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. LorzInstitut 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 . Indicotyledonous species, the ability to generate transgenic plants provides the tools for an understanding of plantgene 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 DNAinto 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. Severaltransformation techniques have been developed but to date only three methods have been found to be suitable forobtaining transgenic cereals : transformation of totipotent protoplasts, particle bombardment of regenerable tissuesand, more recently, tissue electroporation. The current state of transformation methods used for cereals will bereviewed .

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Introduction

The transfer of defined genes is theoretically themost straightforward method for improvement of cropplants. Methods for crop plant transformation haveonly been developed in recent years. Generally themethod of choice for the delivery of genes to dicotyle-donous species is the use of Agrobacterium tumefa-ciens . Transformed plants are being obtained for anincreasing number of species including agronomically-important crops (Hooykaas & Schilperoort, 1992) . TheAgrobacterium vector system is not only used exten-sively for the transfer of various traits to crop plants, butalso for the study of gene function in plants . Applica-tions include the transfer of genes affecting such widelydiverse traits as resistance to pests, diseases or herbi-cides and tolerance to environmental stress. The trans-fer of genes in order to modify metabolic pathwaysto change the quality of plant products for industrialpurposes is also an important goal . Some of the trans-genic crops produced are now ready for marketing, andthus transformation techniques will supplement classi-cal breeding methods.

Cereals, as a major group of crop plants, are impor-tant targets for the application of genetic manipulationtechniques. Unfortunately, most monocotyledons arenot among the natural hosts of Agrobacteria . Onlymembers of the orders Liliales and Arales have provedto be susceptible; all members of the Poales tested haveshown to be nonsusceptible (De Cleene, 1985) . Con-sequently, the prospects for successful genetic engi-neering of cereals utilising Agrobacterium would notseem to be very promising. Nevertheless, there is evi-dence that under certain conditions an Agrobacterium-mediated gene transfer to some monocotyledonousspecies is possible (Bytebier et al ., 1987 ; Raineri etal ., 1990 ; Gould et al ., 1991 ; Mooney et al ., 1991 ;Li et al ., 1992) . However, the regeneration of stably-transformed plants and the inheritance of the trans-ferred gene has been discussed more controversiallyby Langridge et al . (1992) .

Furthermore, several groups have reported thephenomenon of 'agroinfection' where viral genomicsequences have been transferred to cereal meristem-atic cells resulting in systemic viral infection in therecovered plants (Hohn et al ., 1987 ; Grimsley et al .,

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Table 1 . Methods investigated for gene transfer to cereals (forreview see Potrykus, 1990)

Chemically induced DNA uptake into protoplastsElectrically induced DNA uptake into protoplastsBombardment of cells and tissues with DNA-coated particlesElectroporation of tissues with DNAMacroinjection of DNA into floral tillersMicroinjection of DNA into microspores, microspore-derivedcells and tissues with DNA-coated particles

- Electroporation of tissues with DNAMacroinjection of DNA into floral tillersMicroinjection of DNA into microspores, microspore-derivedpro-embryos or zygotic pro-embryosDNA-uptake by germinating pollenImbibition of embryos with DNA

1987; Dale et al ., 1989). As cereals cannot be read-ily transformed by Agrobacterium research activitieshave focussed on the development of alternative genetransfer methods . Various techniques have been tested(Table 1) but presently only three methods have provedto be suitable for obtaining transgenic cereals : tissueelectroporation, transformation of protoplasts and par-ticle bombardment of regenerable tissue cultures .

Tissue electroporation

One successful method is the delivery of DNA to regen-erable tissues by electroporation . Tissue electropora-tion has been used to transfer DNA to enzymatically-or mechanically-wounded zygotic or somatic maizeembryos . Transgenic plants have been obtainedreproducibly and the transferred neo-gene segregatedaccording to Mendelian rules (D'Halluin et al ., 1992) .Although this is a very recent development, the futureprospects of this technique are very promising sincethere has been a further report showing successful genetransfer to scutellum cells of wheat by transient expres-sion experiments (Kloti et al ., 1993) .

Transformation of cereal protoplasts

Another technique which has reproducibly yieldedtransgenic cereals is DNA uptake by protoplasts, stim-ulated either by PEG treatment or induced by elec-troporation. For a long time, cereals seemed to be

recalcitrant in tissue and especially in protoplast cul-ture . However, considerable progress has been made inestablishing reliable and efficient in vitro culture sys-tems. In many dicotyledonous species, plants can beregenerated from mesophyll protoplasts, but in cerealsthere is only scant evidence that protoplasts isolatedfrom leaves are capable of sustained divisions (Hahneet al ., 1990) . There is, however, a recent report of plantregeneration from mesophyll protoplasts of rice (Gupta& Pattanayak, 1993) .

So far, embryogenic suspension cultures are themain source of totipotent cereal protoplasts . Embryo-genic suspensions originating either from immatureembryos or from microspores, have been generated fornearly all important cereals (Vasil & Vasil, 1992) . Theestablishment of embryogenic suspensions suitable forthe release of protoplasts has been an important pre-requisite for the progress achieved in cereal protoplastresearch. Nevertheless, it is very difficult and labour-intensive to initiate and maintain these suspensions .Furthermore, regeneration capacity has been observedto decline gradually during cultivation in cereal sus-pensions (Jahne et al ., 1991 a) . Therefore the long-termavailability of embryogenic suspensions is a limitingfactor in protoplast research . A solution to this prob-lem could be the cryopreservation of suspension cells .Cryopreserved maize suspensions have been shown toprovide a long-term and reliable source of totipotentcells (Shillitoet al ., 1989) . However, efficient freezingprotocols are not available for all cereal cell suspen-sion cultures and considerable time and effort must bespent for the establishment of novel suspension cul-tures (Fretz et al ., 1992) .

Embryogenic suspension cultures have been usedfor the isolation of totipotent protoplasts and regenera-tion of plants from these single cells is possible for mostimportant cereals such as rice, maize, wheat and bar-ley (Table 2) . However, plant regeneration from cerealprotoplasts remains a difficult and often unreliable pro-cess, depending on many parameters not under exper-imental control (Potrykus, 1989) . Protoplast regenera-tion is currently an efficient' and routinely-used methodonly in rice and in specific genotypes of maize .

The use of protoplasts in genetic engineering hasvery significant applications not only for stable trans-formation . For several types of experiment such as theanalysis of promotor function and gene expression, itis possible to use protoplasts for transient expressionstudies .

Protoplasts can be induced to take up DNA either byPEG or by electric pulses . Both methods have proved

to be suitable for stable transformation of cereal pro-toplasts and transformed cell lines could be obtained(Fromm et al ., 1986; Rhodes et al., 1988 ; Lazzeriet al., 1991). Although direct DNA-uptake is a suc-cessful and routinely-used method, the regeneration oftransgenic plants remains difficult. Until now, it hasonly been possible to obtain transgenic plants by pro-toplast transformation in rice and maize, from whichprotoplast-derived fertile plants can be obtained repro-ducibly (Table 2). This underlines that the regenerationof protoplast-derived plants still remains a significantlimiting factor in obtaining transgenic cereals .

In both maize and rice it has now been shownthat the transferred genes are inherited by the proge-ny and are as stable as original plant genes. Further-more, direct DNA-uptake into protoplasts provides thepossibility of co-transformation and thus the recoveryof transgenic plants without selectable marker genescan be achieved by subsequent conventional breedingmethods .

Particle bombardment

Embryogenic cell suspensions represent not only asource of totipotent protoplasts, but can also be used

37

as target cells for an alternative successful transfor-mation technique: particle bombardment . The particlebombardment process is a method for the delivery ofgenes into intact cells and tissues through the use ofDNA-coated microprojectiles (tungsten or gold) . It wasdeveloped by Sanford & co-workers (1987) and hasbecome the second most widely-used method for plantgenetic transformation after Agrobacterium-mediatedgene transfer (Gray & Finer, 1993) . Several labo-ratories have demonstrated that microprojectiles aresuitable for the transfer of genes to a wide range ofplant tissues and species. Apparently, there is no dif-ference in the efficiency of biolistic transformation ofmonocotyledonous and dicotyledonous species (San-ford, 1990) .

For the transformation of cereals, the choice ofappropriate target tissue is of major importance asthere are only a few tissues capable of plant regener-ation. Regenerable tissues of cereals have been testedby transient expression assays and have proved to besuitable for biolistic transformation experiments (Table3). The most common tissues used for this purpose areembryogenic suspension cells and embryogenic calluscultures. For the stable transformation and regenera-tion of maize (Gordon-Kamm et al ., 1990 ; Fromm et

Table 2 . Regenerationcrops

and transformation of protoplasts of cereal Table3 . Biolistic transformation of cereals usingdifferent target tissues

Regeneration Transgenic plants Embryogenic cell suspension culturesof fertile plants Rice Cao et al., 1992

Maize Gordon-Kamm et al., 1990Rice Abdullah et al., 1986 Toriyama et al., 1988 Fromm et al ., 1990

Toriyama et al., 1986 Zhang et al ., 1992 Oat Somerset al ., 1992Kyozuka et al ., 1987 Zhang & Wu, 1988 Embryogenic callus culturesKyozuka et al ., 1988 Shimamoto et al ., 1989 Maize Genovesi et al., 1992Datta et al ., 1990 Datta et al., 1990 Walters et al ., 1992Li & Murai, 1990 Tada et al., 1990 Wheat Vasil et al ., 1992Datta et al ., 1992 Terada et al ., 1993 Barley Wan & Lemaux, 1993

Rathore et al., 1993 Sugarcane Bower & Birch, 1992

Maize Prioli & Sondahl, 1989 Rhodes et al ., 1988 Primary explants

Shillito et al ., 1989 Donn et al ., 1992 Rice Christou et al., 1991Morocz et al ., 1990 Golovkin et al., 1993 Maize Koziel et al ., 1993

Donn et al ., 1992 Omirulleh et al ., 1993 Wheat Weeks et al ., 1993Becker et al., 1994

Barley Jahne et al., 1991b Barley Wan & Lemaux, 1994Funatsuki et al ., 1992 Ritala et al ., 1994Golds et al ., 1993 Jahne et al., 1994

Wheat Ahmed & Sagi, 1993 Tritordeum Barcelo et al ., 1994Triticale Zimny et al., 1995

38

al., 1990), rice (Cao et al ., 1992) and oat (Somers etal., 1992) suspension cells have been used as target tis-sue. However, the morphogenetic competence of cellsis significantly reduced during maintenance and thephenomenon of somaclonal variation limits the suit-ability of this tissue . Accordingly, embryogenic callushas been considered as a target tissue because the timeneeded for establishment of cultures and plant regen-eration is shorter for callus cultures than for suspen-sion cultures. Using callus cultures, it was possibleto regenerate transgenic sugarcane (Bower & Birch,1992), wheat (Vasil et al ., 1992) maize (Genovesi etal., 1992; Walters et al., 1992) and barley (Wan &Lemaux, 1993) .

Although these cultures could be successfully usedfor the production of transgenic cereals, it would bemore desirable to deliver DNA directly into primaryexplants with a high regeneration capacity. The timenecessary for the preparation of the target tissue iscomparatively low and the risk of somaclonal varia-tion is reduced as the period in culture is shortened to afew weeks . In cereals, scutellar tissues of rice (Chris-tou et al ., 1991), maize (Koziel et al ., 1993) and wheat(Weeks et al ., 1993) have been used for the regenerationof stably-transformed plants . In the following section,the progress made in our laboratory towards biolis-tic transformation of cereals using various explants issummarized .

Strategies for the biolistic transformation ofprimary explants

A prerequisite for the production of transgenic cere-als has been the development of efficient in vitro cul-ture systems from which fertile plants can be regen-erated at a high frequency . In our experiments theexplants of choice have been the scutellar tissue ofimmature embryos of wheat and triticale, immatureinflorescences of Tritordeum (Barcelo et al ., 1989)and barley microspores . Currently, the culture of bar-ley microspores is considered superior to anther cul-ture as the regeneration frequency can be significantlyincreased (Olsen, 1991 ; Hoekstra et al ., 1993 ; Mord-horst & Lorz, 1993) . In barley, a spontaneous autoen-doreduplication of the genome during the first divisionof the microspore leads to homozygous, diploid regen-erants. Therefore, this haploid target tissue makes theregeneration of homozygous Ro-plants possible .

Each target tissue has been the subject of individu-al optimization experiments to improve conditions for

particle bombardment. The most convenient methodto measure the efficiency of DNA delivery into intactcells, is the determination of the number of cells whichtransiently express the uidA-gene (,Q-glucuronidase) .Because of the relatively low sensitivity of the his-tochemical GUS-assay, the use of a strong promoterwhich enhances the expression of the marker gene isimportant. A construct harbouring the uidA-gene driv-en by an Act-1-D-promoter (McElroy et al ., 1990)provided best results in all tissues .

For optimization of the bombardment process andfor selection of stably-transformed plants, a plasmid(pDB 1 ; Fig . 1) containing the visualizable marker geneuidA under the control of the Actin I promoter fromrice, and the selectable marker gene bar under thecontrol of the CaMV 35S promoter has been used .

For Tritordeum the method of co-transformationusing the plasmids pAct-1-D/GUS and pCalneo hasbeen successfully demonstrated (S . Lutticke, unpub-lished) .

The aim of these optimization studies is to achieve ahigh frequency of transiently expressing cells . Howev-er, it is also very important that the target tissue does notsuffer significantly from the bombardment. The degreeof tissue damage depends on the type of explant, theparticle density and the acceleration pressure .

Immature embryos of wheat and triticale haveproved to be the most sensitive tissue . In wheat, highparticle densities (116 jug gold particles average size0,4-1,2 pm) per bombardment caused severe tissuedamage, whereas Tritordeum inflorescences were notreduced in their morphogenetic competence under thesame conditions. The viability of barley microsporeshas not been influenced by the amount of particles usedfor bombardment in our studies .

The results from our optimization and selectionexperiments demonstrate that the optimal particle den-sity has to be determined carefully for each type ofexplant, whereas the acceleration pressure can be rel-atively wide-ranging without having a negative effecton tissue viability and competence .

Strategies for the improvement of the biolisticmethod not only concern the parameters of the par-ticle bombardment process, but also the culture condi-tions for explants . In spring varieties of wheat and intriticale, a clear reduction of tissue damage has beenachieved when the immature embryos were precul-tured in vitro for two to seven days . In Tritordeum, apreculture of one day led to an increase in the numberof transformed plants, a finding which reinforces theimportance of the determination of optimal preculture

Acil

conditions (Barcelo et al ., 1994) . In barley, no effecton transient expression of the 0-glucuronidase gene oron viability has been observed when freshly-isolatedor one- to three-day old microspores were bombarded .Under optimal culture and bombardment conditions,an average number of 100 transient GUS-signals perembryo has been counted in wheat and triticale (Fig .2A). In barley, about 1 % of the bombarded mirosporestransiently expressed the uidA-gene (Fig . 2B) .

The establishment of an efficient transformationsystem requires careful choice of an appropriateselectable marker gene . For grasses, antibiotics suchas hygromycin, kanamycin of G-418 have been usedsuccessfully. In comparison, the major advantage ofherbicide selection is that plants can be selected in latestages of development by a simple spray test .

For wheat, triticale and barley, phosphinothricin(PPT) resistance conferred by the bar gene has provedto be a useful selectable marker for the regeneration oftransformed plants. The successful use of PPT selec-tion depends on the cell type and the developmentalstage. Therefore, the selection conditions were opti-mized individually. The optimal selection conditionsvaried ; in wheat a PPT concentration of 0 .5-2 mg/Iwas most suitable, whereas in barley 3-5 mg/1 PPT wasoptimal. Two to three weeks after bombardment thecalli were transferred to selection medium. The selec-tion pressure was applied both during callus inductionand the plant regeneration phase (Fig . 2C) . Further-more, the regenerants were sprayed in a later stage ofdevelopment with a solution containing 150-200 mg/IPPT in order to verify their resistance (Fig . 2D) . Thisselection system represents a fast and highly-efficientmethod for identifying transformed plants .

Using the pDB 1-construct (Fig . 1) it is also possi-ble to screen non-selected regenerants by spraying withPPT or by histochemically assaying the expression of

gus

1KB

Fig.] . Schematic representation of plasmid pDB 1 used in transformation experiments : S : Sal I ; Sa : SacI ; B : BamHl ; N: Ncol.

Sa

S

39

the uidA gene (Fig . 2G). By this method several trans-genic triticale (J . Zinmy, personal communication) andone transformed barley plant have been identified .

The transformation efficiency depended on thequality of the explant material and varied from experi-ment to experiment. In wheat, an average frequency ofone transgenic plant per 83 bombarded embryos couldbe achieved (Becker et al ., 1994) . This frequency issubstantially higher than the 1-2 plants per 1000 bom-barded embryos reported by Weeks et al . (1993) . Inbarley, independent transformation events led to theaverage recovery of one plant per 1 x 107 bombardedmicrospores (Jahne et al ., 1994) . Southern analysis ofselected regenerants demonstrated that in most casesplants contained intact copies of both marker genes(Fig . 3) . Single copy as well as multi-copy integrationof one or both marker genes (uidAlbar or uidAlneo)was detectable . Furthermore larger or smaller frag-ments were observed, suggesting that deletions, rear-rangements and/or methylation at restriction sites hadoccurred .

In our experiments, no phenotypic abnormalities orreduced fertility (Fig. 2H) was observed. This has beenreported for transgenic maize (Gordon-Kamm et al .,1990), wheat (Vasil et al ., 1992) and oat plants (Somerset al ., 1992) obtained from microprojectile bombard-ment of embryogenic suspension or callus cultures.

The segregation of the introduced marker geneuidA was visualized histochemically in pollen grainsof RO-plants (Fig . 2E and F) and in leaves of the proge-ny. The functional activity of the bar gene was testedby spraying the progeny with an aqueous solution ofthe herbicide Basta.

A 1 : 1 segregation as well as segregation of theintroduced marker gene in a non-Mendelian fashionwas observed in wheat, triticale and Tritordeum . Inbarley, the introduced marker genes were inherited by

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

conditions using

5 m

g/l

PPT.

D: Selection of a transformed barley plant by spraying an aqueous solution of 150 mg/l PPT

; left

: sensitive control plant, right

: re

sist

ant

tran

sgen

icplant.

8

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

in leaf tissue of transgenic wheat

. H : Mature transgenic RO-plant (left) and a seed-derived wheat plant (right)

.

42

Fig.3. Integration of the gusgene 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 Idigested with BamHI and Sacl . U : Undigested . 1 : Digested with BamHI/SacI to cut out the gus gene . 2: Digested with Ncol to determine thenumber of integration sites per genome.

all the progeny, indicating the homozygous genotypeof the transformed plants .

Progeny have been further analysed by Southernhybridization. Wheat plants showing a 1 : 1 segregationin pollen grains and a 3 : I inheritance of the markergenes in the progeny, had the same integration patternas the parental line. This indicates a close linkage ofthe introduced marker genes and their inheritance asa genetic unit. However, individuals containing multi-copy insertions did not always inherit the genes in aMendelian fashion .

Conclusion

Although the establishment of protoplast regenerationand transformation systems has improved significant-ly, a routine combination of both systems is not yetpossible .

On the other hand, the development of biolistictransformation systems has allowed rapid progresstowards the recovery of transgenic cereals . Embryo-

kb

PC

1/1

2/6

2/8

4/11

NCT U 1 2 U 1 2 U 1 2 U 1 2 U

2

8,0

4,8

1,8

genic suspension and callus cultures were used initiallybut recent results show that primary explants seem tobe more advantageous for the routine production of fer-tile transgenic cereals . The recently-developed methodof tissue electroporation promises to be another veryattractive approach for the transformation of primaryexplants. In summary, the tools of genetic manipulationof cereals have been significantly improved, provid-ing increased opportunities to transfer agronomically-interesting genes.

Note added in proof

In the meantime several new reports on the transformation of cere-als have been published. Most of these reports present transgeniccereal plants obtained either by particle bombardment or by tissueelectroporation giving further evidence for the suitability of thesemethods .

However, an unexpected publication was the one of Hiei et al .1994 . The Plant J . 6 : 271-282, who reported the regeneration oftransgenic Japonica rice plants from scutellar tissues co-cultivatedwithAgrobacterium tumefaciens.

-.-gus

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