Download Genetic engineering of cereal crop plants: a review

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Euphytica 85: 3 5-44,1995 .
© 1995 Kluwer Academic Publishers . Printed in the Netherlands .
Genetic engineering of cereal crop plants : a review
A. Jahne, D. Becker & H. Lorz
Institut ftir Allgemeine Botanik, Zentrum ftir angewandte Molekularbiologie der Pflanzen, AMP II, Ohnhorststr .
18, D-22609 Hamburg, Germany
Key words : cereals, protoplast transformation, tissue electroporation, particle bombardment
Summary
Many aspects of basic and applied problems in plant biology can be investigated by transformation techniques . In
dicotyledonous species, the ability to generate transgenic plants provides the tools for an understanding of plant
gene function and regulation as well as for the directed transfer of genes of agronomic interest .
For many dicotyledonous plants Agrobacterium tumefaciens can be routinely used to introduce foreign DNA
into their genome . However, cereals seem to be recalcitrant to Agrobacterium-mediated transformation .
In cereals, many efforts have been made in recent years to establish reliable transformation techniques . Several
transformation techniques have been developed but to date only three methods have been found to be suitable for
obtaining transgenic cereals : transformation of totipotent protoplasts, particle bombardment of regenerable tissues
and, more recently, tissue electroporation. The current state of transformation methods used for cereals will be
reviewed .
Introduction
The transfer of defined genes is theoretically the
most straightforward method for improvement of crop
plants. Methods for crop plant transformation have
only been developed in recent years . Generally the
method of choice for the delivery of genes to dicotyledonous species is the use of Agrobacterium tumefaciens . Transformed plants are being obtained for an
increasing number of species including agronomicallyimportant crops (Hooykaas & Schilperoort, 1992) . The
Agrobacterium vector system is not only used extensively for the transfer of various traits to crop plants, but
also for the study of gene function in plants . Applications include the transfer of genes affecting such widely
diverse traits as resistance to pests, diseases or herbicides and tolerance to environmental stress. The transfer of genes in order to modify metabolic pathways
to change the quality of plant products for industrial
purposes is also an important goal . Some of the transgenic crops produced are now ready for marketing, and
thus transformation techniques will supplement classical breeding methods.
Cereals, as a major group of crop plants, are important targets for the application of genetic manipulation
techniques . Unfortunately, most monocotyledons are
not among the natural hosts of Agrobacteria . Only
members of the orders Liliales and Arales have proved
to be susceptible ; all members of the Poales tested have
shown to be nonsusceptible (De Cleene, 1985) . Consequently, the prospects for successful genetic engineering of cereals utilising Agrobacterium would not
seem to be very promising . Nevertheless, there is evidence that under certain conditions an Agrobacteriummediated gene transfer to some monocotyledonous
species is possible (Bytebier et al ., 1987 ; Raineri et
al ., 1990 ; Gould et al ., 1991 ; Mooney et al ., 1991 ;
Li et al ., 1992) . However, the regeneration of stablytransformed plants and the inheritance of the transferred gene has been discussed more controversially
by Langridge et al . (1992) .
Furthermore, several groups have reported the
phenomenon of 'agroinfection' where viral genomic
sequences have been transferred to cereal meristematic cells resulting in systemic viral infection in the
recovered plants (Hohn et al ., 1987 ; Grimsley et al .,
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Table 1 . Methods investigated for gene transfer to cereals (for
review see Potrykus, 1990)
Chemically induced DNA uptake into protoplasts
Electrically induced DNA uptake into protoplasts
Bombardment of cells and tissues with DNA-coated particles
Electroporation of tissues with DNA
Macroinjection of DNA into floral tillers
Microinjection of DNA into microspores, microspore-derived
cells and tissues with DNA-coated particles
- Electroporation of tissues with DNA
Macroinjection of DNA into floral tillers
Microinjection of DNA into microspores, microspore-derived
pro-embryos or zygotic pro-embryos
DNA-uptake by germinating pollen
Imbibition of embryos with DNA
1987 ; Dale et al ., 1989) . As cereals cannot be readily transformed by Agrobacterium research activities
have focussed on the development of alternative gene
transfer methods . Various techniques have been tested
(Table 1) but presently only three methods have proved
to be suitable for obtaining transgenic cereals : tissue
electroporation, transformation of protoplasts and particle bombardment of regenerable tissue cultures .
Tissue electroporation
One successful method is the delivery of DNA to regenerable tissues by electroporation . Tissue electroporation has been used to transfer DNA to enzymaticallyor mechanically-wounded zygotic or somatic maize
embryos . Transgenic plants have been obtained
reproducibly and the transferred neo-gene segregated
according to Mendelian rules (D'Halluin et al ., 1992) .
Although this is a very recent development, the future
prospects of this technique are very promising since
there has been a further report showing successful gene
transfer to scutellum cells of wheat by transient expression experiments (Kloti et al ., 1993) .
Transformation of cereal protoplasts
Another technique which has reproducibly yielded
transgenic cereals is DNA uptake by protoplasts, stimulated either by PEG treatment or induced by electroporation. For a long time, cereals seemed to be
recalcitrant in tissue and especially in protoplast culture . However, considerable progress has been made in
establishing reliable and efficient in vitro culture systems . In many dicotyledonous species, plants can be
regenerated from mesophyll protoplasts, but in cereals
there is only scant evidence that protoplasts isolated
from leaves are capable of sustained divisions (Hahne
et al ., 1990) . There is, however, a recent report of plant
regeneration from mesophyll protoplasts of rice (Gupta
& Pattanayak, 1993) .
So far, embryogenic suspension cultures are the
main source of totipotent cereal protoplasts . Embryogenic suspensions originating either from immature
embryos or from microspores, have been generated for
nearly all important cereals (Vasil & Vasil, 1992) . The
establishment of embryogenic suspensions suitable for
the release of protoplasts has been an important prerequisite for the progress achieved in cereal protoplast
research . Nevertheless, it is very difficult and labourintensive to initiate and maintain these suspensions .
Furthermore, regeneration capacity has been observed
to decline gradually during cultivation in cereal suspensions (Jahne et al ., 1991 a) . Therefore the long-term
availability of embryogenic suspensions is a limiting
factor in protoplast research . A solution to this problem could be the cryopreservation of suspension cells .
Cryopreserved maize suspensions have been shown to
provide a long-term and reliable source of totipotent
cells (Shillitoet al ., 1989) . However, efficient freezing
protocols are not available for all cereal cell suspension cultures and considerable time and effort must be
spent for the establishment of novel suspension cultures (Fretz et al ., 1992) .
Embryogenic suspension cultures have been used
for the isolation of totipotent protoplasts and regeneration of plants from these single cells is possible for most
important cereals such as rice, maize, wheat and barley (Table 2) . However, plant regeneration from cereal
protoplasts remains a difficult and often unreliable process, depending on many parameters not under experimental control (Potrykus, 1989) . Protoplast regeneration is currently an efficient' and routinely-used method
only in rice and in specific genotypes of maize .
The use of protoplasts in genetic engineering has
very significant applications not only for stable transformation . For several types of experiment such as the
analysis of promotor function and gene expression, it
is possible to use protoplasts for transient expression
studies .
Protoplasts can be induced to take up DNA either by
PEG or by electric pulses . Both methods have proved
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Table 2 . Regeneration and transformation of protoplasts of cereal
crops
Rice
Maize
Barley
Wheat
Regeneration
of fertile plants
Transgenic plants
Abdullah et al., 1986
Toriyama et al., 1986
Kyozuka et al ., 1987
Toriyama et al., 1988
Kyozuka et al ., 1988
Datta et al ., 1990
Shimamoto et al ., 1989
Datta et al., 1990
Li & Murai, 1990
Datta et al ., 1992
Tada et al., 1990
Terada et al ., 1993
Rathore et al., 1993
Prioli & Sondahl, 1989
Shillito et al ., 1989
Morocz et al ., 1990
Rhodes et al ., 1988
Donn et al ., 1992
Golovkin et al., 1993
Donn et al ., 1992
Omirulleh et al ., 1993
Zhang et al ., 1992
Zhang & Wu, 1988
Jahne et al., 1991b
Funatsuki et al ., 1992
Golds et al ., 1993
Ahmed & Sagi, 1993
to be suitable for stable transformation of cereal protoplasts and transformed cell lines could be obtained
(Fromm et al ., 1986; Rhodes et al ., 1988 ; Lazzeri
et al ., 1991) . Although direct DNA-uptake is a successful and routinely-used method, the regeneration of
transgenic plants remains difficult. Until now, it has
only been possible to obtain transgenic plants by protoplast transformation in rice and maize, from which
protoplast-derived fertile plants can be obtained reproducibly (Table 2) . This underlines that the regeneration
of protoplast-derived plants still remains a significant
limiting factor in obtaining transgenic cereals .
In both maize and rice it has now been shown
that the transferred genes are inherited by the progeny and are as stable as original plant genes . Furthermore, direct DNA-uptake into protoplasts provides the
possibility of co-transformation and thus the recovery
of transgenic plants without selectable marker genes
can be achieved by subsequent conventional breeding
methods .
Particle bombardment
Embryogenic cell suspensions represent not only a
source of totipotent protoplasts, but can also be used
Table3 . Biolistic transformation of cereals using
different target tissues
Embryogenic cell suspension cultures
Rice
Cao et al., 1992
Maize
Oat
Gordon-Kamm et al., 1990
Fromm et al ., 1990
Somerset al ., 1992
Embryogenic callus cultures
Maize
Genovesi et al., 1992
Wheat
Walters et al ., 1992
Vasil et al ., 1992
Barley
Wan & Lemaux, 1993
Sugarcane
Bower & Birch, 1992
Primary explants
Rice
Maize
Wheat
Barley
Tritordeum
Triticale
Christou et al., 1991
Koziel et al ., 1993
Weeks et al ., 1993
Becker et al., 1994
Wan & Lemaux, 1994
Ritala et al ., 1994
Jahne et al., 1994
Barcelo et al ., 1994
Zimny et al., 1995
as target cells for an alternative successful transformation technique: particle bombardment . The particle
bombardment process is a method for the delivery of
genes into intact cells and tissues through the use of
DNA-coated microprojectiles (tungsten or gold) . It was
developed by Sanford & co-workers (1987) and has
become the second most widely-used method for plant
genetic transformation after Agrobacterium-mediated
gene transfer (Gray & Finer, 1993) . Several laboratories have demonstrated that microprojectiles are
suitable for the transfer of genes to a wide range of
plant tissues and species. Apparently, there is no difference in the efficiency of biolistic transformation of
monocotyledonous and dicotyledonous species (Sanford, 1990) .
For the transformation of cereals, the choice of
appropriate target tissue is of major importance as
there are only a few tissues capable of plant regeneration . Regenerable tissues of cereals have been tested
by transient expression assays and have proved to be
suitable for biolistic transformation experiments (Table
3) . The most common tissues used for this purpose are
embryogenic suspension cells and embryogenic callus
cultures. For the stable transformation and regeneration of maize (Gordon-Kamm et al ., 1990 ; Fromm et
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al ., 1990), rice (Cao et al ., 1992) and oat (Somers et
al ., 1992) suspension cells have been used as target tissue . However, the morphogenetic competence of cells
is significantly reduced during maintenance and the
phenomenon of somaclonal variation limits the suitability of this tissue . Accordingly, embryogenic callus
has been considered as a target tissue because the time
needed for establishment of cultures and plant regeneration is shorter for callus cultures than for suspension cultures . Using callus cultures, it was possible
to regenerate transgenic sugarcane (Bower & Birch,
1992), wheat (Vasil et al ., 1992) maize (Genovesi et
al ., 1992 ; Walters et al ., 1992) and barley (Wan &
Lemaux, 1993) .
Although these cultures could be successfully used
for the production of transgenic cereals, it would be
more desirable to deliver DNA directly into primary
explants with a high regeneration capacity. The time
necessary for the preparation of the target tissue is
comparatively low and the risk of somaclonal variation is reduced as the period in culture is shortened to a
few weeks . In cereals, scutellar tissues of rice (Christou et al ., 1991), maize (Koziel et al ., 1993) and wheat
(Weeks et al ., 1993) have been used for the regeneration
of stably-transformed plants . In the following section,
the progress made in our laboratory towards biolistic transformation of cereals using various explants is
summarized .
Strategies for the biolistic transformation of
primary explants
A prerequisite for the production of transgenic cereals has been the development of efficient in vitro culture systems from which fertile plants can be regenerated at a high frequency . In our experiments the
explants of choice have been the scutellar tissue of
immature embryos of wheat and triticale, immature
inflorescences of Tritordeum (Barcelo et al ., 1989)
and barley microspores . Currently, the culture of barley microspores is considered superior to anther culture as the regeneration frequency can be significantly
increased (Olsen, 1991 ; Hoekstra et al ., 1993 ; Mordhorst & Lorz, 1993) . In barley, a spontaneous autoendoreduplication of the genome during the first division
of the microspore leads to homozygous, diploid regenerants. Therefore, this haploid target tissue makes the
regeneration of homozygous Ro-plants possible .
Each target tissue has been the subject of individual optimization experiments to improve conditions for
particle bombardment . The most convenient method
to measure the efficiency of DNA delivery into intact
cells, is the determination of the number of cells which
transiently express the uidA-gene (,Q-glucuronidase) .
Because of the relatively low sensitivity of the histochemical GUS-assay, the use of a strong promoter
which enhances the expression of the marker gene is
important . A construct harbouring the uidA-gene driven by an Act-1-D-promoter (McElroy et al ., 1990)
provided best results in all tissues .
For optimization of the bombardment process and
for selection of stably-transformed plants, a plasmid
(pDB 1 ; Fig . 1) containing the visualizable marker gene
uidA under the control of the Actin I promoter from
rice, and the selectable marker gene bar under the
control of the CaMV 35S promoter has been used .
For Tritordeum the method of co-transformation
using the plasmids pAct-1-D/GUS and pCalneo has
been successfully demonstrated (S . Lutticke, unpublished) .
The aim of these optimization studies is to achieve a
high frequency of transiently expressing cells . However, it is also very important that the target tissue does not
suffer significantly from the bombardment . The degree
of tissue damage depends on the type of explant, the
particle density and the acceleration pressure .
Immature embryos of wheat and triticale have
proved to be the most sensitive tissue . In wheat, high
particle densities (116 u
j g gold particles average size
0,4-1,2 pm) per bombardment caused severe tissue
damage, whereas Tritordeum inflorescences were not
reduced in their morphogenetic competence under the
same conditions. The viability of barley microspores
has not been influenced by the amount of particles used
for bombardment in our studies .
The results from our optimization and selection
experiments demonstrate that the optimal particle density has to be determined carefully for each type of
explant, whereas the acceleration pressure can be relatively wide-ranging without having a negative effect
on tissue viability and competence .
Strategies for the improvement of the biolistic
method not only concern the parameters of the particle bombardment process, but also the culture conditions for explants . In spring varieties of wheat and in
triticale, a clear reduction of tissue damage has been
achieved when the immature embryos were precultured in vitro for two to seven days . In Tritordeum, a
preculture of one day led to an increase in the number
of transformed plants, a finding which reinforces the
importance of the determination of optimal preculture
39
Sa
Acil
S
gus
1KB
Fig.] . Schematic representation of plasmid pDB 1 used in transformation experiments : S : Sal I ; Sa : SacI ; B : BamHl ; N: Ncol.
conditions (Barcelo et al ., 1994) . In barley, no effect
on transient expression of the 0-glucuronidase gene or
on viability has been observed when freshly-isolated
or one- to three-day old microspores were bombarded .
Under optimal culture and bombardment conditions,
an average number of 100 transient GUS-signals per
embryo has been counted in wheat and triticale (Fig .
2A) . In barley, about 1 % of the bombarded mirospores
transiently expressed the uidA-gene (Fig . 2B) .
The establishment of an efficient transformation
system requires careful choice of an appropriate
selectable marker gene . For grasses, antibiotics such
as hygromycin, kanamycin of G-418 have been used
successfully. In comparison, the major advantage of
herbicide selection is that plants can be selected in late
stages of development by a simple spray test .
For wheat, triticale and barley, phosphinothricin
(PPT) resistance conferred by the bar gene has proved
to be a useful selectable marker for the regeneration of
transformed plants . The successful use of PPT selection depends on the cell type and the developmental
stage. Therefore, the selection conditions were optimized individually. The optimal selection conditions
varied ; in wheat a PPT concentration of 0 .5-2 mg/I
was most suitable, whereas in barley 3-5 mg/1 PPT was
optimal . Two to three weeks after bombardment the
calli were transferred to selection medium . The selection pressure was applied both during callus induction
and the plant regeneration phase (Fig . 2C) . Furthermore, the regenerants were sprayed in a later stage of
development with a solution containing 150-200 mg/I
PPT in order to verify their resistance (Fig . 2D) . This
selection system represents a fast and highly-efficient
method for identifying transformed plants .
Using the pDB 1-construct (Fig . 1) it is also possible to screen non-selected regenerants by spraying with
PPT or by histochemically assaying the expression of
the uidA gene (Fig . 2G) . By this method several transgenic triticale (J . Zinmy, personal communication) and
one transformed barley plant have been identified .
The transformation efficiency depended on the
quality of the explant material and varied from experiment to experiment. In wheat, an average frequency of
one transgenic plant per 83 bombarded embryos could
be achieved (Becker et al ., 1994) . This frequency is
substantially higher than the 1-2 plants per 1000 bombarded embryos reported by Weeks et al . (1993) . In
barley, independent transformation events led to the
average recovery of one plant per 1 x 107 bombarded
microspores (Jahne et al ., 1994) . Southern analysis of
selected regenerants demonstrated that in most cases
plants contained intact copies of both marker genes
(Fig . 3) . Single copy as well as multi-copy integration
of one or both marker genes (uidAlbar or uidAlneo)
was detectable . Furthermore larger or smaller fragments were observed, suggesting that deletions, rearrangements and/or methylation at restriction sites had
occurred .
In our experiments, no phenotypic abnormalities or
reduced fertility (Fig . 2H) was observed. This has been
reported for transgenic maize (Gordon-Kamm et al .,
1990), wheat (Vasil et al ., 1992) and oat plants (Somers
et al ., 1992) obtained from microprojectile bombardment of embryogenic suspension or callus cultures.
The segregation of the introduced marker gene
uidA was visualized histochemically in pollen grains
of RO-plants (Fig . 2E and F) and in leaves of the progeny. The functional activity of the bar gene was tested
by spraying the progeny with an aqueous solution of
the herbicide Basta.
A 1 : 1 segregation as well as segregation of the
introduced marker gene in a non-Mendelian fashion
was observed in wheat, triticale and Tritordeum . In
barley, the introduced marker genes were inherited by
conditions using 5 mg/l PPT . D : Selection of a transformed barley plant by spraying an aqueous solution of 150 mg/l PPT ; left : sensitive control plant, right: resistant transgenic
plant.
Fig . 2 . 2A + B: Transient GUS activity in scutellar tissue of wheat (A) and in barley microspores (B) 48 h after bombardment . C: Regeneration of a barley plant under selection
8
in leaf tissue of transgenic wheat . H: Mature transgenic RO-plant (left) and a seed-derived wheat plant (right) .
Fig . 2 . 2E + F: GUS activity in pollen of transgenic RO-plants . A segregation of I : I in a wheat plant (E) and pollen of a homozygous barley plant (F) are shown . G: GUS activity
42
kb
PC
T
1/1
2/8
4/11
NC
2/6
U 1 2 U 1 2 U 1 2 U 1 2 U
2
8,0
4,8
-.-gus
1,8
Fig.3. Integration of the gus gene in four GUS positive Ro-plants of wheat (1 / 1, 2/6 .2/8 and 4/11). Southern blots of genomic DNA (25 µg/lane) .
Hybridization was carried out using a DIG- l 1-dUTP-labelled gus probe . NC: Negative wheat control, PC : Positive control ; plasmid pDB I
digested with BamHI and Sacl . U : Undigested . 1 : Digested with BamHI/SacI to cut out the gus gene . 2: Digested with Ncol to determine the
number of integration sites per genome.
all the progeny, indicating the homozygous genotype
of the transformed plants .
Progeny have been further analysed by Southern
hybridization. Wheat plants showing a 1 : 1 segregation
in pollen grains and a 3 : I inheritance of the marker
genes in the progeny, had the same integration pattern
as the parental line . This indicates a close linkage of
the introduced marker genes and their inheritance as
a genetic unit . However, individuals containing multicopy insertions did not always inherit the genes in a
Mendelian fashion .
genic suspension and callus cultures were used initially
but recent results show that primary explants seem to
be more advantageous for the routine production of fertile transgenic cereals . The recently-developed method
of tissue electroporation promises to be another very
attractive approach for the transformation of primary
explants . In summary, the tools of genetic manipulation
of cereals have been significantly improved, providing increased opportunities to transfer agronomicallyinteresting genes.
Note added in proof
Conclusion
Although the establishment of protoplast regeneration
and transformation systems has improved significantly, a routine combination of both systems is not yet
possible .
On the other hand, the development of biolistic
transformation systems has allowed rapid progress
towards the recovery of transgenic cereals . Embryo-
In the meantime several new reports on the transformation of cereals have been published . Most of these reports present transgenic
cereal plants obtained either by particle bombardment or by tissue
electroporation giving further evidence for the suitability of these
methods .
However, an unexpected publication was the one of Hiei et al .
1994 . The Plant J . 6 : 271-282, who reported the regeneration of
transgenic Japonica rice plants from scutellar tissues co-cultivated
with Agrobacterium tumefaciens.
43
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