La digitalización como herramienta para la preservación

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.

179 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.

181 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 and 25 μg/mL rifampicin for EHA105/ pBINm-gfp5-ER). Adjust to pH 7.0 with NaOH.

2. Induction medium for A. tumefaciens : ABB salts [ 27 ] (20 g/L of NH 4 Cl, 12.3 g/L of MgSO 4 · 7H 2 O, 3.0 g/L of KCl, 0.265 g/L of CaCl 2 · 2H 2 O, 0.5 g/L of FeSO 4 · 7H 2 O, 5 μg/L 2.1 Plant Material 2.2 Agrobacterium Strains 2.3 Culture Media for A. tumefaciens and A. rhizogenes

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of biotin), 2 mM NaH 2 PO 4 at pH 5.6, 40 mM 2-( N -morpholino)

ethanesulfonic acid (MES), 0.5 % glucose, and 100 μM acetosyringone.

All stock solutions were sterilized through a 0.2 μm fi lter and stored at 4 °C unless otherwise stated.

1. Half-strength Murashige and Skoog (MS) macroelements (10×, [ 28 ]): 16.5 g/L of NH 4 NO 3 , 4.4 g/L of CaCl 2 · 2H 2 O,

3.7 g/L of MgSO 4 · 7H 2 O, 19.7 g/L of KNO 3 , and 1.7 g/L

of KH 2 PO 4 .

2. NN macroelements (10×, [ 29 ]): 7.2 g/L of NH 4 NO 3 , 9.5 g/L

of KNO 3 , 4.4 g/L of CaCl 2 · 2H 2 O, 3.7 g/ L of MgSO 4 · 7H 2 O,

and 1.7 g/L of KH 2 PO 4 . 3. GNBC macroelements (10×, [ 30 ]): 10 g/L of KNO 3 , 5 g/L of Ca(NO 3 ) 2 · 4H 2 O, 1.6 g/L of NH 4 NO 3 , 1.25 g/L of MgSO 4 · 7H 2 O, and 1.25 g/L of KH 2 PO 4 . 4. MS microelements (1,000×, [ 28 ]): 6.2 g/L of H 3 BO 3 , 22.3 g/L of MnSO 4 · 4H 2 O, 8.6 g/L of ZnSO 4 · 7H 2 O, 0.83 g/L of KI, 0.25 g/L of Na 2 MoO 4 · 2H 2 O, 25 mg/L of

CuSO 4 · 5H 2 O, and 25 mg/L of CoCl 2 · 6H 2 O.

5. GNBC microelements (1,000×, [ 30 ]): 460 mg/L of MnSO 4 · 4H 2 O, 250 mg/L of KI, 58 mg/L of ZnSO 4 · 7H 2 O,

25 mg/L of H 3 BO 3 , 25 mg/L of CuSO 4 · 5H 2 O, 25 mg/L of

NiCl 2 · 6H 2 O, 25 mg/L of CoCl 2 · 6H 2 O, and 25 mg/L of

Na 2 MoO 4 · 2H 2 O. 6. LG0 macroelements [ 21 ]: 1.5 g/L KNO 3 , 150 mg/L (NH 4 ) SO 4 , 150 mg/L CaCl 2 · 2H 2 O, 250 mg/L MgSO 4 · 7H 2 0, 250 mg/L NaH 2 PO 4 · 2H 2 O. 7. MS/2 macroelements [ 21 ]: 950 mg/L KNO 3 , 825 mg/L NH 4 NO 3 , 220 mg/L CaCl 2 · 2H 2 O, 185 mg/L MgSO 4 · 7H 2 O, 85 mg/L KH 2 PO 4 .

8. Fe-EDTA (200×, [ 28 ]): Dissolve 7.44 g of Na 2 EDTA · 2H 2 O

in 900 mL of nanopure water. Heat the solution to almost boiling point and gradually add 1.86 g of FeSO 4 · 7H 2 O. Make

up to 1 L with nanopure water.

9. Ferric citrate (200×, [ 30 ]): Dissolve 4 g ammonium ferric citrate in 1,000 mL nanopure water.

10. Vitamins T (1,000×, [ 31 ]): 50 g/L of mesoinositol, 1 g/L of nicotinic acid, 1 g/L of thiamine HCl, 1 g/L of pyridoxine HCl, 1 g/L of calcium pantothenate, and 0.01 g/L of biotin.

11. Vitamins B5 (1,000×, [ 32 ]): 100 g/L of mesoinositol, 10 g/L of thiamine HCl, 10 g/L of nicotinic acid, 1 g/L of pyridoxine HCl.

2.4 Stock Solutions and Other Supplies

183 12. Amino acid mix (1,000×, [ 17 ]): 100 g/L of glutamine, 10 g/L

of phenylalanine, and 2 g/L of glycine.

13. 2,4-Dichlorophenoxyacetic acid (2,4-D, 1 mM): Dissolve 44.2 mg in 1 mL 10 N NaOH. Add nanopure water and heat until completely dissolved. Make up the volume to 200 mL in nanopure water.

14. 6-Benzylaminopurine (BAP, 1 mM): Dissolve 45.04 mg in 1 mL of 10 N NaOH. Add nanopure water to make a volume of 200 mL.

15. Thidiazuron (TDZ, 500 μM): Dissolve 22 mg in 1 mL Dimethyl sulfoxide (DMSO). Add nanopure water to make a volume of 200 mL.

16. 3-Naphthoxyacetic acid (NOA, 1 mM): Dissolve 40.44 mg in 1 mL 10 N NaOH. Add nanopure water to make a volume of 200 mL.

17. Indole-3-acetic acid (IAA, 1 mM): Dissolve 35.0 mg in 1 mL 10 N NaOH. Add nanopure water to make a volume of 200 mL.

18. α-Naphthaleneacetic acid (NAA, 1 mM): Dissolve 37.2 mg in 1 mL 10 N NaOH. Add nanopure water to make a volume of 200 mL.

19. Timentin ® _{(Smith-Kline Beecham, Boronia, Australia).}

20. Augmentin: Dissolve in nanopure water at a concentration of 250 mg/mL. Store as aliquots at −20 °C.

21. Claforan or cefotaxime stock solution: Dissolve in nanopure water at a concentration of 250 mg/mL. Store as aliquots at −20 °C.

22. Kanamycin monosulfate: Dissolve in nanopure water at a con- centration of 100 mg/mL. Store as aliquots at −20 °C. 23. Hygromycin B: Dissolve in nanopure water at a concentration

of 25 mg/mL.

24. Rifampicin: Dissolve in dimethyl sulfoxide (DMSO) at a con- centration of 25 mg/mL. Store as aliquots at −20 °C.

25. Acetosyringone: Dissolve in absolute ethanol at a concentration of 100 mM. Store as aliquots at −20 °C.

All the media are sterilized by autoclaving for 30 min at 110 °C. 1. Culture medium for initiation of embryogenic calli from

anthers depending on genotype. For PIV medium [ 12 ]: NN macroelements, MS microelements, Fe-EDTA, vitamins B5, 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 8.9 μM BAP, 60 g/L sucrose, and 3 g/L Phytagel ® _{(Sigma) as the gelling}

agent; adjust pH to 5.7 with 1 M KOH. For Harst medium [ 18 ]: NN macroelements, MS microelements, Fe-EDTA, vitamins

2.5 Plant Material and Tissue Culture