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Commercial production of European chestnuts is now possible.
Protocols for the different micropropagation steps from in vitro establishment up to plantlet regeneration of European chestnut are defi ned [ 13 ].
2. Specifi c genes for resistance to European chestnut ink disease have not yet been identifi ed, but the use of pathogenesis- related (PR) proteins such as thaumatin-like proteins would be an interesting alternative approach when the production of ink disease-tolerant/disease-resistant plants is the fi nal objective.
Specifi c details for the construction of pK7WG2D-TAU used in this work are reported elsewhere [ 10 ]. Any other binary v ector containing antifungal genes can also be used with the protocol defi ned in the present work.
3. When possible, the gfp should be used as a visual marker gene in the vector, as this simplifi es and improves evaluation of transformation events in real time. Selection of whole fl uores-cent embryos facilitates the proliferation of transgenic embryos, limiting the subculturing of possible escape tissues, as occurs when GUS expression is used as a selection marker.
Although there is some evidence that gfp may occasionally be cytotoxic to plant cells, the data obtained to date suggest that gfp is not cytotoxic for European chestnut material [ 10 ].
4. The frequency of SE induction from immature zygotic embryo explants is higher than that obtained from leaf explants, rang-ing from 2.2 to 10 %. The induction rate was clearly affected by both genotype and year of seed collection. Between 1 and 20 somatic embryos at different stages of development can be obtained from a single explant [ 12 ].
5. Stock shoot multiplication cultures of C. sativa should be maintained by sequential subculture of shoot tips and nodal segments every 4–5 weeks [ 13 ]. Only apical leaves from healthy and vigorous shoots should be used as explants to initiate the embryogenic process.
6. When somatic embryos are originated from leaf tissues of C. sativa , the explants initially respond by enlargement fol-lowed by a small callus formation, which is mainly differentiated on the cut leaf surfaces. A greenish callus subsequently arises from the midvein, spreading to the rest of the explant. In some cases, translucent globular structures and somatic embryos begin to grow from this callus tissue. The process is not syn-chronized, and embryos at various developmental stages will be present in the embryogenic explants. The SE induction fre-quency in leaf explants is generally lower (around 1 %) than that obtained when zygotic embryos are used as initial explants.
Furthermore, the time required for the appearance of the fi rst somatic embryos is longer (3–6 months) in leaf explants [ 12 ].
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7. In chestnut, the multiplication and maintenance of embryo-genic capacity can be carried out by either of two methods:
(1) secondary or repetitive embryogenesis from isolated somatic embryos at torpedo-cotyledonary stages which develop secondary embryos from the root-hypocotyl zone and (2) sub-culture of nodular embryogenic masses or proembryogenic masses (PEMs) [ 21 ]. PEMs are produced from the surface of somatic embryos. Despite the low induction rates from origi-nal explants, chestnut embryogenic cultures show a high capacity for secondary embryogenesis, which ensures the maintenance of embryogenic competence.
8. As the induction of somatic embryogenesis, the transformation effi ciency is clearly genotype dependent [ 9 , 10 ], and the use of different genotypes is highly recommended for these purposes.
9. The coculture period is clearly genotype dependent and should be evaluated with the specifi c target material used, although in our experience with European chestnut, 4–5 days of coculture is suffi cient for all the genotypes tested [ 8 – 10 ].
10. The concentration of kanamycin was selected on the basis of previous results, but should be reevaluated for a specifi c chest-nut material. The original creamy-yellowish color of the embryo clumps became brownish/blackish after 2–3 weeks of culture in selection medium. However, the addition of antioxi-dants such as cysteine, ascorbic acid, or acetosyringone in either cocultivation or selective media is not necessary in European chestnut and even may be detrimental for the trans-formation effi ciency [ 8 , 10 ].
11. Each transformation event should be taken as the start of a putative transgenic line. Each transgenic embryogenic line should be derived from one somatic embryo to confi rm that the line is the consequence of a unique transformation event.
The line should be routinely maintained by secondary embryo-genesis separated from the other transgenic lines produced.
12. Regardless of the genotype, the rate of embryo conversion (shoot + root development) is very low for both European and American chestnut. The limited number of plantlets produced makes subsequent analyses that should be carried out with these plants diffi cult. However, some of the embryos cultured on germination medium develop only a shoot as a “partial”
germination response, and this provides an opportunity to multiply and root these shoots to obtain an unlimited number of transgenic European chestnut plants by proliferation of axil-lary shoot cultures. Efforts should obviously be made to increase plantlet conversion from transgenic somatic embryos;
however, at present this method (axillary shoot proliferation) is the only realistic alternative. At this stage, the plants may be used for both molecular analyses and/or for testing the resis-tance to fungal attacks.
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References
1. Conedera M, Manetti MC, Giudici F et al (2004) Distribution and economic potential of the sweet chestnut ( Castanea sativa Mill.) in Europe. Ecol Mediter 30:47–61
2. Bellini E (2005) The chestnut and its resources:
images and considerations. Acta Hort 693:
85–96
3. Vannini A, Natili G, Anselmi NA et al (2010) Distribution and gradient analysis of ink dis-ease in chestnut forests. For Pathol 40:73–86 4. Heiniger U, Rigling D (1994) Biological con-trol of chestnut blight in Europe. Annu Rev Phytopathol 32:581–599
5. Vieitez E, Vieitez ML, Vieitez FJ (1996) El castaño. Edilesa, León
6. Rodríguez L, Cuenca B, López CA et al (2005) Selection of Castanea sativa Mill. gen-otypes resistant to ink disease in Galicia (Spain). Acta Hort 693:645–651
7. Maynard CA, Powell WA, Polin-McGuigan LD et al (2008) Chestnut. In: Kole C, Hall TC (eds) Compendium of transgenic crop plants:
transgenic forest tree species. Blackwell, Chichester, pp 169–192
8. Corredoira E, Montenegro D, San-José MC et al (2004) Agrobacterium -mediated transfor-mation of European chestnut embryogenic cultures. Plant Cell Rep 23:311–318
9. Corredoira E, San-José MC, Vieitez AM et al (2007) Improving genetic transformation of European chestnut and cryopreservation of transgenic lines. Plant Cell Tiss Org Cult 91:
281–288
10. Corredoira E, Valladares S, Allona I et al (2012) Genetic transformation of European chestnut somatic embryos with a native thaumatin- like protein ( CsTL1 ) gene isolated from Castanea sativa seeds. Tree Physiol 32:1389–1402 11. Vanblaere T, Szankowski I, Schaart J (2011)
The development of a cisgenic apple plant.
J Biotechnol 154:304–311
12. Corredoira E, Ballester A, Vieitez FJ et al (2006) Somatic embryogenesis in chestnut.
In: Mujib A, Samaj J (eds) Plant cell mono-graphs, vol 2, Somatic Embryogenesis.
Springer, Berlin, pp 177–199
13. Vieitez AM, Sánchez C, García-Nimo ML et al (2007) Protocol for micropropagation of Castanea sativa . In: Jain SM, Häggaman H (eds) Protocols for micropropagation of woody trees and fruits. Springer, Heidelberg, pp 299–312
14. Corredoira E, Valladares S, Vieitez AM et al (2008) Improved germination of somatic embryos and plant recovery of European chest-nut. In Vitro Cell Dev Biol Plant 44:307–315 15. Hood EE, Gelvin SB, Melchers LS et al (1993)
New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218 16. García-Casado G, Collada C, Allona I et al
(2000) Characterization of an apoplastic basic thaumatin-like protein from recalcitrant chest-nut seeds. Physiol Plant 110:172–180 17. Hoagland DR, Arnon DI (1941) The water
culture method for growing plants without soil. Miscellaneous publications N° 3514.
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18. Sambrook J, Russell D (2001) Molecular clon-ing: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 19. Gresshoff PM, Doy CH (1972) Development
and differentiation of haploid Lycopersicon esculentum . Planta 107:161–170
20. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:
473–497
21. Vieitez FJ, Merkle SA (2005) Castanea spp.
chestnut. In: Litz RE (ed) Biotechnology of fruit and nut crops. CAB International, Wallingford, pp 265–296
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Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224, DOI 10.1007/978-1-4939-1658-0_15, © Springer Science+Business Media New York 2015
Chapter 15
Grapevine ( Vitis vinifera L . )
Laurent Torregrosa , Sandrine Vialet , Angélique Adivèze , Pat Iocco- Corena , and Mark R. Thomas
Abstract
Grapevine ( Vitis ) is considered to be one of the major fruit crops in the world based on hectares cultivated and economic value. Grapes are used not only for wine but also for fresh fruit, dried fruit, and juice pro-duction. Wine is by far the major product of grapes, and the focus of this chapter is on wine grape cultivars.
Grapevine cultivars of Vitis vinifera L. have a reputation for producing premium quality wines. These premium quality wines are produced from a small number of cultivars that enjoy a high level of consumer acceptance and are fi rmly entrenched in the market place because of varietal name branding and the asso-ciation of certain wine styles and regions with specifi c cultivars. In light of this situation, grapevine improve-ment by a transgenic approach is attractive when compared to a classical breeding approach. The transfer of individual traits as single genes with a minimum disruption to the original genome would leave the traditional characteristics of the cultivar intact. However, a reliable transformation system is required for a successful transgenic approach to grapevine improvement. There are three criteria for achieving an effi cient Agrobacterium -mediated transformation system: (1) the production of highly regenerative transformable tissue, (2) optimal cocultivation conditions for both grapevine tissue and Agrobacterium , and (3) an effi cient selection regime for transgenic plant regeneration. In this chapter, we describe a grapevine transformation system that meets these criteria. We also describe a protocol for the production of transformed roots suitable for functional gene studies and for the production of semi-transgenic grafted plants.
Key words Agrobacterium , Antibiotic sensitivity , Embryogenic callus , Grapevine , Hairy roots , Reporter genes , Plant regeneration , Semi-transgenic grafted plants , Selectable markers , Transformation effi ciency , Transgenic , Vitis
1 Introduction
Most of the known grapevine wine varieties have been vegetatively propagated for several centuries. The reasons for the persistence of traditional European grapevine ( Vitis vinifera L.) cultivars for wine production are many with both plant and human factors involved [ 1 ]. All V. vinifera cultivars are highly heterozygous and do not breed true from seed. The combination of genes in a heterozygous genome responsible for wine quality is conserved by vegetative propagation. Thus, classical breeding programs, particularly those
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that have attempted to improve disease resistance and maintain wine quality, have had limited success in the past. Consumer pressure for the same cultivars and the specie’s lack of amenability to classical breeding has focused research attention on producing improved transgenic plants of established cultivars as the approach causes minimum disturbance to the original heterozygous genome [ 2 ].
Moreover, the ability to produce transgenic plants is also an invalu-able tool for understanding gene function and biological processes in grapevine [ 3 ]. A reliable transformation system is required for a successful transgenic approach to grapevine improvement espe-cially for disease and stress resistance or tolerance [ 4 ]. There are several prerequisites for achieving an effi cient Agrobacterium -mediated transformation system.
First, the production of highly regenerative transformable tis-sue is critical. Since the fi rst report of successful grapevine transformation [ 5 ], the methods for the production of transgenic grape plants have been based on the use of embryogenic cultures [ 6 ]. Previously, the lack of success in combining Agrobacterium - mediated transformation with direct shoot organogenesis from leaf explants was explained by the fact that the cells competent for regeneration were not competent for transformation [ 7 ]. However, recent work indicates that techniques based on regeneration by organogenesis can be effi cient [ 8 ]. The explant source, type, and quality of embryogenic cultures are a key factor in successful trans-formation, and improved conditions for initiation and maintenance of cultures suitable for genetic transformation have been defi ned [ 9 ]. Second, it is important to optimize cocultivation conditions for both Agrobacterium strains and grapevine tissue. The infection of cells with any given Agrobacterium strain and successful T-DNA integration is affected by several factors, such as strain, bacterial culture conditions, bacterial density, cocultivation time, and media used. Finally, one needs an effi cient selection regime for transgenic plant regeneration. The use of the selectable marker gene npt II that induces resistance to kanamycin has been widely reported in transformation experiments in Vitis . As an alternative to the npt II gene, the hpt gene has also been used effi ciently with hygromycin as the selective agent [ 10 – 13 ]. The bar ( pat ) gene encoding phos-phinothricin acetyltransferase (PAT) has also been used with the herbicide Basta ® as the selective agent [ 11 ], but its effi ciency is debated [ 14 , 15 ]. In addition initial attempts to use the phospho-mannose isomerase ( pmi ) gene as an alternate selectable marker have been reported as disappointing [ 14 , 16 ].
The transformation procedure described below uses embryogenic cultures obtained from immature anthers. This tissue type is widely used to induce somatic embryogenesis in grapevine [ 11 , 17 , 18 ].
Transformation can also be used with embryogenic cultures obtained from other tissues such as ovaries, nucelli, embryos, hypocotyls, or young leaves from in vitro plantlets. Maintenance of embryogenic cultures in a state suitable for transformation appears Laurent Torregrosa et al.
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dependent on the interaction of genotype and culture medium and in many cases represents a greater challenge than the initiation step [ 2 ]. Most importantly, two distinct types of embryogenic cultures are usually obtained on semisolid media: a culture designated type I which consists mostly of small globular embryos and undifferen-tiated calli and a type II culture which often develops from type I calli and consists entirely of somatic embryos at various stages of development from heart shape to torpedo stage, which proliferate by secondary embryogenesis [ 12 ].
When the response of different culture types to Agrobacterium tumefaciens transformation was examined, it was found that culture type did not have a major effect on initial rates of transformation.
This can be determined by the level of green fl uorescent protein (GFP) in cell clusters after cocultivation (Fig. 1c ) and measured by
Fig. 1 Successive stages of the plant transformation procedure in grapevine. ( a ) Infl orescence with immature fl ower buds produced on cutting and ready for anther culture (bar = 5 mm). ( b ) Embryogenic calli grown on GS1CA prior to cocultivation (bar = 1 mm). ( c ) Bright clusters of gfp -expressing cells observed 5 weeks after cocultivation (bar = 50 μm). ( d ) Clusters of kanamycin-resistant embryogenic cells visually selected among dead tissue 2 months after cocultivation (bar = 1 mm). ( e ) Germination of a kanamycin-resistant embryo on MG1 medium (bar = 5 mm). ( f ) Well-developed embryo before trimming of cotyledons and roots (bar = 5 mm).
( g ) Growth and axillary branching of the embryo apical meristem on BFe2 medium with 50 μg/mL kanamycin (bar = 5 mm). ( h ) Rooted transformed plantlets on micropropagation medium. Both plants from the left are transformed by VlmybA1-2 gene that activates anthocyanidin pigmentation in all vegetative organs, both plants from the right being untransformed controls
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the number of positive units recorded per plate [ 19 ]. However, different culture types have a signifi cant effect on the recovery of transgenic plants, with more plants recovered from type I cultures [ 12 ]. The reasons for the difference in recovery rate are complex.
In V. vinifera , the data for optimal transformation and selection conditions are confl icting as they may not only result from differ-ences in genotypes and selection strategies, but also from the embryogenic state of the cultures at the time of transformation.
The number of transgenic plants that can be recovered from an experiment is important when evaluating transgene expression.
Aberrant expression patterns of a transgene under the control of a constitutive promoter such as CAMV35S can reach a frequency of 35 % in transgenic plants evaluated [ 18 ] and need to be taken into account when seeking to introduce a trait which has commercial potential or for gene function analysis.
With the procedure described below, the number of embryos growing and rooting on germination medium under kanamycin selection and showing uidA or gfp expression is variable, depending on the cultivar and the A. tumefaciens strain. From 1 g of coculti-vated embryogenic calli, it may range from 10 to 100 or more embryos. A bottleneck in the transformation procedure still remains at the stage of shoot and plantlet development. The percentage of transgenic plantlets regenerated from transgenic embryos using this procedure ranges from 10 to 33 %, depending on the cultivar.
Among these transgenic plants, those regenerated from independent transformation events, as determined by Southern blot analysis, range from 77 to 100 %. The fi nal transformation effi ciency obtained with the procedure described below can range from 1 to 33 or more independent transgenic lines obtained from 1 g of embryo-genic calli. For wine grape cultivars, some of them such as Chardonnay are easy to transform [ 18 ]. Others such as Pinot Noir are more recalcitrant despite it being a parent of Chardonnay [ 20 ].
More recently, this protocol has been successfully applied to the microvine model using hygromycin for selection of transformed plantlets suitable for rapid forward and reverse genetic studies in small controlled environments due to the plant’s small stature and rapid fl owering phenotypes [ 13 ].
An alternative to stable grapevine transformation for gene function analysis is the production of transformed hairy root cul-tures by co-transformation with A. tumefaciens and A. rhizogenes . Transgenic hairy roots are quicker and easier to regenerate from stems and petioles than transgenic plantlets from embryogenic cal-lus and therefore a more suitable system when the regeneration of transgenic plants or a reproductive organ evaluation is not necessary.
Hairy roots are obtained by the inoculation of in vitro plantlets with non-disarmed A. rhizogenes strains. The oncogenes are located on the pRI plasmid inducing the formation of adventitious root, Laurent Torregrosa et al.
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which can be propagated in axenic cultures [ 21 ]. To avoid the tedious construction of co-integrated vectors, a system based on the co-inoculation with A. rhizogenes and A. tumefaciens was proposed [ 21 ]. Recombinant binary plasmids can also be introduced into wild-type A. rhizogenes strains where pRi virulence genes act in trans with T-DNA binary plasmid. A large number of genotypes of Vitis vinifera cultivars were found suitable for hairy root induction, with the cultivar “Maccabeu” being one of the most appropriate.
Microvines were also found suitable for hairy root experiments, with some lines such as 04C023V0002 and 04C023V0007 obtained from the cross Picovine 00C001V008 × Grenache, being found the most suitable [ 13 ].
Thus, the use of A. rhizogenes as part of the grapevine root transformation system provides an easy and effi cient way to gener-ate large amounts of transformed roots within a few weeks. This approach has proved very useful for gene function studies with the grapevine [ 22 ]. Furthermore, gene function studies involving the grafting of transgenic hairy roots onto scions of choice is now a possibility [ 21 ].
2 Materials
Somatic embryogenic cultures are initiated from immature anthers from many grapevine cultivars including Sultana, Portan, Shiraz, Chardonnay, Cabernet Sauvignon, Riesling, and Sauvignon Blanc.
The A. tumefaciens strain EHA101 [ 23 ] and its derivative EHA105 that contain a binary plasmid are used for transformation. They are more effi cient for grapevine transformation than the widely used strain LBA4404 and other strains [ 19 ]. The A. rhizogenes strain A4 is very effi cient for hairy root induction [ 24 ]. No antibiotic selection is required because pRi from the strain is co-transformed with the T-DNA from the binary vector [ 21 , 25 ].
Media are sterilized by autoclaving for 30 min at 110 °C.
1. Modifi ed MG/L medium [ 26 ]: 5 g/L of mannitol, 1 g/L of
L -glutamate, 5 g/L of tryptone, 2.5 mL/L of Fe-ethylene-diamine tetracetic acid (EDTA) stock solution, 5 g/L of NaCl, 150 mg/L of KH 2 PO 4 , 100 mg/L of MgSO 4 · 7H 2 O, 2.5 g/L yeast extract, 20 μg/L of biotin, and antibiotics depending on the bacterial strain and binary vector (100 μg/mL kanamycin
L -glutamate, 5 g/L of tryptone, 2.5 mL/L of Fe-ethylene-diamine tetracetic acid (EDTA) stock solution, 5 g/L of NaCl, 150 mg/L of KH 2 PO 4 , 100 mg/L of MgSO 4 · 7H 2 O, 2.5 g/L yeast extract, 20 μg/L of biotin, and antibiotics depending on the bacterial strain and binary vector (100 μg/mL kanamycin