GENETIC MODIFICATION OF ALFALFA (MEDICAGO SATIVA L.) FOR ...

GENETIC MODIFICATION OF ALFALFA (MEDICAGO SATIVA L.) FOR QUALITY IMPROVEMENT AND PRODUCTION OF NOVEL COMPOUNDS M. Vlahova, G. Stefanova, P. Petkov, A. Barbulova, D. Petkova, P. Kalushkov, A. Atanassov AgroBioInstitute, Sofia, Bulgaria

Introduction

formation, was also achieved through microinjection (45) and electroporation ( 30).

Alfalfa, a perennial herbaceous forage crop, known as lucerne, extensively grown in the world, is utilised as hay, silage and for pasture. It is valuable feed for many classes of livestock, because of a large dry biomass yield, high protein, minerals and vitamins content. Due to its ability for nitrogen fixation alfalfa enriches the soil, reduces the need of fertilization and supports the growth of other plants. This legume crop is of considerable agronomical importance for Bulgaria, where presently occupies about 160 000 hectares. Improvement of some genetic characters like resistance to abiotic and biotic stress, protein quality and digestibility, could significantly increase the yield and nutritional value of alfalfa. Due to its highly heterozygosity and almost self-incompatibility such improvement is definitely difficult by classical means. Possessing good regeneration ability, this fodder crop is a suitable candidate for genetic modification via unconventional approaches. Agrobacterium - mediated gene transfer has been basically used for transformation of embryogenic alfalfa genotypes (11, 16, 36, 42). It has been revealed that the transformation efficiency through Agrobacterium is genotype dependent (19) and in order to overcome this limitation as an alternative technique a direct gene transfer was applied. Transformed alfalfa plants were obtained by particle bombardment of calli (40) and pollen (44). Efficient transBiotechnol. & Biotechnol. Eq. 19/2005 Special Issue

Herbicide resistance Most of the trials, carried out with alfalfa, are focused on resistance to herbicides and diseases, but the quality aspects are also considered. Weed problems are one of the most serious and difficult to control. The use of herbicides for elimination of weeds, which compete with crops for water and nutrient compounds and lower yields, has become a wide applied practice. One of the most destructive weeds in many alfalfa seed fields is Dodder (Cuscuta spp.) – an annual parasitic weed, which cause 20% reduction of forage yield and blocks the seed production. The most effective and ecologically friendly is integrated management, which reduces significantly the infection, but it is not economically beneficial for the farmers. This is one major reason that biotechnologically - derived herbicide resistant crops have become so useful. Genes, conferring resistance to herbicides, were among the first, which have been identified and used for this purpose. The first alfalfa transgenic plants, expressing a gene of practical interest – bar, encoding resistance to the herbicide glufosinate-ammonium, were obtained by Agrobacterium Ti plasmid-mediated transformation and analyzed under greenhouse and field conditions (22). In order to promote alfalfa herbicide tolerance we optimized procedures for Agrobacterium tumefaciens 56

20th Anniversary AgroBioInstitute - R&D

mediated transformation - leaf disk and vacuum infiltration of seedlings of a highly regenerable line (R4) of the economically important Bulgarian cultivar Obnova 10 (6). Two different bacterial strains – LBA 4404 and GV3101, containing respectively binary vectors, carrying bar gene (for contact non-selective herbicide Basta) and ahas-3Rgene (for selective herbicide Glean), were utilized. The transgenic nature of the putative transformants were early determined by chlorophenol-red assay and further screened by bar- and npt II-specific PCR amplification and Southern blot hybridisation. The expression of used transgenes was proved by spaying of transgenic plants with the respective herbicide at greenhouse conditions (7). Probably, alfalfa “Roundup-Up Ready” (RR) will join very soon corn, soybean, and cotton as one of the glyphosate-resistant crops, as a part of the biotech cropping system, because in 2004 Monsanto submitted a federal petition for commercialization. It has been found that glyphosate applied at the appropriate growth stage generates at least 95% weed control of nearly all weed species invading alfalfa.

cDNA clone, encoding chicken ovalbumin, was introduced into three different alfalfa varieties (47). Transgenic alfalfa commercial varieties, expressed chimeric genes, encoding a rumen stable, sulphur amino acid-rich protein from sunflower seeds (50). This significantly increased the level to which this protein accumulated in the leaves of transformed plants. B-phaseolin gene under the control of 35S promoter was also introduced in alfalfa and the performed analyses revealed that the mature seeds showed significant accumulation of this protein (4). The gene, coding for zein, a major reserve protein in maize kernels, is extensively utilised in genetic engineering programmes (5, 48). Digestibility, one of the most important characteristics of the forage crops such as alfalfa, negatively correlates with the degree of lignification of the cell wall. Lignin – the major structural polymer of plant vascular tissue and fibers, increases with maturity in stems, inhibits rumen fermentation of cell wall polysaccharides, and reduces the utilization of forage by ruminant animals (28). By reduction of lignin content or alteration of its internal cross-linking, forage digestibility could be improved, which is expected to have significant impact on forage quality and respectively on animal productivity. Genes, encoding several enzymes from lignin biosynthetic pathway have been cloned and characterized (10, 18, 26). The achieved success in the application of the antisense strategy with the genes, involved in lignifications, open up the possibility for creation of new varieties with better properties of practical interests. Down-regulation of genes, encoding key enzymes of phenylpropanoid pathway, resulted in alteration of lignin composition and/or reduction in overall lignin levels in different species even in a case of gene expression in heterologous system (20, 9, 17, 43). Transgenic alfalfa with reduced activity of Cinnamyl Alcohol Dehydro-

Improvement of alfalfa forage quality It is well known that forage legumes are comparatively low in sulphur-containing amino acids (14) and their availability to ruminants is further adversely affected during rumen digestion. This leads to the reduction of the optimum for animal growth level of essential amino acids. Plant genetic modification with genes encoding for a sulphur amino acid-rich proteins, resistant to rapid rumen degradation can compensate this deficiency. A small number of proteins, including bovine serum albumin, chicken ovalbumin and bovine sub-maxillary microprotein (37), are known to be relatively resistant to degradation by rumen microbes and can be assimilated by the sheep. 20th Anniversary AgroBioInstitute - R&D

57

Biotechnol. & Biotechnol. Eq. 19/2005 Special Issue

genese (CAD), catalyzing the last step in the biosynthesis of the lignin precursors, possess altered lignin composition and exhibiting improved in situ cell wall degradability (8). All studies demonstrated that the plant system tolerates changes in their lignin pattern, which is very important when lignin modification aims application in the agricultural practice. Promising results for improvement of alfalfa digestibility have been also obtained, using a gene encoding for caffeoyl - CoA-O-methyltransferase (CCoAOMT) (21). This enzyme has been initially identified as involved in the pathogen defence response (39), but later on it was found to be considerably induced during lignification (58). Eleven independent alfalfa transgenic lines with normal growth and fertility, possessing reduced lignin content up to 20% and better digestibility, were selected after transformation of highly embryogenic genotype A70/3k cv. Rangelander with full- length CCoAOMT cDNA of poplar in antisense orientation (53, 54). Totally 729 clones of eight S1 self-pollinated transgenic lines were tested at field conditions. The breeding assessment of the lines in the same development stage were performed on the basis of productivity (green mass weight, g/plant-1), height (cm), stem number, chemical composition, digestibility and resistance to two diseases - root rot and Alfalfa Mosaic Virus (AMV). The data of the carried out evaluation at the conditions of polycross, revealed that three of the tested lines, could be suitable candidates for creation of synthetic populations, because of their good productivity, forage quality, persistence and tolerance to the above mentioned economically important diseases (41).

kind of utilization of plant biotechnology, known as “bio-pharming”, has been successfully developed and a large number of important pharmaceuticals, such as monoclonal antibodies for diagnostic use (24, 29), blood substitudes (32), have been produced. Genetically modified forage species, which are commonly used in the diet of domestic animals, are also very suitable for producing of edible vaccines (51). Transgenic alfalfa plants, expressing structural protein VP1 of foot and mouth disease virus (FMDV) were applied for immunisation of mice. It has been shown that freshly harvested leaves in their diet induced a virus-specific response (55). Like other crops, transgenic alfalfa can be also used as a bioreactor for producing of industrial enzymes, an approach significantly cheaper and conventional in contrast to the expenses for constructing new fermentation facilities (3, 15). An iron-binding glycoprotein - lactoferrin (Lf), major component of human colostral whey proteins is active against a large number of pathogenic microorganisms. Its ability to inhibit bacterial growth due to sequestration of the iron in the medium required for microbial metabolism. More recently, antibacterial direct killing effect has been described, which is independent of iron-binding and involves the basic N-terminal region of lactoferrin. This mechanism was clarified by studies showing that lactoferrin can disrupt or even penetrate bacterial cell membranes (57). Lactoferrin also shows antiviral activity and it is capable of inhibiting replication of a wide range of human and animal both RNA- and DNA- viruses. Mechanism probably involves blocking of cell-virus interactions. Infection of the target cell is prevented by direct binding to virus particles or binding to host cell molecules that the virus uses as a receptor or co-receptor. Lactoferrin is a powerful immunemodulating protein and also has anti-oxidant ef-

Alfalfa - an expression system for producing of novel compounds Plant cells provide an inexpensive and safe system for manufacturing commercially useful recombinant proteins. Recently, this Biotechnol. & Biotechnol. Eq. 19/2005 Special Issue

58


fects. It plays a role in the cellular defence system, most probably by regulating the macrophage activity and stimulating the proliferation of lymphocytes. Lf was also shown to regulate the antibody-dependent cytotoxity, cytokine production, and growth of some cells in vitro. It is a potent activator of natural killer cells, which may play a role in antitumor defence. As an iron scavenger, Lf helps preventing the formation of free radicals that trigger the oxidation process and thus may reduce susceptibility to disease. The potential for lactoferrin to act both as an antimicrobal and an imune regulatory agent in addition to its nutritional and pharmaceutical value has stimulated interest in development of an expression system which can provide large amounts of biologically active recombinant lactoferrin protein. It has already been expressed in lower eukaryotes, such as Saccharomyces cerevisiae (31) and in a variety of mammalian systems, including baby hamster cells (49), transgenic cows and mice (38). High levels of recombinant lactoferrin expression have been obtained as a fusion protein in Aspergillus (54). These expression systems require expensive purification processes and are not suitable for very large scale production. Plant biologists have successfully been able to express recombinant hLf in various crops. By using strong promoters, high levels of expression can be achieved. It is also possible to direct the process of production of recombinant proteins and specific parts of the plant can be utilized: fruit (tomatoes), seeds (rice, barley), leaves (tobacco, lucerne) and tubers (potatoes). Mitra & Zhang (1994) (34) first reported expression of the hLf gene under control of the CaMV 35S promoter in tobacco (Nicotiana tabacum) cultured cells, which contained approx. 1.8% of total cellular protein. Using oxidative stress-inducible peroxidase (SWPA2) promoter has increased accumulation of hLf in transgenic tobacco 20th Anniversary AgroBioInstitute - R&D

cell lines up to 4.3% (12). Lactoferrin has also been produced in transgenic plants at a maximal level of 0.3% of total cellular protein (46, 13, 2). Transgenic tobacco plants (46) have showed low expression levels and the protein needs to be purified extensively before it could be considered for any food application. This makes it unlikely as a commercially viable product. Potato (Solanum tuberosum) plants have been used for the production of hLf (13) and an expression level of 0.1% of total cellular protein has been obtained. This system is attractive because potatoes are a normal part of human diet but it is uncertain if the protein will have any biological activity after the extensive boiling that is used for potatoes. A serious disadvantage of tobacco and potato plants is high level of produced toxic compounds. Lactoferrin has also been expressed in rice grain for use in infant formula (2, 35), barley and tomato plants (2). Rice has several advantages as an expression system: 1) it has low allergenicity, because does not contain any toxic compounds and is one of first “nonmilk” foods introduced to infants; 2) expression can be directed so the protein is either expressed as a storage protein (in seed) or expressed only during germination. Glycosylation studies have shown that alfalfa is capable of producing recombinant glycoproteins with homogenous glycosylation patterns. In order to achieve a new expression system for producing recombinant human lactoferrin we chose one of the main fodder crops - alfalfa (Medicago sativa L.). Our goal is to develop an effective system for obtaining both - hLf producing transgenic alfalfa plants for forage use and fast growing suspension culture as a source of recombinant protein for the need of pharmacy. For production of hLf in transgenic alfalfa plants highly embryogenic clone R4 from the commercial Bulgarian cultivar Obnova 10 (Medicago sativa L.) (6), was 59


utilized as initial plant material. Agrobacterium tumefaciens-based gene transfer system for stable transformation (25) of alfalfa with a binary vector, carrying human lactoferrin cDNA under the control of 35S promoter from CaMV and selectable bar gene, were applied. The selection of transgenic plants resistant to phosphinotricin and producing recombinant human lactoferrin are in progress. Non embryogenic variety Furez (M.sativa L.) was used for producing recombinant hLF from cell cultures. Described above Agrobacterium tumefaciensbased gene transfer system and gene construct was utilized. Currently we determine the conditions for fast growing transgenic suspension culture.

ronmental risks as a whole and particularly the impact on the beneficial fauna. One of the most discussed environmental effects, associated with the use of transgenic plants is the possible flow of genes to wild relatives. The performed analyses for risk assessment of GM alfalfa deal mainly with pollen dispersal and herbicide resistance (1, 56). Some authors suppose that any alterations in lignin biosyntesis might affect feeding and population growth rates of defoliators (27). Considerable ongoing research attention has focused on the secondary ecological effect (on non-target or beneficial insects to food webs and the integrity of populations of soil biota) of insect-resistant GM crops, mainly on beneficial insects that collect pollen and are therefore heavily exposed. A number of studies have investigated the possible impacts of GM plants and recombinant proteins on bees revealed that direct toxicity is extremely rare and evidence from the most widely grown commercial crops has found no effect on colony performance (33). Under controlled (laboratory) and at field conditions we evaluated the eventual direct effect of transgenic alfalfa plants with reduced lignin content on phytophagues organisms or indirectly on the beneficial fauna through the feeding chain. In respect to different insects the ecosystem of alfalfa (Medicago sativa L.), is one of the richest, where more than 10 injurious arthropods could be recognized and dozens other arthropodes that play beneficial role (ladybird beetles, predatory buds, lacewings, spiders and synphid flies), also can be found. The preliminary results from the performed comparative investigations between 11 transformed alfalfa lines, possessing reduced lignin content (53) and respective control plants revealed that there was no significant difference in rate of larval development between individuals fed with both diets. The period for larva's development lasted 19 – 23 days and larval mortal-

Biosafety assessment of genetically modified alfalfa Nowadays, number of field trials with GM plants is increasing exponentially. This calls for a necessity of a new responsibility to produce and release them in the environment in safe manner. At present, the use of genetic engineering methods for modifying plants is highly controversial. Critics point several reasons to oppose this technology, because of risk for health (allergenicity, toxicity, antibiotic resistance), environment (impact on non-target organisms, unintended expression and effects, gene flow) and others with social and ethic characteristics. An assessment of risk of GM plants will minimize the likelihood of negative ecological effect, which can be manifested under certain circumstances. Making assessment of the risk, needs to be considered sexual compatibility, geography (origin), presence of outcrossing plants in the area of transgenic crops production, and mechanisms of pollination (e.g. wind, bees, animals etc.) in order to avoid "gene escape" (23). The implementation of field trials with GM cross-pollinated plants such as alfalfa necessitates profound evaluation of enviBiotechnol. & Biotechnol. Eq. 19/2005 Special Issue

60


ity was lower than 20 % at 25 ± 2 C°, at a relative humidity 65-85 % and 16L: 8D photoperiod. Summarizing the obtained data from the I st year of assessment at field conditions could be concluded that there aren't any indication for negative effect of the field released GM alfalfa with genetically modified lignin biosynthesis on insect populations. This study will be deployed on additional beneficial organisms from the food chain if some negative influence of GM plants will be established in the coming experiments. In order to take full advantage of the selected GM alfalfa lines our efforts will be focused on: • Optimization of the conditions for routine utilization of alfalfa expression system for producing pharmaceuticals compounds; • Investigation the effect of suppression or overexpression of transgenes involved in metabolic pathways on plant behavior under biotic and abiotic stresses; • “Gene –flow” risk evaluation. This data provide valuable information that will allow building up the necessary measures considering GM alfalfa field trials.

Opsomer C., Van Montagu M., Botterman J. (1999) Plant Mol. Biol., 39, 437-447. 9. Baucher M., Chabbert B., Pilate G., Doorsselaere J.V., Tollier M.T., Petit-Conil M., Cornu D., Monties B., Montagu M.V., Inzè D., Jouanin L., Boerjan W. (1996) Plant Physiol., 112, 1479-1490. 10. Boudet A., Grima J. (1996) Mol. Breeding, 2, 25-39. 11. Chaband M., Passiatore J., Cannon F., Buchanan-Wollaston V. (1988) Plant Cell Rep., 7, 512516. 12. Choi S.M., Lee O.S., Kwon S.Y., Kwak S.S., Yu D.Y., Lee H.S. (2003) Biotechnology Letter, 25, 213-218. 13. Chong D., Langridge H.R. (2000) Transgenic research, 9, 71-78. 14. Croissant G., Meton B., Miller D., Kellog W. (1976) New Mexico State Univ.Agricult. Experiment station, Las Cruces. 15. D`Aoust M.A., Lerouge P., Busse U., Bilodeau P., Trépanier S., Gomord V., Faye L., Vézina L.P. (2004) In: Molecular Farming, (R. Fischer, S. Schillberg, Eds.), 1-12. 16. Deak M., Kiss G., Konz C., Duditz D. (1986) Cell Rep., 5, 97-100. 17. Dixon R.A., Lamb C.J., Masond S., Sewalt V.J.H., Paiva N.L. (1996) Gene, 179, 61-71. 18. Douglas C. (1996) Trends in Plant Sci., 1(6), 171-178. 19. Du S., Erickon L., Bowley S. (1994) Plant Cell Rep., 13(6), 330-334. 20. Dwivedi U.N., Campbell W.H., Yu J., Dalta R.S.S., Bugos R.C., Chiang V.L., Podila G.K. (1994) Plant Mol. Biol., 26, 61-71. 21. Guo D., Chen F., Inoue K., Blount J.W., Dixon R.A. (2001) The Plant Cell, 13, 73-88. 22. Halluin K., Botterman., De Greef W. (1990) Crop Sci., 30(4), 866-871. 23. Hardy R., Segelken J. (Eds.) (1998) NABC Report, 10, 23-29. 24. Hiatt A. (1990) Nature, 344, 469-470. 25. Horsch R., Fry J., Hoffman N., Neidermeyer J., Rogers S.G., Fraley R.T. (1988) Plant Molecular Biology Manual, A5, 1-9. 26. Inone K., Sewalt V., Balance G., Sturzer C., Dixon R. (1998) Plant Physiol., 117, 761-770. 27. James C. (1999) Commersialized transgenic crops preview ISSAAA, 12. 28. Jung H. (1989) Agron. J., 81, 33-38. 29. Khoudi H., Laberge S., Ferullo J.M., Bazin R., Darveau A., Castonguay Y., Allard G., Lemeieux R., Vezina L.P. (1999) Biotech. and Bioeng., 64,

REFERENCES 1. Amand St.P.C., Skinner D.Z., Peaden R.N. (2000) Theor. Appl. Genet., 101, 107-114. 2. Anzai H., Takaiwa F., Katsumata K. (2000) Elsevier science, in Lactoferrin: Structure, Function and Application. 3. Austin B., Bingham E., McKersie B., Brown D. (1997) Biotechnology in Agriculture, 17, 409-424. 4. Bagga S., Sutton D., Kemp J.D., SenguptaGopalan C. (1992) Plant Mol. Biol., 19(6), 951-958. 5. Bagga S., Adams H., Kemp J.D., SenguptaGopalan C. (1995) Plant Physiol., 107 (1), 13-23. 6. Barbulova A., Iantcheva A., Zhiponova M., Vlahova M., Atanassov A. (2002a) Biotechnol. & Biotechnol. Eq., 16(1), 55-63. 7. Barbulova A., Iantcheva A., Zhiponova M., Vlahova M., Atanassov A. (2002b) Biotechnol. & Biotechnol. Eq., 16(2), 21-27. 8. Baucher M., Bernard V., Chabbert B., Besle J.,


61


135-143. 30. Kuchuk N., Komarnitski I., Shakhovsky A., Gleba Y. (1990) Plant Cell Reports, 8, 660-663. 31. Liang Q., Richardson T. (1993) J.Agric Food Chem., 41, 1800-1807. 32. Magnuson N.S., Linzmaier P.M., Reeves R., An G., HayGlas K., Lee J.M. (1998) Protein Expr. Purif., 13, 45-52. 33. Malone L.A., Pham-Delègue M.H. (2001) Apidologie, 32, 278-304. 34. Mitra A., Zhang Z. (1994) Plant Physiol., 106, 977-981. 35. Nandi S., Suzuki Y.A., Huang J., Yalda D., Pham P., Wu L., Bartley G., Huang N., Lönnerdal B. (2002) Plant Science, 163, 713-722. 36. Ninkovic S., Miljus-Djukis J., Neskovic M. (1995) Plant Cell Tissue and Organ Culture, 42(3), 255-260. 37. Nugent J., Jones W., Jordan D., Mangan J. (1983) British J. of Nutrition, 50, 357-368. 38. Nuijens J.H., van Berkel P.H., Geerts M.E., Hartevelt P.P., de Boer H.A., van Veen H.A., Pieper F.R. (1997) J. Biol. Chem., 272, 8802-8807. 39. Pakusch A.E., Matern U. (1991) Plant Physiology., 96, 327-330. 40. Pereira L., Erickson L. (1995) Plant Cell Rep., 14, 290-293. 41. Petkova D., Vlahova M., Marinova D., Petkov P., Carlier L., Atanassov A. (2004) Proceedings of 5th European Conference on Grain Legumes, p.105. 42. Pezzotti M., Pupilli F., Damiani F., Arcioni S. (1991) Plant Breeding, 106, 39-46. 43. Piquemal J., Chamay O.U.S., Nadaud I., Beckert M., Barriere J., Mila I., Lapierre C., Rigan J., Boudet A-M, Goffner D., Pichon M. (2002) Plant Phisiol., 130(4), 1675-1685. 44. Ramaiah S., Skinner D. (1997) Current Sci., 73(8), 674-682.


45. Reich T., Iyer V., Miki B. (1986) Bio/Technology, 4, 1001-1003. 46. Salmon V., Legrand D., Slomianny M.C., Yazidi I., Spik G., Gruber V., Bournat P., Olagnier B., Mison D., Theisen M., Merot B. (1998) Protein expression and purification, 13, 127135. 47. Schroeder H., Rafique M., Khan I., KuibbW., Spencer D., Higgins T. (1991) Aust. J. Plant Physiol., 18, 495-505. 48. Sharma S., Hancock K., Ealing P., White D. (1998) Mol. Breeding, 4, 435-448. 49. Stowel K.M., Rado T.A., Funk W.D., Tweedie J.M. (1991) Biochem J., 276, 349-355. 50. Tabe L., Wardley R., Ceriotti A., Aryan A., McNabb W., Moore A., Higgins T. (1995) J. of Animal Sci., 73(9), 2752-2759. 51. Tuboly T., Yu W., Baily A., Erickson L., Nagy E. (2000) In: Molecular Farming, (J.P. Toutant, Balazs, Eds.), Proceedings of the OECD Workshop, La grande Motte, France, 3-6 September 2000, 239248. 52. Vlahova M., Carlier L, Bockstaele E.V., Boerjan W., Atanassov A. (2001) Proceedings: XVIth EUCARPIA Congress, Edinburgh, Scotland, 10-14 September. 53. Vlahova M., Petkov P., Atanassov A., Carlier L., Van Waes C., Van Bockstaele E. (1998) Rastenievadni Nauki, 35(9), 679-685. 54. Ward P.P., May G.S., Headon D.R., Coneely O.M. (1992) Gene, 122, 219-223. 55. Wigdorovitz A., Carrillo C., Dus Santos M., Trono K., Peralta A., Gomez M., Rio R., Franzone, Sadir A., Escribano J., Borca M. (1999) Virology, 255, 347-353. 56. Williamson M. (1993) Experientia, 49, 219-224. 57. Yamauchi K., Tomita M., Giehl T., Ellison R. (1993) Infection and Immunity, 61, 719-728. 58. Ye Z. H. (1997) Plant Physiol., 115, 1341-1350.

62
