have
indicated that seed specificity is conferred by re- gions upstream of a minimal promoter in the
approximate region − 100 to − 250 with en- hancers further upstream regulating quantitative
control only see for example [29]. However, data on grain legume seed-specific promoters that are
derived from studies in non-legume transgenic plants may not correspond exactly with data ob-
tained in a homologous background [30]. It is also possible that sequences upstream of the promoter
used suppress its induction in roots under drought conditions and that the induced TI RNA Fig. 5j
and l reflects activity of the second TI gene only. It is more probable, however, that both the genes
of this class behave in a similar fashion and that the non-isolation of cDNAs homologous to the
TI
1
gene in previous work was a statistical anomaly of the small number of cDNAs isolated
from the root library [10]. The existence of a G-box element with adjacent
CE1 and ‘C-repeat’ elements within the promoter Fig. 1C may explain the drought responsiveness
of TI
1
; combinations of G-box and CE1-like ele- ments are implicated in responses to drought in
many Lea ‘late embryogenesis abundant’ genes population 1:C:14 and the control. These seeds
were sown and T
3
plants analysed by painting leaves with herbicide. All the plants from the four
transgenic populations were resistant to the herbi- cide and thus all seeds assayed contained the
transgenes.
4. Discussion
