Grape possesses the basic prerequisites for Agrobacterium-mediated transformation it is a host for Agrobacterium and plant regeneration can be induced from cultured grape explants. Leaf explants were cocultivated with disarmed Agrobacterium vectors carrying kanamycin resistance and GUS genes and cultured on shoot-inducing medium containing kanamycin. After 21 d, intense and sharply-defined blue regions were observed, including some blue organized meristematic structures, consistent with plant-driven GUS gene expression. No GUS activity was detected in control explants. Among single leaf tips excised from over 200 regenerated shoots, one was GUS positive. The recovery of transgenic shoots might be improved by increasing the frequency or modifying the site of transformation and/or regeneration

Bandman, E. B.

Colby, S. M.

Dandekar, A. M.

Meredith, C. P.

Stamp, J. A.

English

JKI Open Journal Systems (Julius Kühn-Institut)

408 Section 5 Progress toward the production of transgenic grapevines by Agrobllcterium-mediated transformation C. P. MEREDITH 1), S. M. CotBY 1), J. A. STAMP 3), A.M. DA~EKAR 2) and E. B. BANOMAS 1) 1) DepartmentofViticulture and Enology, Universicy ofCalifomia, Davis, Califomia 95616, USA 2) Department ofPomology, Universicy ofCalifomia, Davis, Califomia 95616, USA ') Agritope lnc., 15425 S.W. Koll Parkway, Beaverton, Oregon 97006, USA Summary: Grape possesses the basic prerequisites for Agrobacterium·mediated transfonnation- it is a host for Agrobacterium and plant regeneration can be induced from cultured grape explants. Leaf explants were cocultivated with disanned Agrobacterium vectors carryi.ng kanamycin resistance and GUS genes and cultured on shoot·inducing medium containing kanamycin. After 21 d, intense and sharply·defmed blue regions were observed, including some blue organized meristematic structures, consistent with plant-driven GUS gene expression. No GUS activity was detected in control explants. Among single leaf tips excised from over 200 regenerated shoots. one was GUS positive. The recovery of transgerne shoots might be improved by increasing the frequency or modifYing the site oftransfonnation and/ or regeneration. K e y wo r d s : transgenic grapevine, Agrobacterium, crown gall, tissue culrure, kanamycin resistance, ß · glucuronidase, gene expression, genetic engineering, genetics. lntroduction Ofthe several different Iransfonnation strategies that have now been successfully employed to produce transgenic plants, the most common is Agrobacterium-mediated transfonnation (KLEE et al. 1987). This approach exploits the natural ability ofthis bacterium to introduce genesintoplant cells. Agrobacterium strains from which the disease-causing properties are removed ('disanned') can be endowed with new genes of academic or agricultural interest from virtually any other organism. When such a strain is allowed to infect a plant cell, the new gene is introduced and in-tegrated into the plant's own genetic material and becomes a pennanent part of the genetype of the recipient plant cell. Because the descendents of such a transfonned cell inherit the new gene, a plant regenerated from transfonned cells will be t r an s g e n i c : it will carry the new gene in every cell. The basic requirement for successful Agrobacterium-mediated transfonnation is an explant that is both transfonnahte and regenerable. The plant species must be a host for Agrobacterium and the particular explant tissue used must be susceptible to infection. The recovery of a transgenic plant is dependent on a means toregenerate an entire plant from the explant. These requirements have been fulfilled in systems a.~ diverse as tomato leaf disks from which adventitious shoots develop (HoR.'\cH et al. 1985) and walnur somatic embryos that produce secondary embryos on their surface (McGRANAHA~ er al. 1988). Grape (Vitis l'inifera L) is a well-known host for Agrobacterium and, in fact. crown gall disease is a serious -viticultural problern (PE.>.RSON and GoHEEN 1988). Emire plants can also be regenerated from a variety of grape explant types via either adventitious shoots or somatic embryos (MoxETTE 1988). Because the ba.<;ic requirell)ent is met, it is reasonable to expecr that Agrobacterium-mediated Iransfonnation can be successfully adapted to grape. The AGROBAcTERIUM strains a.-;sociated with grape-vines in nature are predominantly of the biovar 3 type (PEARSON and GoHEEl' 1988). However, the strains generally used as plant gene vectors are biovar 1, a distinctly different group with different properties (KNAUF et al. 1982). To establish whether the biovar 1 strains in general use might be employed a.'> gene vectors for grape, we investigated the ability of several biovar l strains to incite galls on in vitro-grown plants of Tissue and cell culture 409 Results of a typical cocultivation experiment with 'Thompson Seedless' leaf explants. Data was collected after 15 d of incubation, at which time all tissue was sacrificed for analysis and nonewas allowed to develop funher Number of explants Percentage Total 193 Producing adventitious buds and shoots 120 62 Containing GUS-positive regions 183 95 GUS expression in meristematic structures 14 7 Structures partially GUS-positive 7 4 Structures entirely GUS-positive 7 4 several vinifera cultivars. A.lthough ditferences were observed among strains, galls were produced an all cultivars and the presence and expression ofimroduced genes could be detected in gall tissue, thus demonstrating that biovar 1 Agrobacrerium strains could infect and transform grape cells (MARTI!'\ 1987). Materials aitd methods Employing disanned Agrobacrerium vectors carrying chimeric genes encoding arnino-glycoside phosphotransfera~e (kanamycin resistance) and ß -glucuronidase (GUS), an enzyme whose activity can be detectecd as a blue color in a histochemical assay (JEFFERS0:-1 er al. 1987), we conducted a series of studies with leaf explams from which the development of adventitious shoots can be induced. Leaves of V. vinifera cvs Thompson Seedless (syn. Sultanina) and French Colombard were obtained from in l'irro nodal cultures and either cultured intact or bisected perpendicular to the midrib. Under the appropriate conditions. this explant type will produce adventitious shoots an the petiolar stub and at the basal end ofsevered veins (SrAMP er al. 1989). Leaf explants were cocultivated with Agrobacrerium and then incubated on shoot-inducing medium containing carbenicillin and kanamycin. At periodic intervals, explants were sacrificed and sectioned for histochemical analysis of GUS activity. Other explants were incubated umil advemitious shoots developed. A single leaf-tip was removed from each adventitious shoot for GUS assay and the shoots were also excised and tested for their ability to root in kanamycin-containing medium. 410 Section 5 Results and discussion Analysis of GUS expression by both stereo· and compound microscopy at 4 d post cocultivation revealed diffuse pale blue patches on the Iamina and petiole regions and wirhin the leaf veins, suggestive of Agrobacterium-driven GUS gene expression. After 11 d, however, intense and sharply defined blue-staining regions could be seen ·with the aid ofa stereomicroscope. Closer examination of these regions. with a compound microscope revealed both individual cells and groups of cells containing dark blue cytoplasm, consistent with plant-driven GUS expression in clones of transfonned plant cells. Many of these regions were internal but some occurred on the explant surface. lnfrequently, organized meristematic structures associated with the early Stages of shoot fonnation were obseryed that were either partially or entirely intensely blue-staining, suggesting that, in these explants, cells that can be transfonned (or their descendants) can also participate in regeneration (Table). No GUS activity was detected in control explants that had not been cocultivated with Agrobacterium. Explants that were allowed to continue until adventitious shoots fonned (28-56 d) exhibited bleaching typical ofkanamycin toxicity. Islands ofgreen tissue and, less commonly, green shoots were observed on some cocultivated explants. Histochemical analysis ofleaftips from over 200 of these green shoors revealed one GUS·positive leaftip. The putatively transgenic shoot from which this leaftip was obtained was desrroyed in a Iabaratory accident before a Southern analysis could be perfonned. >-u z w :::l Cll w Ct: u... TRANSFORMATION REGENERATION Improving the recovery of transgenic plants. The relative number of cells in the explant (E) that participate in both transformation (T) and regeneration (R) is indicated by the shaded area. Increasing the frequency or changing the site of either transformation or regenerationwill increase the size ofthe shaded area. Tissue and cell culture 411 Although the approach employed here possessed the two requisite componems - an explant that is both transformable and regenerable- it ha.<; not yet resulted in the production oftransgenic plants at an acceptable frequency. There are a number of avenues by which the results might be improved. A transgerne plant is the result ofthe coincidence oftransfonnation and regeneration in the same cells (Fig.). The !arger the number of transformed cells that also panicipate in regeneration, the higher the probability of recovering a transgenic plant. Both transformation and regeneration are phenomena that are amenable to manipulation. Critical parameters of both processes are f r e q u e n c y and s i t e . An increase in frequency or a change in the site of either transformation or regeneration may increase the size of the coincidem cell population (Fig.). The site and/or frequency of transfonnation might be improved by choice of A.grobacterium strain, cocultivation conditions, or explant type. The site and/ or frequency of regeneration might be modified by choice of explant or culture conditions. A histological study of adventitious shoot developmem from leaf explants has revealed that the coincidence of transformation and regneration is likely very small under our conditions (CoLsv, S. M.; Ju!'cosA, A.; Sn.MP, J. A.; MEREDITH, C. P.; in preparation) and we are curremly investigating several approaches by which we hope to improve oursuccess. Literature cited HORSCH, R. B,; FRY, J. E.; HOFFMANN, N.; EICHHOL TZ, D.; ROGERS, S. G.; FRALEY, R. T.; 1985: A simple and general method fortransferring genes into plants. Science 227, 1229·1231. JEFFERSOX, R. A.; KAVANAGH, T. A.; BEVAN, M. W.; 1987: GUS fusions: ß ·glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901· 3907. KLEE, H.; HORSCH, R.; ROGERS, S.; 1987: A.grobacrerium·mediated plant transformation and its further applications to plant biology. Ann. Rev. Plant Physiol. 38,467-486. KNAUF, V. C.; PASAGOPOULOS, C. G.; NESTER, E. W.; 1982: Genetic factors controlling the host range of Agrobacrerium rumejaciens. Phyropathology 72, 1545·1549. MARTI:-<, L. A.; 1987: Genetic Transformation and Foreign Gene Expression in Tissue of Different Woody Species. Master ofScience Thesis, Univ. of California, Davis, USA. McGRANAH.AN, G. H.; LESLIE, c. A.; URATSU, s. L.; MARTI:\, L. A.; DA!>.'DEK.AR., A. M.; 1988: Agrobacterirlm-mediated transformation ofwalnut somatic embryos and regeneration oftransgenic plants. Bio-Technology 6, 800-804. MO~ETTE, P. L.; 1988: Grapevine.ln BA1Al, Y. P. S. (Ed.): Biotechnology in Agriculrure and Forestry, Vol6, Crops Il, 3-37. Springer-Verlag, Berlin. PEARSOI', R. C.; GOHEEN, A. C. (Eds.); 1988: Grape Disease.Compendium. American PhyropatholOgical Society, St. Paul, Minnesota, USA. STAMP, J. A.; CoLBY, S. M.; MEREDITH, C. P.; 1989: Direct shoot organogenesis and plant regeneration from leaves of grape (Fitis vinifera L.). Plant Cell Tissue Organ Culture (submitted). 

Progress toward the production of transgenic grapevines by Agrobacterium-mediated transformation

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Progress toward the production of transgenic grapevines by Agrobacterium-mediated transformation

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