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Agriculture
Canada

uanadian Agriculture LiDrary
Bibliotheque canadienne de I'agriculture
Ottawa K1 A 0C5

2-7

Research Branch
Technical Bulletin 1993-7E

Wheat

transformation

technologies

Canada

Cover illustration

The images represent the Research Branch's objective:

to improve the long-term competitiveness of the Canadian

agri-food sector through the development and transfer of new

technologies.

Illustration de la couverture

Les dessins illustrent I'objectif de la Direction generate de la
recherche : ameliorer la competitivite a long terme du secteur
agro-alimentaire canadien grace a la mise au point et au transfer!

de nouvclles technologies.

Conception par le Service awe programmes de recherches.

Wheat

transformation

technologies

JOHN SIMMONDS and DAINA SIMMONDS

Plant Research Centre

Agriculture Canada

Ottawa, Ontario

K1A0C6

Technical Bulletin 1993-7E

Research Branch

Agriculture Canada

1993

Copies of this publication are available from

Director

Plant Research Centre

Research Branch, Agriculture Canada

Central Experimental Farm

K.W. Neatby Building

Ottawa, Ont. K1A0C6

Produced by Plant Research Centre

CONTENTS

Summary/ Resume iv

INTRODUCTION 1

AGROBACTERIUM-MEDIATED TRANSFORMATION 3

DIRECT DNA TRANSFER AND PROTOPLASTS 5

IMBIBITION OF DNA ACROSS THE CELL WALL 6

DIRECT DNA TRANSFER INTO CELLS THROUGH

RUPTURED CELL WALLS \ . . 8

CONCLUSIONS 11

REFERENCES 13

1 1 1

SUMMARY

Rapidly developing technologies in plant molecular biology and cell biology
have created the possibility of introducing novel genes into crop plants in order
to affect specific agronomic traits. Wheat is one of the most important food
crops in the world and it can be anticipated that genetic engineering to affect
quality, productivity or sustainabil ity will have a significant impact on
agricultural economies. The development of transformation systems for the
graminaceous cereals has been particularly difficult because Agrobacterium-
mediated gene transfer is not applicable. This review described progress with
methods of direct DNA delivery to cereals; evaluates the evidence for claims of
integration of foreign genes into the plant genome and discusses the potential
of developing tissue culture independent transformation systems to eliminate
somaclonal damage and to provide genotype independent technology applicable
directly to elite cultivars.

RESUME

La progression rapide des techniques de biologie moleculaire et cellulaire
vegetale ouvre la voie a 1 ' introduction, chez des productions vegetales, de genes
nouveaux qui modifient des carateres agronomiques particul iers. Le ble est l'une
des cultures vivrieres les plus importantes au monde. On prevoit que les
manipulations de genie genetique, qui modifient la qualite, la productivity et
la durability, auront un impact considerable sur les economies agricoles. La
mise au point de systemes de transformation chez les cereales de la famille des
graminees a ete particul ierement difficile, parce que, dans ce cas particulier,
Agrobacterium ne peut servir a transferer des genes. Le present document decrit
les progres realises dans les methodes d' introduction direct d'ADN dans des
cereales, evalue les donnees etayant les allegations d' integration de genes
etrangers au genome vegetal et examine les possibilites de mettre au point des
systemes de transformation n'ayant pas recours aux cultures de tissus, afin
d'eliminer les dommages somaclonaux et d'utiliser une technique sans lien avec
le genotype, applicable directement aux cultivars elites.

INTRODUCTION

Wheat with an annual world production on 226 million hectares of about 550
billion tonnes valued at nearly $80 billion, is clearly one of the most important
food crops in the world. Furthermore improvement in quality, productivity, or
sustainability of such a major source of nutrition can be expected to have a
significant impact on agriculture.

During the past few decades, advances in plant cell culture and recombinant
DNA technology have created the potential to transfer genes from any organism
into plant cells. In principle, a gene influencing a single trait can be
isolated and transferred to a superior cultivar without concomitant transfer of
undesirable traits, which normally occurs using classical breeding methods. As
such, this technology would complement and enhance the efficiency of wheat
breeding.

The most successful procedures for gene transfer into plants have been
based on Agrobacterium-mediated gene delivery. Agrobacterium tumefaciens and A.
rhizogenes are pathogenic bacteria that have evolved the capacity of delivering
DNA from their Ti or Ri plasmids, respectively, into cells of a wide variety of
dicotyledonous and some monocotyledonous plants. Incorporation of the
transferred DNA (T-DNA) into the nuclear genome of the plant cell and its
resultant expression causes a pathogenic response that includes tumor formation.
To take advantage of nature's way of putting foreign genes into plant cells,
molecular biologists have disarmed the Ti plasmid by removing the genes
responsible for the tumorigenic response. It was then possible to incorporate

any desirable gene into the T-DNA, which could then be transferred to plant cells
and integrated into the host genome. Provided that these transformed cells can
be induced to regenerate into plants, novel transgenic plants can be produced.
While the Ti-based system is currently the method of choice for delivering DNA
into many plants, the limited host range of Agrobacterium infection precludes its
use in a large number of species, in particular the graminaceous cereals.

To overcome this host range limitation, numerous procedures have been
developed for delivering DNA directly into plant cells, i.e., without a
biological vector. These procedures include,

• imbibing cells or tissues in DNA solutions,

• transferring DNA into protoplasts by destabilizing the plasmamembrane
either with polyethylene glycol (Armstrong et al . 1990) or by
electroporation (Fromm et al . 1986),

• rupturing the cell wall with laser beams, microprojectile bombardment, or
by microinjection to overcome the cell wall barrier.

To date, the most promising transformation systems for wheat are by bombardment
of DNA-coated microprojectiles into embryogenic callus cultures (Vasil et al .
1992) or by microinjection of DNA into apical meristem cells, which subsequently
develop into pollen or ovules (Simmonds et al . 1992).

The development of transformation systems for the graminaceous cereals
remains fraught with problems (Potrykus 1989). Numerous transformation systems
have been attempted with wheat and some have yielded potentially encouraging
results. However, in many studies claims of transformation are ambiguous because
strict criteria for proof of integration of the foreign gene were not applied

(Potrykus 1991). Proof of integration must include the following criteria:

• controls for treatment and analysis

• good correlation between physical (e.g., Southern blot) and phenotypic
(e.g., enzyme assay) data

t complete Southern analysis containing

(a) the predicted signals in high molecular weight DNA

(b) hybrid fragments containing host DNA and the foreign gene

(d) controls showing evidence of the absence of contaminating DNA
fragments

• molecular and genetic analysis of the offspring populations.

In this review the various approaches to transforming wheat are examined and
evaluated with respect to these criteria.

AGROBACTERIUM-MEDIATED TRANSFORMATION

Although the graminaceous cereals show limited susceptibility to
Agrobacterium encouraging progress with this method has been made by the
demonstration of agroinfection of maize with cloned DNA of maize streak gemini
virus (Grimsley et al . 1987) and wheat with wheat dwarf gemini virus DNA (Hayes
et al . 1988). Agroinfection is defined as the introduction of plant infectious
agents via Agrobacterium and is limited to those cases involving molecules that
can replicate independently of the plant chromosomal DNA. Agroinfection of
cereals is an important discovery as it provides evidence that Agrobacterium can

delivered DNA can be integrated into the plant genome of cereals. Mature rice
embryos, inoculated with a supervirulent strain of Agrobacterium, formed
tumorigenic callus tissue. A disarmed strain carrying genes for 6-glucoronidase
(GUS) activity and kanamycin resistance produced kanamycin resistant callus
showing GUS activity. Molecular evidence indicated that T-DNA had been
integrated into the rice genome (Raineri et al . 1990). Similarly, molecular
analyses of wheat plants produced from florets inoculated with Agrobacterium just
prior to anthesis suggested that T-DNA had been incorporated into the plant
genome (Hess et al . 1990). However, without any evidence from progeny analyses
indicating that the transgenes are heritable, such claims should be treated with
caution. A critical assessment of Agrobacteri urn-mediated transformation of wheat
(Langridge et al . 1992) provided only superficial evidence of DNA transfer from
the bacterium to the plant. Embryos derived from florets inoculated with
Agrobacterium carrying an antibiotic resistant gene showed elevated resistance
to the antibiotic. Southern hybridization analysis of resistant plants showed
positive signals. However, identical banding patterns were seen for every
putative transformant. The generation of only one pattern of DNA fragments
implies that the foreign DNA had inserted at the identical site in each
transformation, a most improbable event. Furthermore, the progeny of these
putative transformants were negative in Southern analysis. In conclusion,
Agrobacter i u/n-mediated transformation has not been successful in wheat.
Nevertheless, DNA has been transferred to the plant and has persisted for one
generation. A possible explanation is that T-DNA was transferred to an
endophytic bacterium present at the time of inoculation.

DIRECT DNA TRANSFER AND PROTOPLASTS

The cell wall, a physical and chemical barrier, can be removed by enzymatic
digestion to produce viable protoplasts (Kao et al . 1971). Membranes can then
be permiabilized with polyethylene glycol (Armstrong et al . 1990) or by
electroporation (Fromm et al . 1986) to allow DNA uptake by protoplasts. These
procedures have been used initially with Triticum monococcum protoplasts (Lorz
et al . 1985) and subsequently with T. aestivum (Oard et al . 1989) to identify
constructs most suitable for wheat transformation studies. Transient expression
assays using chloramphenicol acetyl transferase (CAT) and neomycin
phosphotransferase II (NPT II) in T. monococcum protoplasts showed that the
kinetics of delivery of DNA were. similar to those for dicot protoplasts but the
expression was generally 10-100 times lower in monocots (Hauptmann et al . 1988).
It was also demonstrated that the CaMV 35s promoter gave higher levels of
expression that nopal ine synthase promoter. Subsequently, 35s-NPT II fusions
were used to generate stable transformants of T. monococcum nonregenerable calli
(Hauptmann et al . 1988). An important advance was made with the demonstration
that the inclusion of an ADH 1 intron in gene fusions significantly increased
gene expression in maize (Call is et al . 1987). Inclusion of an intron near the
5' end of the mRNA increased the expression of CAT, NPT II, and firefly
luciferase coding regions by 200-fold. The importance of including introns in
gene constructs for wheat transformation was established by the demonstration
that the inclusion of a maize intron in a 35s-CAT construct gave a 185-fold
increase in activity in T. aestivum protoplasts (Oard et al . 1989). Similarly,
T. monococcum and T. aestivum protoplasts were used to show that the maize ADH
1 promoter gave higher expression than the CaMV 35s promoter. Inclusion of the

ADH 1 intron and various enhancer elements with the ADH promoter (pEmu)
consistently produced 40-fold more GUS activity in wheat cells than did a similar
35s fusion construct (Last et al . 1991).

The generation of transgenic wheat from protoplasts remains elusive because
of the difficulty of obtaining regenerable cultures. Harris et al . (1988)
regenerated plants from protoplasts of an anther-derived cell suspension, but
they did not survive to maturity. Plants have been regenerated from protoplasts
isolated from suspension cultures of immature embryo callus (Vasil et al . 1990,
Wang and Nguyen 1990, Chang et al . 1991). These plants showed a considerable
degree of somaclonal variation resulting in arrested development and both male
and female sterility. Nevertheless, nonregenerable cultures can be exploited for
gene expression studies and for developing selection protocols (Simmonds and
Grainger 1993). In view of major cytological problems associated with wheat
protoplast cultures (Karp et al . 1987), a plethora of alternative methods to
deliver DNA into cells or tissues were explored.

IMBIBITION OF DNA ACROSS THE CELL WALL

Pollen-mediated transformation resulting from the incubation of mature
pollen or of germinating pollen in DNA solutions prior to pollination has been
investigated in a number of laboratories. Genomic DNA (Ohta 1986) or constructs
with reporter genes have been used with maize pollen (Booy et al . 1989) and with
wheat pollen (Picard et al . 1988), but despite claims of integrative
transformation (De Wet et al . 1985), definitive proof has not been established.

It would be expected that nuclease activity associated with germinating pollen
would be a major problem with this approach. Complete degradation of large
amounts of plasmid DNA by nucleases released from germinating maize pollen could
be demonstrated within minutes of mixing the DNA and pollen (Booy et al . 1989).

The transfer of DNA into the zygote through pollen tubes in cut pollinated
pistils was claimed to be an effective transformation system for rice (Luo and
Wu 1988) and wheat (Picard et al . 1988) but the molecular evidence for
transformation was ambiguous. Although this approach appears to be very simple
and direct, it has not been reproducible in other laboratories. The presence of
callose plugs and nuclease activity in the pollen tube are some problems
associated with this technique.

A simple procedure of soaking isolated dry embryos of wheat, barley, rye,
triticale, oat, and maize in DNA solutions resulted in the transient expression
of reporter genes (Topfer et al . 1989), but there was no molecular evidence of
stable integration into the plant genome. A modification of this approach, in
which an attempt was made to electrophoretically transfer DNA into germinating
barley seeds also failed to demonstrate integrative transformation (Ahokas 1989).

Electroporation of DNA into leaf-base segments of rice, maize, barley, and
wheat was used to obtain transient gene expression (Dekeyser et al . 1990).
Electroporation of immature zygotic embryos and embryogenic callus cultures of
maize produced transgenic plants. Integration of the foreign DNA into the maize
genome was clearly demonstrated and the gene was transmitted to the progeny of
the transformants in a Mendel ian fashion. The transgenic maize were not

chimeric, which suggested that regeneration had occurred from single transformed
cells. The application of this procedure to wheat requires a regeneration system
from single cells of the superficial cell layer. To date, no such system is
available. Macroinjection, a process that involves the injection of DNA through
large needles to deliver DNA into extracelluar spaces, has been proposed as a
transformation procedure. DNA would then have to move across the cell wall into
intact cells. Attempts to macroinject ovules and embryos have failed, but
success was reported with a floral meristem system. A reporter gene, when
injected into the stem below the developing spike of rye, 14 days prior to
meiosis, was subsequently expressed in selected offspring (De la Pena et al .
1987). Unfortunately, numerous large-scale experiments using this technique with
barley, maize, rice, rye, and wheat have failed to produce transgenic plants.

Transformation procedures that require DNA solutions to pass through the
cell wall are limited because DNA will bind readily to cell walls and because of
the presence DNA degrading enzymes. The plasmamembrane will also remain a very
effective barrier unless disrupted chemically or electrically.

DIRECT DNA TRANSFER INTO CELLS THROUGH RUPTURED CELL WALLS

Laser beams can be focused to dimensions of less than 1 /xm and used to cut
holes in plant cell walls and membranes. DNA uptake through the micropuncture
can be facilitated along an osmotic gradient by placing the recipient cells in
hypertonic buffer. Transient expression of reporter genes has been demonstrated
(Weber et al . 1990). This technology is relatively new and its potential for

producing transgenic cereals cannot be assessed at this time.

Silicon carbide fibers can facilitate the delivery of DNA into plant cells.
Transient expression of reporter genes was obtained after vortexing maize
suspension culture cells with DNA and silicon carbide fibers. Penetration of the
cells by the fibers was observed by scanning electron microscopy and it seems
likely that DNA adhering to the fibers could be carried into the cells.
Subsequently, stable transformed herbicide-resistant maize cell lines were
selected (Kaeppler et al . 1991). It appears that the application of this
technique to embryogenic cultures could provide a relatively simple procedure for
generating transgenic plants.

Another relatively new technology, using a biol istics process (Klein et al .
1987), has received considerable attention. This technique involves the
acceleration of high-density micron-sized particles to velocities sufficient to
penetrate through plant cell walls. DNA adhering to such microprojectiles is
carried into the cell and it is either released directly into the nucleus or is
transported there from the cytoplasm. In graminaceous cereals, transient
expression of genes has been reported in maize (Klein et al . 1988), rice (Wang
et al. 1988), barley (Kartha et al . 1989), and wheat (Oard et al . 1990). This
procedure is limited by the low frequencies of transient (10"3) and integrative
10"6) events. Nevertheless, the high-velocity microprojectile process has been
used to obtain stable transformed nonregenerable maize (Klein et al . 1989) and
wheat cells (Vasil et al . 1991). More significantly, this procedure has yielded
transformed embryogenic cultures of maize (Fromm et al . 1990) and wheat (Vasil
et al . 1992); in maize numerous fertile transgenic plants have been established

but in wheat only one male sterile plant was produced, which was rescued by
outcrossing to establish a transgenic line. These examples offer the most
encouraging approach to the transformation of cereals. However, it should be
recognized that the process relies on efficient regenerable tissue culture
systems; unfortunately tissue culture is genotype dependent. All the maize
transgenics were obtained from cell lines derived from the B73 x A188 hybrid
genotype, which have exceptionally high embryogenic responses in culture.
Similarly, the wheat line was selected for its high level of regeneration.

DNA can be delivered directly into plant cells by the process of
microinjection. Glass microcapillaries are used to mechanically deliver DNA
solutions into cells in such a way that the injected cell survives and
proliferates. Stable transformation frequencies as high as 20% have been
reported with tomato callus cells (Toyoda et al . 1988). However, microinjection
requires a high degree of technical skill and in comparison to particle
bombardment only relatively few cells can be handled. The power of
microinjection is in the ability to deliver DNA precisely into a cell of choice.
This transformation method could be exceptionally effective if DNA could be
targeted into gametes or their precursor cells so that transgenic plants could
be produced by normal zygotic embryogenesis. This method would eliminate the
need for genotype dependent regeneration and cell selection systems and would
avoid problems of somaclonal variation. Transformation of sperm or egg cells and
in vitro fertilization to generate transgenics by zygotic embryogenesis is
appealing, but the micromanipulation techniques for this method are still
preliminary (Kranz and Lorz 1990). Shoot apical meristems contain cell initials
that generate the floral cell lineages; the transformation of such cells could

result in the production of transformed gametes. Histological analysis of wheat
apical meristems showed that the floral meristems are initiated from cells in the
hypodermal (L2) layer (Sharmen 1983). When a cell in L2 divides, the orientation
of the spindle is normally parallel to the outer surface of the apex, thus
maintaining a single file of cells. This file of cells is generated by a few
apical initial cells, possibly only three (Fig. 1). Transformation of an L2
apical cell in a vegetative apex would establish a transgenic sectorial chimera,
which would contribute to the numerous floral meristems in the developing spike.
Vegetative apical meristems of wheat, dissected for micromanipulation so that the
LI and L2 layers can be viewed in bright-field microscopy (Fig. 2), will
regenerate phenotypically normal fertile plants (Simmonds et al . 1992).
Micropipettes could be inserted into cells of the L2 layer (Fig. 3) and
fluorescein isothiocyanate labelled dextran (MW 1700) was used as a marker to
develop microinjection technology for delivering DNA solutions into L2 cells
(Fig. 4). The feasibility of this approach for transformation of wheat was
demonstrated by the expression of reporter genes microinjected into apical cells
(Figs. 5,6). The continued development of these microinjected cells into
germline tissues offers the prospect of a tissue culture and genotype-independent
transformation system that will have universal application.

CONCLUSIONS

The fundamental problem in the production of transgenic wheat lies not so
much in the delivery or even in the integration or expression of the foreign DNA,
but rather in the recognition of cells that can regenerate phenotypically normal

fertile plants, and the ability to target DNA into these cells. Gametic tissues
(pollen and ovules) are obvious targets and regenerable somatic tissues
(embryogenic callus, Magnusson and Bornman 1985, or leaf-base tissue, Wernickle
and Milkovits 1984) also have potential. Shoot apical meristems contain cells
that generate the cell lineages of germ-like tissues and consequently these cells
are also interesting targets for transformation studies.

Genetic transformation of wheat has been limited by the lack of an
Agrobacteri urn-mediated gene transfer system. Evidence that Agrobacterium can
transfer DNA into wheat cells is promising, especially if mechanisms controlling
the integration of T-DNA are discovered. Recent improvements in the technology
of delivering genes into cells by microprojectile bombardment and microinjection,
and the availability of gene constructs optimized for expression in monocots,
e.g., monocot promoters and introns, have resulted in significant progress in
cereal transformation. Cell culture technology remains the major obstacle to
routine transformation of cereals, but increased efforts to target DNA into germ-
line cells will eliminate tissue culture alterations and provide a genotype-
independent means of transformation.

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Figs. 1-6. Wheat apical moristem transformation system,
apical domes is 100 jum.)

(The mean diameter of

Fig. 1. Optical sectionof a differential interference contrast micrograph shows
the cell layers of the vegetative apex of wheat which are targets for DNA
delivery. Apical initial cells {arrowheads) generate the L2 cell linkage from
which floral meristems are derived. Note cell divisions in the L2 layer and
central region of the apex {arrows).

Fig. 2. Bright-field micrograph of wheat apex with the micropipette positioned
for injecting. Note that the LI and L2 cell layers can be distinguished.

Fig. 3. Micrograph shows the micropipette inserted into an apical initial of the
L2 cell layer.

Fig. 4. A combination bright-field and fluorescence microscopy was used to show
the successful delivery of a fluorescent dextran marker co-injected with DNA
solution into both an LI and L2 {arrow) cells.

Fig. 5. Micrograph shows the wheat apex following the destructive histochemical
assay for expression of B-glucuronidase (GUS) activity 48 h after injection with
the GUS gene. The blue cells are evidence of foreign gene activity.

Fig. 6. Micrograph of a live wheat apical meristem showing red cells, a non-
destructive assay for expression of anthocyanin. Corn genes, which regulate the
biosynthesis of anthocyanin pigment, were injected into wheat apical meristem
cells. Three days later these cells had produced the expected pigment {arrow).

BIBVIOTHtQVJE

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