by inserting genes into Greenfeast, using a method modified from that used at CSIRO. The transgenic
plants grow well and the inserted genes are func- tional and inherited [5]. However, Greenfeast is
not suitable for western Canadian growing condi- tions or markets. Thus, an important question is:
can we transform cultivars that are more suitable for the western Canadian industry, or is the ability
to transform peas limited to a few genotypes?
To help answer this question, seven advanced breeding lines were obtained from three pea breed-
ing programs and used for transformation experi- ments, with Greenfeast included for comparison.
Three Agrobacterium vectors with different chemi- cal selection systems were used in these transfor-
mation experiments. The purpose of this study was to examine the effects of pea genotype, gene con-
struct and developmental stage of pea seeds on the regeneration of transgenic pea.
2. Materials and methods
2
.
1
. Construction of binary Agrobacterium transformation 6ectors for pea transformation
2
.
1
.
1
. LBG
66
A bi-functional fusion gene uidA
::
nptII confer- ring both b-glucuronidase GUS and neomycin
phosphotransferase NPTII activities [6], with a 35S35SAMV promoter [7], a NOS terminator and
an intron [8] was cloned as a HindIII – EcoR1 fragment into pPBI3008 Fig. 1 to produce the
binary plasmid pPBI3010.
2
.
1
.
2
. LBG
71
An intron-containing
uidA gene
with a
35S35SAMV promoter and a NOS terminator was introduced into the pBIN19 binary transformation
vector [11]. A phosphinothricin N-acetyltrans- ferase pat gene [12] with a 35S35SAMV pro-
moter and a 35S terminator was inserted 5 to the uidA gene, in the same transcriptional orientation,
to produce the binary vector pPBI3011.
2
.
1
.
3
. LBG
75
A mutant acetohydroxy acid synthase ahas
3
r gene [13] with a 35S35SAMV promoter and its
own terminator sequence, was introduced into pPBI3010, 3 to the uidA
::
nptII gene and in the opposite transcriptional orientation, to produce
the binary vector pPBI3020. Each of these binary plasmids was electropo-
rated into the EHA105 disarmed Agrobacterium strain [14]. The Agrobacterium cultures were
grown in darkness at 28°C on a rotary shaker in 2YT medium containing l
− 1
: yeast extract 10 g, tryptone 1 g and NaCl 5 g, pH 7.0 with appro-
priate antibiotics. Overnight cultures were cen- trifuged and resuspended in 2YT to a final
concentration of 10
8
bacteria cells ml
− 1
of culture A
660
= 0.06.
2
.
2
. Plant germplasm used for transformation and plant culture
Eight pea Pisum sati6um L. genotypes were used in the transformation experiments Table 1.
A line selected from the cultivar Greenfeast, from which the recovery of transgenic peas has already
been demonstrated [4,5], was used as the control genotype.
Pea seeds were planted in 15 cm plastic pots filled with a 1:1 mixture of commercial potting soil
Sunshine Mix c 1 and vermiculite. Plants were grown in a controlled environment chamber at
2015°C with a 16 h photoperiod at 200 mmol quanta m
− 2
s
− 1
supplied by mixed fluorescent-in- candescent lamps. At the 4 – 6 leaf stage, Nutricote
Type 100 14 – 14 – 14 slow release fertilizer was
Fig. 1. Construction of binary plasmid pPBI3008. The binary plasmid pPBI3008 was constructed from: a 2332 bp HindIII –
PstI fragment with a gene for gentamicin resistance from pXS1 [9] subcloned into pUC8, a 5119 bp HindIII – PstI
fragment from pMON505 containing the RK2 and pBR322 origins of replication [10] and a 1986 bp BglII fragment of
pBIN19 [11] containing Agrobacterium tumefaciens borders LB, RB, the lacZa gene and the multiple cloning site. An
1836 bp ClaI – SauI fragment containing the nptII gene was removed and the free ends of the pBIN19 fragment were
ligated together. Asterisks indicate restriction sites eliminated during construction.
Table 1 Pea genotypes used in pea transformation experiments: descriptions and sources
Label Description
Genotype Source
Selected line, wrinkled green seed, normal leaf Greenfeast
CSIRO
a
GF Austrian winter pea cultivar, speckled seed, semi-leafless
VI CDC
b
CDC Vienna Breeding line, green seed, normal leaf, high yield, early maturing
S2-90-25E CDC
25E 93-4-18G
18G Breeding line, green seed, semi-leafless
CDC Breeding line, yellow seed, normal leaf, high yield
MP1338 AA-FC
c
M38 Breeding line, yellow seed, semi-leafless
M82 AA-FC
MP1382 Breeding line, green seed, semi-leafless
AWPNZ66 AWP
d
NZ66 AWP1512
1512 Breeding line, green seed, semi-leafless
AWP
a
H.E. Schroeder, Plant Industry Division, CSIRO, Australia.
b
A.E. Slinkard, Crop Development Centre, University of Saskatchewan, Saskatoon, Sask., Canada.
c
T.D. Warkentin, Agriculture and Agri-Food Canada, Morden Man., Canada.
d
T. Ferguson, Alberta Wheat Pool, Calgary, Alta, Canada.
added to each pot. The closed flower of pea makes it difficult to determine anthesis without poten-
tially causing damage to the flower. Therefore, three times per week, all newly opened flowers
were tagged with coloured tape so that stages of seed development could be identified, based on the
number of days after flowering DAF.
2
.
3
. Preparation of embryo explants Seeds, aged between 23 and 34 DAF, were
collected in groups of the same age. For the purpose of reporting, the explants were combined
into three age groups of: 23 – 26, 27 – 30 and 31 – 34 DAF. The seeds were removed from the pods and
surface sterilized for 1 min in 70 ethanol and 8 min in 1 wv NaClO, and then rinsed thor-
oughly with sterile distilled water. The seed coat and one cotyledon were removed from each seed
and the radicle was excised. The remaining em- bryo axis tissue was sliced longitudinally into five
slices, with a blade dipped in the Agrobacterium, as described by Schroeder et al. [4]. The slices were
placed on a co-cultivation medium which was modified from Brown and Atanassov [15] and
contained l
− 1
: KNO
3
3 g, CaCl
2
770 mg,
L
-glutamine 800 mg, MgSO
4
·7H
2
O 500 mg,
L
-serine 100 mg, glutathione 10 mg, adenine 1 mg, sucrose 30 g, 2,4-D 1 mg, kinetin 0.2 mg
and agar 0.8. In addition, acetosyringone was added to the co-cultivation medium after autoclav-
ing, to a final concentration of 100 mM. A 5 ml droplet of Agrobacterium suspension culture was
added to each of 35 thin slices in a 60 × 15 mm plastic Petri dish.
2
.
4
. Co-culti6ation of explants and reco6ery of transgenic plants
The explants were incubated at 25°C with a 16 h photoperiod of fluorescent light at 35 – 55 mmol
quanta m
− 2
s
− 1
for 4 days and were then rinsed in 300 mg l
− 1
Timentin
®
, a mixture of the antibi- otic ticarcillin and clavulanic acid [16]. The ex-
plants were transferred to a callusing medium, P1 [4], with 150 mg l
− 1
Timentin
®
, and the appropri- ate selection chemical
L
-phosphinothricin L- PPT, 10 mg l
− 1
; kanamycin, 40 mg l
− 1
; or chlorsulfuron, 10 mg l
− 1
. The explants were re- turned to the same incubation conditions for 14
days. For an early indication of cell transforma- tion, random samples of five slices per Petri dish
were collected for a quantitative determination of GUS activity.
The remaining explants were transferred to a shoot induction P2 medium [4], also with Ti-
mentin
®
and the appropriate selection chemical. Any shoots that developed were excised and dis-
carded as they probably arose from pre-existing meristems. Surviving explants were transferred to
fresh P2 medium every 3 weeks. Any shoots, or clusters of shoots produced thereafter were excised
from the explants and transferred to 100 × 25 mm plastic Petri dishes with MS7T medium, a MS [17]
medium with B5 vitamins [18], 3 sucrose, 150 mg l
− 1
Timentin
®
and benzylaminopurine BAP; 1 mg l
− 1
, to enhance shoot elongation. The concentra- tion of selection chemicals was increased L-PPT,
15 mg l
− 1
; kanamycin, 50 mg l
− 1
; chlorsulfuron, 50 mg l
− 1
. Elongated shoots 1.5 – 2 cm were removed from the explants and transferred to
B52T rooting medium, with half-strength B5 salts and vitamins, 3 sucrose, 150 mg l
− 1
Timentin
®
, and NAA 0.185 mg l
− 1
. The selection chemicals remained at the same level as in the shoot elonga-
tion MS7T medium. Once roots were estab- lished, the young transformants were transferred
to pots of soil and grown as described for the original plants.
2
.
5
. Analysis of putati6e transgenic plants for presence and expression of inserted genes
2
.
5
.
1
. b-Glucuronidase assays A b-glucuronidase X-Gluc histochemical reac-
tion [19] was used on small amounts of tissue leaf, stipule, tendrils for visual identification of GUS
activity in young regenerated plants. For a quanti- tative determination of GUS activity, assays using
4-methyl umbelliferyl b-
D
-glucuronide MUG as a substrate [20] were performed. Tissue equivalent
to one axis five slices was homogenized in 500 ml of GUS extraction buffer [20]. The samples were
centrifuged at 5°C 14 000 rpm for 2 min and a 100 ml sample was removed for measurement of
protein concentration using a dye-binding Brad- ford assay [21] BioRad Richmond, CA. This
volume was replaced with 100 ml of a 5 mM solution of MUG and the samples were incubated
at 37°C. After 15 min and again after a further 4 h, the samples were vortexed and centrifuged as
above. A 100 ml sample of the supernatant was added to tubes containing 900 ml of stop buffer
0.2 M Na
2
CO
3
. The 4-methyl umbelliferone MU concentration was determined with a Perkin
Elmer LS50 fluorometer and GUS activity was expressed as pmol MU axis
− 1
min
− 1
. The same procedure was used with two leaf discs 8 mm
from putative transformants, once established in the soil.
2
.
5
.
2
. Southern hybridization analysis To confirm the incorporation of inserted genes,
determine the number of inserts, and identify inde- pendent transgenic events, Southern blot analysis
was performed on each putative transgenic plant. Genomic DNA was extracted from four leaf discs
of each plant using a CTAB method modified from Murray and Thompson [22]. The DNA was
digested with restriction enzymes as follows: LBG66, HindIII for gus; LBG71 HindIII for gus,
EcoRI for pat; LBG 75, EcoRI for both gus and ahas. The gus probe was an 1800bp BamH1Sst1
fragment from pBI121 [19]. The pat probe was a 550 bp SalI fragment of the plasmid pPAT2,
acquired from AgrEvo Canada. The ahas probe was a mixture of 2 – 300 bp HindIII fragments
from ahas
3
r [23]. The probes were labelled with [
32
P]dCTP using random primers. Hybridization was done with the QuikHyb system Stratagene.
2
.
5
.
3
. Segregation analysis To examine the inheritance of the gus gene, the
T
1
progeny from
different plant
genotype Agrobacterium vector combinations were planted
under the same conditions as the donor plants. Leaf discs 8 mm were collected for analysis by
both X-Gluc and MUG assays.
3. Results and discussion
