Plant Science 153 2000 161 – 170
The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions
P.L. Polowick , J. Quandt
1
, J.D. Mahon
Plant Biotechnology Institute, National Research Council of Canada,
110
Gymnasium Pl., Saskatoon, Sask., Canada S
7
N W
9
Received 9 September 1999; received in revised form 1 December 1999; accepted 3 December 1999
Abstract
Transgenic pea plants can be produced by Agrobacterium-mediated transformation of thin slices from developing embryo axes. To determine if the method is effective for different pea genotypes, seven pea breeding lines adapted to western Canadian growing
conditions were tested, using three different Agrobacterium tumefaciens transformation vectors. All vectors contained the gus uidA gene coding for the b-glucuronidase GUS protein, but with different chemical selection genes. In total, 323 transgenic
plants were recovered from 39 independent transformation events. Transgenic plants were recovered from each genotype and each selection system, but not from all combinations. GUS-positive explants were obtained from seeds harvested between 24 and 31
days after flowering. The mean time from Agrobacterium treatment to planting into soil averaged 186 days. Based on the initial number of seeds used, the transformation frequency was 0.6 i.e. six independent transgenic events per 1000 axes sliced. The
inserted genes were functional and inherited in a Mendelian fashion. Although more plants were recovered by selection on chlorsulfuron, GUS activity was generally greater in plants selected on kanamycin. GUS activity in the leaves of the original
plants varied, but GUS activity in the second generation was correlated with that of the original transformants. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Plant transformation; Agrobacterium tumefaciens; Pea Pisum sati6um; Genotype independence; GUS b-glucuronidase www.elsevier.comlocateplantsci
1. Introduction
Grain legumes are important world-wide, as a source of human and animal food and an impor-
tant component in crop rotations. The demand for these crops is expanding but they are susceptible
to weed competition and pathogen attack. There- fore, there is widespread interest in incorporating
novel genetic traits, such as herbicide tolerance and disease and insect resistance, which can often
be introduced only via genetic transformation technology. The application of plant biotechnol-
ogy to crop improvement has developed rapidly and transgenic ‘genetically engineered’ cultivars
are available. However, the potential benefits of transgenics are not yet available for pulse crop
improvement. Like many other grain legumes, peas are recalcitrant to regeneration in vitro.
Moreover, regeneration of peas from transformed tissue culture explants has been reported to pro-
duce such genetic abnormalities as polyploidy and loss of introduced traits [1], chimaeras and escapes
[2] and infertility [3].
In 1993, scientists in the Division of Plant In- dustry, at the Commonwealth Scientific and In-
dustrial Research
Organization CSIRO,
Australia reported successful, reproducible trans- formation of peas using Agrobacterium and devel-
oping embryo
axes of
two pea
genotypes, Greenfeast and Rondo [4]. Since 1995, the Legume
Biotechnology Group of the Plant Biotechnology Institute has been producing transgenic pea plants
NRCC c 43778. Corresponding author. Tel.: + 1-306-9755584; fax: + 1-306-
9754839. E-mail address
:
patricia.polowicknrc.ca P.L. Polowick
1
Present Address: Aventis Cropscience c 203 – 407 Downey Road, Saskatoon, Sask., Canada S7N 4L8.
0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 6 7 - 8
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
