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35 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 ., 36 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 37 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 38 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 References Abdullah, B ., E.C. Cocking & J.A. Thompson, 1986 . Efficient plant regeneration from rice protoplasts through somatic embryogenesis . Bio/Technology 4 : 1087-1090. Ahmed. K .Z . & F Sagi, 1993 . Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.) . Plant Cell Rep. 12 : 175-179 . Barcelo, P, A. Vazquez & A . Martin, 1989 . Somatic embryogenesis and plant regeneration from Tritordeum . Plant Breeding 103 : 235-240 . Barcelo, P, C . Hagel, D. Becker, A. Martin & H . Lorz, 1994. Transgenic cereal (Tritordeum) plants obtained at high efficiency by microprojectile bombardment of inflorescence tissue . Plant J . 5(4) : 583-592 . Becker, D ., R . Brettschneider & H . Lorz, 1994. Fertile transgenic wheat from microprojectile bombardment of scutellar tissue . Plant J . 5(2) : 299-307 . Bower, R. & R .G. Brich, 1992 . Transgenic sugarcane plants via microprojectile bombardment. Plant J . 2(3): 409-416 . Bytebier, B,, F. Deboeck, H . De Greve, M . Van Montagu & J.-P. Hemalsteens, 1987. T-DNA organization in tumour cultures and transgenic plants of the monocotyledon Asparagus officinalis . Proc . Natl . Acad. Sci . USA 84: 5345-5349. Cao, J., X. Duan, D. McElroy & R. Wu, 1992. Regeneration of herbicide resistant transgenic rice plants following microprojectilemediated transformation of suspension culture cells. Plant Cell Rep. 11 : 586-591 . Christou, P., T.L. Ford & M . Kofron, 1991 . Production of transgenic rice (Oryza sativa L.) plants from agronomically important Indica and Japonica varieties via electric discharge acceleration of exogenous DNA into immature zygotic embryos . Bio/Technology 9:957-962 . Dale, PJ ., M.S. Marks, M.M . Brown, C .J . Woolston, D .F. Chen, D .M. Gilmour & R .B . Flavell, 1989. Agroinfection of wheat: Inoculation of in vitro seedlings and embryos . Plant Sci . 63 : 237245 . Datta, S .K ., K . Datta & I. Potrykus, 1990. Fertile Indica rice plants regenerated from protoplasts isolated from microspore-derived cell suspensions . Plant Cell Rep . 9 : 25 3-256 . Datta, S .K ., A. Peterhans, K. Datta & I . Potrykus, 1990 . Genetically engineered fertile Indica rice recovered from protoplasts . Bio/Technology 8 : 736-740. Datta, K., I . Potrykus & S .K . Datta, 1992 . Efficient fertile plant regeneration from protoplasts of the Indica rice breeding line IR72 (Oryza saliva L.) . Plant Cell Rep. 11 : 229-233 . De Cleene, M ., 1985 . The susceptibility of monocotyledons to Agrobacterium tumefaciens. Z. Phytopathol . 113 : 81-89 . D'Halluin, K., E . Bonne, M . Bossut, M . De Beuckeleer & J . Leemans, 1992 . Transgenic maize plants by tissue electroporation . Plant Cell 4: 1495-1505 . Donn, G ., P. Eckes & H. Muller, 1992 . Gentibertragung auf Nutzpflanzen. BioEngineering 8 : 40-46 . Fretz, A ., A . Jhne & H . Lorz, 1992. Cryopreservation of embryogenic suspension cultures of barley (Hordeum vulgare L.) . Botanica Acta 105: 140-145 . Fromm, M., L .P. Taylor & V. Walbot, 1986. Stable transformation of maize after gene transfer by electroporation . Nature 319 : 791793 . Fromm, M .E., F. Morrish, A. Armstrong, R. Williams, J. Thomas & T.M. Klein, 1990. Inheritance and expression of chimeric genes in the progeny of transgenic maize plants . Bio/Technology 8: 833-839. Funatsuki, H., H. Lorz & P.A . Lazzeri, 1992. Use of feeder cells to improve barley protoplast culture and regeneration . Plant Sci . 85 : 179-187 . Genovesi, D ., N . Willetts, S . Zachwieja, M . Mann, T. Spencer, C . Flick & W. Gordon-Kamm, 1992 . Transformation of an elite maize inbred through microprojectile bombardment of regenerable embryogenic callus . In Vitro Cell Dev. Biol . 18 : 189-200 . Golovkin, M .V., M. Abraham, S. Morocz, S. Bottka, A . Feder & D. Dudits, 1993 . Production of transgenic maize plants by direct DNA uptake into embryogenic protoplasts . Plant Sci. 90 : 41-52 . Golds, T.J., J . Babczinsky, A .P. Mordhorst & H .-U . Koop, 1993 . Protoplast preparation without centrifugation: plant regeneration from barley (Hordeum vulgare L .). Plant Cell Rep . 13 : 188-192. Gordon-Kamm, WJ., T.J . Spencer, M.L . Mangano, T.R. Adams, R .J . Dames, W.G . Start, J .V O'Brien, S .A . Chambers, W.R. Adams, N.G . Willetts, T.B . Rice, C.J. Mackey, R.W. Krueger, A.P. Kausch & PG. Lemaux, 1990 . Transformation of maize cells and regeneration of fertile transgenic plants . Plant Cell 2: 603-618 . Gould, J ., M . Devey, O. Hasegawa, E .C . Ulian, G . Peterson & R .H. Smith, 1991 . Transformation ofZea mays L . using Agrobacterium tumefaciens and the shoot apex. Plant Physiol. 95 : 426-434 . Gray, D .J . & J.J . Finer, 1993 . Development and operation of five particle guns for introduction of DNA into plant cells . Plant Cell Tiss. & Org . Cult. 33 : 219 . Grimsley, N., T. Hohn, J .W. Davies & B . Holm, 1987 . Agrobacterium-mediated delivery of infectious maize streak virus into maize plants . Nature 325 : 177-179. Gupta, H.S . & A. Pattanayak, 1993 . Plant regeneration from mesophyll protoplasts of rice (Oryza sativa L.) . Bio/Technology 11 : 90-94 . Hahne, B ., H . Lorz & G . Hahne, 1990. Oat mesophyll protoplasts: their response to various feeder cultures. Plant Cell Rep. 8 : 590593 . Hoekstra, S., M.H. van Zijderveld, F. Heidekamp & F van der Mark, 1993 . Microspore culture of Hordeum vulgare L. : the influence of density and osmolarity. Plant Cell Rep. 12: 661-665 . Hohn, B., T. Hohn, M .I . Boulton, J .W. Davies & N . Grimsley, 1987. Agroinfection of Zea mays with maize streak virus DNA . p. 459-468 .1n : N .-H . Chu (Ed) . Plant Molecular Biology . Plenum Publishing Corporation, New York. Hooykaas, PJJ . & R.A . Schilperoort, 1992 . Agrobacterium and plant genetic engineering. Plant Mol . Biol . 19: 15-38 . Jahne, A ., P.A . Lazzeri, M . Jager-Gussen & H. Lorz, 1991a. Plant regeneration of embryogenic cell suspensions of barley (Hordeum vulgare L.) . Theor. Appl . Genet. 82 : 74-80 . Jahne, A ., PA . Lazzeri & H . Lorz, 1991b . Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.) . Plant Cell Rep . 10 : 1-6 . Jahne, A ., D . Becker, R . Brettschneider & H. L6rz, 1994. Regeneration of transgenic, microspore-derived, fertile barley . Theor. Appl. Genet . 89 : 525-533 . Kloti, A ., V.A. Iglesias, J . Wunn, PK . Burkhardt, S.K. Datta & I . Potrykus, 1993 . Gene transfer by electroporation into intact scutellum cells of wheat embryos . Plant Cell Rep. 12: 671-675 . Koziel, M .G ., G .L . Beland, C . Bowman, N.B. Carozzi, R . Crenshaw, L. Crossland, J. Dawson, N . Desai, M . Hill, S . Kadwell, K . Launis, K. Lewis, D. Maddox, K . McPherson, M .R. Meghji, E . Merlin, R . Rhodes, G .W. Warren, M . Wright & S .E . Evola, 1993. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Bio/Technology 11 : 194-200 . 44 Kyozuka, J ., Y. Hayashi & K . Shimamoto, 1987 . High frequency plant regeneration from rice protoplasts by novel nurse culture methods . Mot . Gen . Genet. 206 : 408-413 . Kyozuka, J ., E . Otoo & K . Shimamoto, 1988 . Plant regeneration from protoplasts of Indica rice : genotypic differences in culture response . Theor. Appl . Genet. 76 : 887-890 . Langridge, P, R . Brettschneider, P. Lazzeri & H . Lorz, 1992 . Transformation of cereals via Agrobacterium and the pollen pathway : a critical assessment. Plant J . 2: 631-638 . Lazzeri, PA ., R . Brettschneider, R . Liihrs & H . LOrz, 1991 . Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts. Theor. Appl . Genet. 81 : 437-444. Li, X .-Q., C: N . Liu, S .W. Ritchie, J .-Y. Peng, S.B . Gelvin & T.K. Hodges, 1992 .Factors influencing Agrobacterium-mediated transient expression of gusA in rice . Plant Mot . Biol . 20 : 1037-1048 . Li, Z. & N. Murai, 1990 . Efficient plant regeneration from rice protoplasts in general medium . Plant Cell Rep . 9 : 216-220 . McElroy, D ., W. Zhang, J . Cao & R . Wu, 1990 . Isolation of an efficient Actin promotor for use in rice transformation . The Plant Cell 2 : 163-171 . Mooney, PA ., PB . Goodwin, E.S . Dennis & D .J . Llewllyn, 1991 . Agrobacterium tumefaciens-gene transfer into wheat tissues . Plant, Cell Tiss. & Org . Cult. 25 : 209-218 . Mordhorst, A .P. & H. LOrz, 1993 . Embryogenesis and development of isolated barley (Hordeum vulgare L .) microspores are influenced by the amount and composition of nitrogen sources in culture media . J . Plant Physiol . 142 : 485-492. Morocz, S., G . Donn, J . Nemeth & D . Dudits, 1990 . An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture . Theor. Appl . Genet . 80 : 721-726 . Olsen, FL ., 1991 . Isolation and cultivation of embryogenic microspores from barley (Hordeum vulgare L.) . Hereditas 1 15: 255-266 . Omirulleh, S ., M . Abraham, M . Golovkin, I . Stefanov, M .K . Karabaev, M . Mustardy, S . Morocz & D . Dudits, 1993 . Activity of a chimeric promoter with the doubledCaMV 35S enhancerelement in protoplast-derived cells and transgenic plants in maize . Plant Mot . Biol . 21 : 415-428 . Petersen, W.L., S . Sulc & C .L . Armstrong, 1992 . Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts . Plant Cell Rep. 10 : 591-594. Potrykus, I ., 1989. Gene transfer to cereals: an assessment . Tibtech . 7 :269-273 . Potrykus, I ., 1990. Gene transfer to plants: assessment and perspectives . Physiol. Plant 79 : 125-134 . Prioli, L.M. & M .R. Sdndahl, 1989 . Plant regeneration and recovery of fertile plants from protoplasts of maize (Zea mays L.) . Bio/Technology 7 : 589-594 . Raineri, D .M., P Bottino, M .P. Gordon & E.W. Nester, 1990 . Agrobacterium-mediated transformation of rice (Oryza saliva L .) . Bio/Technology 8 : 33-38 . Rathore, K .S ., V.K . Chowdhury & T.K . Hodges, 1993 . Use of bar as selectable marker gene and for the production of herbicideresistance in rice plants from protoplasts. Plant Mot . Biol . 21 : 871-884 . Rhodes, C .A., K .S . Lowe & K .L . Ruby, 1988 . Plant regeneration from protoplasts isolated from embryogenic maize cell cultures . Bio/Technology 6 : 56-60 . Ritala, A ., K . Aspegren, U . Kurten, M . Salmenkallio-Marttila, L. Mannonen, R. Hannus, V. Kauppinen, T.H . Teeri & T.M . Enari, 1994. Fertile transgenic barley by particle bombardment of immature embryos . Plant Mot. Biol . 24: 317-325 . Sanford, J.C., T.M . Klein, E .D . Wolf & N. Allen, 1987 . Delivery of substances into cells and tissues using a particle bombardment process . J . Part. Sci. Tech . 5 : 27-37. Sanford, J.C ., 1990 . Biolistic plant transformation . Physiol. Plant 79 :206-209. Shillito, R .D ., G .K . Carswell, C.M . Jonsons, J .J . DiMaio & C .T. Harms, 1989 . Regeneration of fertile plants from protoplasts of elite inbred maize . Bio/Technology 7: 581-587. Shimamoto, K ., R . Terada, T. Izawa & H . Fujimoto, 1989 . Transgenic rice plants regenerated from transformed protoplasts . Nature 338: 274-276 . Somers, D.A ., H .W. Rines, W. Gu, H.F. Kaeppler& W.R . Bushnell, 1992. Fertile transgenic oat plants . Bio/Technology 10 : 15891594. Tada, Y., M . Sakamoto & T. Fujimura, 1990. Efficient gene introduction into rice by electroporation and analysis of transgenic plants : use of electroporation buffer lacking chloride ions . Theor. Appl . Genet. 80 : 475-480. Terada, R ., T. Nakayama, M . Iwabuchi & K . Shimamoto, 1993 . A wheat histone H3 promotor confers cell division-dependent and -independent expression of the gusA gene in transgenic rice plants . Plant J . 3 : 241-252 . Toriyama, K., K . Hinata & T. Sasaki, 1986. Haploid and diploid plant regeneration from protoplasts of anther callus in rice . Theor. Appl . Genet. 73 : 16-19. Toriyama, K ., Y. Arimoto, H. Uchimiya & K . Hinata, 1988 . Transgenic rice plants after direct gene transfer into protoplasts . Bio/Technology 6: 1072-1074. Vasil, 1K . & V. Vasil, 1992 . Advances in cereal protoplast research . Physiol . Plant 85 : 279-283 . Vasil, V., A .M . Castillo, M.E . Fromm & 1K . Vasil, 1992. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10 : 667-674. Walters, D .A ., C .S . Vetsch, D .E . Potts & R.C . Lundquist, 1992. Transformation and inheritance of a hygromycin phosphotransferase gene in maize plants . Plant Mot. Biol . 18 : 189-200 . Wan, Y., & PG . Lemaux, 1994. Generation of large numbers of independently transformed fertile barley plants . Plant Physiol . 104: 37-48 . Weeks, J .T., O.D. Anderson & A .E. Blechl, 1993 . Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiol. 102 : 1077-1084 . Zhang, W. & R ., Wu, 1988 . Efficient regeneration of transgenic rice plants from rice protoplasts and correctly regulated expression of foreign genes in the plants . Theor. Appl. Genet . 76 : 835-840 . Zhang, H.M., H . Yang, E.L. Rech, T.J. Golds, A .S . Davis, B .J . Mulligan, E .C . Cocking & M .R. Davey, 1988 . Transgenic rice plants produced by electroporation-mediatedplasmid uptake into protoplasts . Plant Cell Rep . 7 : 379-384 . Zimny, J., D . Becker, R . Brettschneider & H . LOrz, 1995 . Fertile, transgenic Triticale (x Triticosecale Wittmack) . Molecular Breeding 1 : 155-164 .