ORIGINAL PAPER

Improved somatic embryogenesis of grapevine (Vitis vinifera)with focus on induction parameters and efficient plantregeneration

J. R. Vidal Æ J. Rama Æ L. Taboada Æ C. Martin ÆM. Ibanez Æ A. Segura Æ M. E. Gonzalez-Benito

Received: 27 March 2008 / Accepted: 2 October 2008 / Published online: 12 October 2008

� Springer Science+Business Media B.V. 2008

Abstract A study of four parameters (induction medium,

floral explant, developmental stage and year) was carried

out to determine the best combination for the embryogen-

esis induction of eight grapevine (Vitis vinifera L.)

cultivars. Anthers and ovaries were extracted from flower

buds at three developmental phases and incubated in two

induction media over two consecutive years. As average,

the percentage of embryogenesis on Nitsch and Nitsch-

derived medium (9.1%) was higher than in Murashige and

Skoog-derived medium (5.9%) and embryogenesis from

ovaries (10.1%) was 2-fold higher than from anthers

(4.9%). Earlier flower developmental stages (II–III) favored

embryogenic induction from anthers, while later stages

(III–V) did it from ovaries. Induction of embryogenic cul-

tures was genotype dependent. Two years after the

establishment of the embryogenic lines, an average of 48.0%

of the pro-embryogenic masses were viable and suitable to

initiate cell suspensions. Embryogenic cultures of four

genotypes showed a high percentage of conversion from

embryos to plants: Albarino (61.8%), Garnacha (48.8%),

Tempanillo (71.0%) and Sultanina (69.0%). Moreover, cell

suspensions were competent for transient transformation

based on b-glucuronidase assay, as up to 6,387 blue spots per

Petri plate after Biolistic bombardment were obtained. Here,

we present the advantage of ovaries over anthers for the

embryogenesis induction of several grapevine cultivars. This

is the first report of embryogenesis from the cultivars Alba-

rino, Verdejo and Muscat Hamburg as well as transient

transformation of Albarino and Tempranillo.

Keywords Biolistic transformation �Embryogenic cell suspensions � Plant regeneration �Somatic embryogenesis � Vitis vinifera

Abbreviations

2, 4-D 2,4-Dichlorophenoxyacetic acid

4-CPPU N-(2-chloro-4-pyridyl)-N0-phenylurea

BAP 6-Benzylaminopurine

NOA b-Naphthoxyacetic acid

MS Murashige and Skoog medium

NN Nitsch and Nitsch medium

PEM Pro-embryogenic masses

Introduction

Grapevine (Vitis sp.), the most important fruit crop grown

worldwide, has been genetically transformed with both

Agrobacterium and Biolistics systems and regenerated

thereafter (Martinelli and Mandolino 1994; Perl et al. 1996;

Kikkert et al. 1996; Vidal et al. 2003). In addition to the

initial molecular breeding purpose for improving agro-

nomic traits such as disease resistance (Mauro et al. 1995;

Yamamoto et al. 2000; Vidal et al. 2006a), nowadays, in a

post-genomic era and after the recently published

J. R. Vidal (&) � J. Rama � L. Taboada � A. Segura

Department of Plant Physiology, Universidad de Santiago de

Compostela, Campus Sur, 15782 Santiago de Compostela, Spain

e-mail: joseramon.vidal@usc.es

J. R. Vidal

Department of Biotechnology, Universidad Politecnica de

Madrid, Ciudad Universitaria, 28040 Madrid, Spain

C. Martin � M. E. Gonzalez-Benito

Department of Plant Biology, Universidad Politecnica de

Madrid, Ciudad Universitaria, 28040 Madrid, Spain

M. Ibanez

Department of Statistics, Universidad Politecnica de Madrid,

Ciudad Universitaria, 28040 Madrid, Spain

123

Plant Cell Tiss Organ Cult (2009) 96:85–94

DOI 10.1007/s11240-008-9464-z

grapevine genome (Jaillon et al. 2007), the genetic trans-

formation of grapevine is also an indispensable tool to

study gene function.

The main plant material of grapevine used as a target for

genetic transformation and regeneration are somatic

embryogenic cultures due to their high capacity to regen-

erate into plants (Gray 1995). High regeneration frequency

is important, because stable transformation of grapevine

cells occurs at relatively low frequency (Kikkert et al.

1996; Iocco et al. 2001). The establishment of embryogenic

suspensions was first described by Coutos-Thevenot et al.

(1992) and soon it was shown to be the most suitable plant

material for grapevine genetic transformation (Hebert et al.

1993; Bornhoff and Harst 2000). However, the establish-

ment and maintenance of somatic cell suspensions in

woody species is particularly difficult (Jayasankar et al.

1999). The induction of somatic embryogenesis is not yet

routine in most laboratories and the percentage of

embryogenesis differs among grapevine genotypes. The

explant type and induction medium have been reported to

be critical for establishment of embryogenic cultures

(Martinelli and Gribaudo 2001). Anthers have been widely

employed for initiation of somatic embryogenesis in

V. vinifera (Mauro et al. 1986; Franks et al. 1998; Tor-

regrosa 1998; Perrin et al. 2001). Also leaves (Stamp and

Meredith 1988; Nakano et al. 1997), ovaries (Martinelli

et al. 2001; Lopez-Perez et al. 2005; Kikkert et al. 2005) as

well as whole flowers (Gambino et al. 2007) have been

successfully used. Previous reports focused mainly on two

induction media, MS- (Murashige and Skoog 1962) and

NN- (Nitsch and Nitsch 1969) derived ones, both supple-

mented with different plant growth regulators (PGRs).

More recently, the physiological stage of flower buds

(mainly focused on anther developmental stages) was

considered crucial for the success of embryogenesis

induction (Gribaudo et al. 2004). Most studies of grapevine

embryogenesis induction report the occurrence of

embryogenesis without comparison of several parameters

at the same time. After the initiation, browning of

embryogenic cultures is frequently observed, leading to

loss of cell viability and plant regeneration.

The goal of this work was to develop a somatic

embryogenesis protocol that could be widely applicable for

different genotypes by studying the effect of four param-

eters on the induction and establishment of embryogenesis

of nine grapevine cultivars. Two plant explants (anthers

and ovaries) harvested at three different physiological

phases and incubated on MS- and NN-derived media in

two consecutive years were investigated. Also, embryo

development from both callus masses on solid media and

cell suspensions on liquid media was compared. Moreover,

the percentage of plant regeneration and the efficiency of

genetic transformation of cell suspensions were studied.

Materials and methods

Plant material, explant preparation and induction media

Eight elite Vitis vinifera genotypes in two consecutive

years were tested: Albarino, Verdejo, Chardonnay [as

white grapes]; Garnacha (Grenache), Tempranillo, and

Cabernet Sauvignon clon 15 [as red grapes]; and Moscatel

de Hamburgo (Muscat Hamburg) and Sultanina (Thomp-

son seedless) as table grapes. In the second year, Pinot

Meunier (V. vinifera) was also included in the study.

Mature vines from vineyards at ‘‘EL ENCIN’’ (IMIDRA)

in Alcala de Henares, Spain, were the source of plant tis-

sue. Clusters of flower buds were collected during spring

12 ± 2 days pre-bloom (when most anthers were translu-

cent green-yellow during uninucleate microspore

formation), washed and stored as described by Gray

(1995). Flower buds of each cultivar were collected

according to four floral developmental stages: Phase

A (II–III), B (III–IV) and C (IV–V) (Mullins et al. 1992;

Gribaudo et al. 2004). Immature anthers and ovaries were

extracted from unopened flowers after surface sterilization

as previously described (Kikkert et al. 2005). Approxi-

mately 50 anthers or 10–20 ovaries per Petri dish were

plated on two different MS- and NN-derived induction

media. MS-derived medium (pH 5.8) contained MS salts

and vitamins supplemented with 4.52 lM 2,4-D (2,4-

dichlorophenoxyacetic acid), 4.44 lM BAP (6-benzyl-

aminopurine), 2.0% (w/v) sucrose, 0.01% (w/v) inositol,

0.3% (w/v) Phytagel (Perrin et al. 2004). NN-derived

medium (pH 5.7) contained NN salts and MS vitamins

supplemented with 2.5 lM 2,4-D (2,4-dichlorophenoxy-

acetic acid), 2.5 lM NOA (b-naphthoxyacetic acid),

5.0 lM 4-CPPU N-(2-chloro-4-pyridyl)-N0-phenylurea,

0.05% (w/v) glutamine, 3.0% (w/v) sucrose, 0.01% (w/v)

inositol, 0.3% (w/v) Phytagel (Kikkert et al. 2005).

Because of the large number of explants plated, extractions

were done during a 2-week period each year by up to six

people (Table 1). Some explants were lost to contamina-

tion and were not included in Table 1. Petri dishes with

explants were wrapped with Parafilm and incubated at

25 ± 1�C in the dark. Embryogenesis induction procedure

was the same in the two consecutive years.

Embryogenesis induction, culture maintenance

and statistical analysis

Explants were transferred to fresh medium of the same

formulation every 2 weeks during the first month and every

4 weeks during five additional months. At each subculture,

explants were examined under a dissecting microscope at

109 for callogenesis initiation and embryogenesis induc-

tion. Most explants, both anthers and ovaries, grew during

86 Plant Cell Tiss Organ Cult (2009) 96:85–94

123

the six first months and showed callusing responses.

Thereafter, explants that had formed friable and whitish

callus (that appeared pro-embryogenic) were transferred to

the same medium for six additional months, and dead

explants as well as non-embryogenic tissues were dis-

carded. Thereafter, pro-embryogenic masses (PEM) were

maintained on the original embryogenic initiation medium

with transfer to fresh medium every 6–8 weeks. The

number of explants forming PEM was recorded every

4 weeks over the first 6 month period after induction. Cell

viability of established PEM was determined by using the

triphenyltetrazolium chloride (TTC) reduction assay

(Steponkus and Lanphear 1967). The percentages of

embryogenic callus were analysed using a logit model

(Agresti 1996) and treatment means were separated using a

Least Significant Difference (LSD) test. All analyses were

undertaken using the function General Lineal Model of R

(Ihaka and Gentleman 1996).

Table 1 Embryogenesis percentage from anthers and ovaries of eight grapevine cultivars incubated on MS- (Murashige and Skoog) and NN-

(Nistch and Nistch) derived media in two consecutive years

Genotype Year Anthers Ovaries

Total no.

explants

Embryogenic

callus

Estimation (%)

of embryogenesis

Total no.

explants

Embryogenic

callus

Estimation (%)

of embryogenesis

MS-derived medium

Albarino 2005 606 12 1.98 a 178 28 15.70 ef

Albarino 2006 616 8 1.30 a 158 10 6.33 bcd

Cabernet Sauvignon 2005 600 2 0.33 a 178 1 0.56 a

Cabernet Sauvignon 2006 562 1 0.18 a 138 0 0.00 a

Chardonnay 2005 550 7 1.27 a 137 6 4.38 abcd

Chardonnay 2006 698 45 6.45 b 212 38 17.90 f

Garnacha 2005 590 23 3.90 ab 161 12 7.45 cd

Garnacha 2006 469 68 14.50 c 102 9 8.82 cde

Muscat Hamburg 2005 500 0 0.00 a 134 1 0.75 a

Muscat Hamburg 2006 386 1 0.26 ab 105 4 3.81 abcd

Sultanina 2005 400 51 12.80 c 92 29 31.50 g

Sultanina 2006 538 146 27.10 d 131 14 10.70 def

Tempranillo 2005 800 1 0.12 a 221 4 1.81 a

Tempranillo 2006 418 0 0.00 a 127 5 3.94 abc

Verdejo 2005 700 0 0.00 a 178 1 0.56 a

Verdejo 2006 500 0 0.00 a 125 2 1.60 ab

NN-derived medium

Albarino 2005 700 9 1.29 a 201 30 14.90 cde

Albarino 2006 676 16 2.37 a 187 23 12.30 bcde

Cabernet Sauvignon 2005 550 4 0.73 a 173 0 0.00 a

Cabernet Sauvignon 2006 484 8 1.65 a 129 0 0.00 a

Chardonnay 2005 504 18 3.57 abc 125 18 14.40 bcde

Chardonnay 2006 698 54 7.74 bcd 197 30 15.20 cde

Garnacha 2005 600 56 9.33 cd 166 35 21.10 e

Garnacha 2006 534 64 12.00 d 134 26 19.40 e

Muscat Hamburg 2005 450 7 1.56 ab 119 7 5.88 abcd

Muscat Hamburg 2006 370 17 4.59 abcd 117 5 4.27 abc

Sultanina 2005 400 47 11.70 d 96 52 54.20 f

Sultanina 2006 501 117 23.04 e 138 24 17.40 de

Tempranillo 2005 800 2 0.25 a 213 11 5.16 ab

Tempranillo 2006 514 0 0.00 a 150 1 0.67 a

Verdejo 2005 650 1 0.15 ab 174 22 12.60 bcde

Verdejo 2006 550 20 3.64 abc 180 3 1.67 a

The estimation of the embryogenesis was analysed using a logit model and treatment means were separated using a Least Significant Difference

test. Values within a column for each induction medium that are followed by the same letter are not significantly different at the 5% level

Plant Cell Tiss Organ Cult (2009) 96:85–94 87

123

Initiation of cell suspensions cultures

Embryogenic cell suspensions of Albarino, Garnacha,

Tempranillo and Sultanina were initiated on liquid

GM ? NOA medium [MS salts and vitamins with 0.37%

(v/v) glycerol, 1.8% (w/v) maltose and 5.0 lM b-naphth-

oxyacetic acid (NOA); Mauro et al. 1995] from PEM grown

on solid medium. A three-step procedure was followed. First,

cell masses (approx. 200 ± 50 mg) were transferred to

125 ml Erlenmeyer flasks that contained 15 ml of GM ?

NOA medium (Kikkert et al. 2005). Flasks were placed on a

gyratory shaker at 120 rpm and 25 ± 1�C in the dark. One

half of the culture medium was removed and replaced with

fresh medium each week during at least 2 weeks. Second,

when the initial cell density had doubled, the suspension was

doubled with fresh medium to a final volume of 30 ml in the

same flask and the medium was refreshed each week as above

for two additional weeks. Third, when the cell density had

doubled again, cultures were transferred to 250 ml Erlen-

meyer flasks and fresh medium was added to a final volume of

60 ml for establishment of cell suspensions. Thereafter, at

weekly intervals, one-half of the medium (30 ml) was dis-

carded and replaced with fresh medium. When cell density

increased, suspensions were either divided in two 250 ml

flasks (30 ml of suspension plus 30 ml of fresh medium) or

brought to a final volume of 120 ml in a 500 ml Erlenmeyer

flask by adding 60 ml of fresh medium. Cell suspensions were

filtered as needed through a mesh (1.0 mm2 pore size) to

eliminate large clumps.

Embryo development and plant regeneration

Four embryogenic lines (Albarino, line 18; Garnacha,

line 9; Tempranillo, line 7 and Sultanina, line 16) were

selected for regeneration and transformation experiments

due to their high proliferation activity. Embryogenic cul-

tures, from both cell suspensions in liquid medium and

callus masses in solid medium, were spread onto filter paper

(Whatman No. 2, 8 cm diameter) pre-dampened with

1.0 ml GM ? NOA medium, and cultured using conditions

developed for regeneration of plants after biolistic trans-

formation (Kikkert et al. 2004). Cell suspensions at

exponential phase were filtered through a mesh (1.0 mm2

pore size) and the cell density adjusted to 0.2 ml packed cell

volume (PCV) per 10 ml suspension. One ml of adjusted

suspension was spread as a single layer onto a filter paper.

Callus masses were weighted and approx. 250 mg of callus

was also spread as a single layer onto a filter paper using

sterile forceps with flat ends. Filter papers containing

embryogenic cultures were then placed on PGR-free

embryo induction medium [MS medium with half-strength

macro- and micro-elements, full-strength vitamins and

0.01% (w/v) inositol, 3.0% (w/v) sucrose, 0.3% (w/v)

activated charcoal and 0.25% (w/v) Phytagel (Kikkert et al.

1996)]. Petri plates were wrapped with Parafilm and incu-

bated in the dark at 25 ± 1�C. A total of three plates per

cultivar and embryogenic culture type were incubated

during 3 months. Cells on the original filter paper support

were transferred to fresh embryo induction medium every

4 weeks. At each transfer, individual emerged embryos

(1–2 cm long radicle) were counted and removed from the

plates and discarded. Data were adjusted to 1 ml of PCV for

cell suspensions and to 1 g of weight for callus mass. Sig-

nificant difference in the number of embryos developed

between gram of callus and ml of cell suspension was

analysed by Student’s t-test. Some embryos were saved and

used for plant regeneration tests (Table 4). Selected

embryos were transferred to Petri plates for embryo ger-

mination and plant regeneration as previously described

(Vidal et al. 2003). The number of embryos that regenerated

plants within 3 months was recorded. The development and

regeneration experiments were repeated at least three times.

Plant cell transformation

Six to 12-month-old embryogenic cell suspensions of cul-

tivars Albarino, Garnacha, Tempranillo and Sultanina were

used for Biolistic transformation. Preparation of plant cells

on filter paper support, coating gold particles with DNA as

well as particle bombardment using the PDS-1000/He

biolistic device (BioRad, Hercules, CA) were performed

following the step by step procedure described in Kikkert

et al. (2004). The transformation competency of the

grapevine cell suspensions was determined by the b-glu-

curonidase (GUS) assay after bombardment with vector

pBI221 [containing the gus gene] (Clontech) or vector

pBI426 [containing a gus/npt-II gene fusion] (Vidal et al.

2006b). Albarino, Tempranillo and Sultanina were bom-

barded with pBI426 and Garnacha with pBI221. Density of

the cell suspension was adjusted to 0.2 ml of settled cell

volume per 10 ml of suspension and then 5 ml of cells were

spread as a fine layer onto a filter paper support (Kikkert

et al. 2004). A total of three plates containing cells per

cultivar were bombarded and at least three repetitions were

made. Transient GUS expression assay was carried out as

described elsewhere (Vidal et al. 2003) and the number of

blue spots per plate was counted with a stereomicroscope by

using a plastic sheet with an imprinted grid.

Results

Initiation of somatic embryogenesis

All genotypes formed somatic pro-embryogenic callus

from anthers and/or ovaries in both MS and/or NN media

88 Plant Cell Tiss Organ Cult (2009) 96:85–94

123

in at least one of the 2 years they were tested (Table 1).

Most of explants had callogenesis response (Fig. 1a, b).

There were four main types of response after 6 months of

incubation on induction media: anthers or ovaries with no

growth (dead explants, Fig. 1c), explants with compact

non-embryogenic callus (Fig. 1d), explants with friable

soft (foam type) callus (that appeared non-embryogenic)

(Fig. 1e), and obvious embryogenic callus with a fine

granular appearance (Fig. 1f). First embryogenic masses

were observed from Sultanina explants after 6 weeks of

culture. Embryogenesis induction was followed over a

6 months period for all genotypes. Thereafter, independent

embryogenic lines with friable and whitish appearance

were transferred to individual Petri plates containing media

of the same formulation for embryogenic mass growth and

later callus propagation.

All cultivars formed embryogenic callus on both

media; however, Pinot Meunier that was only tested the

second year, formed pro-embryogenic masses only from

anthers on NN medium (data not shown). For most

grapevine cultivars, the first embryogenic callus appeared

10 ± 2 weeks after plating and new embryogenesis

events on incubated explants appeared over the 6 months

induction period. The percentage of embryogenesis was

significantly dependent of the genotype (Table 1). The

highest frequencies of embryogenesis were observed in

Sultanina, Garnacha, Chardonnay and Albarino for both

explants and media and the lowest rates of embryogenesis

occurred in Cabernet Sauvignon, Tempranillo, Muscat

Hamburg and Verdejo. There was significant variation in

the percentage of embryogenesis within genotypes

between the 2 years (P \ 0.0001). Overall, as average for

cultivars, media and years, there was more than a 2-fold

increase in embryogenesis from ovaries (10.14%) com-

pared to anthers (4.87%) (P \ 0.0001). In general,

averaging over cultivars, explant type and years the per-

centage of embryogenesis induced on MS medium

(5.91%) compared to NN medium (9.10%) was signifi-

cantly different (P = 0.0008). The two-way interaction

between genotype and media, and between year and

medium was significant (P = 0.005 and P = 0.017,

respectively). The three-way interaction among genotype,

year and explant type was highly significant (P = 0.009)

but not the rest of three-way interactions [genotype,

medium and explant type (P = 0.153), as well as year,

medium and explant type (P = 0.484)]. Although there

was a predominant increase of embryogenesis from ova-

ries compared to anthers, there were particular instances

Fig. 1 Responses of anthers and ovaries incubated on embryogenic

induction media for obtaining competent pro-embryogenic cultures

for plant regeneration and genetic transformation: a, early callogen-

esis response on anther; b, early callogenesis response on ovary; c,

dead ovary 1 month after plating; d, example of compact non-

embryogenic callus from anther; e, example of friable (foam type)

non-embryogenic callus from ovary; f, friable (granular type)

embryogenic callus mass from Sultanina anther; g, developed

embryos from pro-embryogenic cultures of Albarino on germination

medium; h, germination and regeneration of Albarino plantlets

1 month after germination induction; i, blue spots on cell suspensions

of Tempranillo 2 days after bombardment of cells with plasmid

pBI426

Plant Cell Tiss Organ Cult (2009) 96:85–94 89

123

in which anthers responded a little more favorably than

ovaries (e.g., Sultanina in second year). In general, the

frequency of embryogenesis for all genotypes was higher

in NN-derived medium than in MS-derived medium, with

few exceptions (anthers of Sultanina and ovaries of

Tempranillo in the second year); however, differences

between both media were low (Table 1).

A statistical analysis considering separately the three

developmental phases of the flowers was performed

(Table 2). The two-way interaction between explant and

phase was significant (P B 0.0001). In general, the per-

centage of embryogenesis from anthers was higher in phase

A (6.26%) than phases B (4.99%) and C (3.55%). From

ovaries, phases B (10.71%) and C (9.51%) promoted a

higher frequency of embryogenesis compared to phase A

(8.89%). This tendency was common for most genotypes in

both MS- and NN-derived medium with the exception of

Sultanina, that showed the highest percentage from anthers

in phase C and from ovaries in phase A (Table 2). The

three-way interaction among genotype, explant and

phase was significant (P B 0.0001). The two-way interac-

tion between medium and phase was also significant

(P = 0.03). In this analysis, NN-derived medium also

promoted the best percentage of embryogenesis for all

phases considered. Phase B showed the highest difference

between MS (5.29%) and NN (9.91%) media. The three-

way interactions among explant, medium and phase

(P = 0.21) and among genotype, medium and phase

(P = 0.83) were not significant.

Establishment and maintenance of embryogenic callus

One year after floral explant culture, established embryo-

genic lines were selected for callus multiplication based on

regular pro-embryogenic mass growth and whitish color

(Table 3). Embryogenic masses were transferred to fresh

media of the same formulation every 7 ± 1 weeks by

selecting whitish PEM and removing brown parts (if any)

of the callus with forceps under a stereomicroscope. In

addition to the proliferation activity of the established

embryogenic lines, cell viability was determined by a TTC

assay following a three-category scale (Table 3). Based on

the TTC test, the viability of the embryogenic lines 2 years

after the establishment of the embryogenic cultures ranged

from 35% of efficiency for Garnacha to 80% of efficiency

for Muscat Hamburg (Table 3).

Table 2 Estimation of embryogenesis from anthers and ovaries at three developmental phases of eight grapevine cultivars incubated on MS-

(Murashige and Skoog) and NN- (Nistch and Nistch) derived media

Genotype Estimation of embryogenesis (%) at three developmental phases

Anthers Ovaries

A [II–III] B [III–IV] C [IV–V] A [II–III] B [III–IV] C [IV–V]

MS-derived medium

Albarino 2.35 d 1.19 cd 1.44 cd 11.10 fgh 10.30 efg 12.50 fgh

Cabernet Sauvignon 0.21 ab 0.44 abc 0.11 ab 1.08 abcd 0.0 a 0.00 a

Chardonnay 7.30 e 5.59 e 0.65 bc 11.70 fgh 14.40 gh 12.30 fgh

Garnacha 13.80 f 5.98 e 6.89 e 7.74 defg 5.31 bcde 11.90 fgh

Muscat Hamburg 0.20 bc 0.04 a 0.06 ab 3.74 abcd 1.52 ac 0.00 a

Sultanina 22.80 g 15.80 f 29.80 h 25.10 i 18.50 hi 11.60 efgh

Tempranillo 0.30 abc 0.00 a 0.00 a 1.10 a 5.62 cdef 2.24 ab

Verdejo 0.00 a 0.00 a 0.00 a 0.74 a 1.46 abc 0.83 a

NN-derived medium

Albarino 2.10 b 1.55 ab 1.81 b 10.70 defg 14.00 efgh 16.30 gh

Cabernet Sauvignon 0.67 ab 2.05 b 0.51 ab 0.00 a 0.00 a 0.00 a

Chardonnay 8.99 d 9.84 de 1.15 ab 10.70 cdefg 18.40 gh 15.30 fgh

Garnacha 14.10 ef 8.72 d 9.67 d 16.30 fgh 16.10 gh 30.80 i

Muscat Hamburg 4.59 c 1.40 ab 1.94 abc 7.59 cdef 4.56 abcd 0.0 a

Sultanina 16.20 f 15.30 f 28.40 g 34.80 i 34.70 i 22.90 hi

Tempranillo 0.52 ab 0.00 a 0.00 a 0.97 a 7.15 cde 2.77 ab

Verdejo 5.00 c 0.22 a 0.00 a 4.07 abc 11.00 defgh 6.34 bcd

Data are from two consecutive years

The estimation of the embryogenesis was analysed using a logit model and treatment means were separated using a Least Significant Difference

test. Values within a same organ (columns A, B and C) for each medium that are followed by the same letter are not significantly different at the

5% level

90 Plant Cell Tiss Organ Cult (2009) 96:85–94

123

Cell suspensions and plant regeneration

Liquid cell suspension cultures of Albarino, Garnacha,

Tempranillo and Sultanina were initiated and established

on GM ? NOA medium from PEM with a three-step

procedure. Garnacha and Sultanina suspensions grew and

multiplied their cell density faster than Albarino and

Tempranillo suspensions. It was also observed that

Tempranillo and Sultanina cell cultures were finer sus-

pensions than those from Albarino and Garnacha.

Moreover, cell suspensions aged after a year of culture and

weekly multiplication of suspensions was needed to keep

optimal exponential phase (data not shown). Embryogenic

cultures from both cell suspensions and callus masses of

those four cultivars [Albarino, line 18; Garnacha, line 9;

Tempanillo, line 7 and Sultanina, line 16] were plated and

grown under conditions to promote embryo development

and plant regeneration. Three months after plating, the

emergence of embryos from one milliliter of cell suspen-

sions was higher than from 1 g of callus masses for each

cultivar assayed (Table 4). Albarino was the cultivar that

showed a lower difference in embryo development

(Fig. 1g) between gram of callus (1,350 ± 378 per plate)

and ml of suspension (4,683 ± 905 per plate). A total of

365 elongated embryos from these four cultivars were

selected for conversion to plantlets. Embryos were firstly

grown on germination and then on plant regeneration

media (Fig. 1h). The percentage of plant regeneration was

62.5% for all four cultivars at 6 months after initial plating

(only 3 months for germination and regeneration) and

ranged from 48.8% for Garnacha to 71% for Tempranillo

(Table 4).

Transient transformation

Embryogenic cell suspensions were evaluated by transient

GUS expression following biolistic transformation with a

construct containing the gus gene (Fig 1i). GUS expres-

sion 2 days after particle bombardment yielded a high

number of blue spots per plate for all four cultivars assayed

[Albarino (5,411 ± 849, average ± standard error), Gar-

nacha (1,895 ± 376), Tempranillo (6,387 ± 766) and

Table 3 Viability percentage of embryogenic callus 2 years after establishment of embryogenic lines

Genotype No. of established

embryogenic linesaNo. of viable lines by TCC testb Viability (%)

efficiencyTotal (?) (??) (???)

Albarino 20 8 0 1 7 40.0

Cabernet Sauvignon 5 2 1 1 0 40.0

Chardonnay 20 9 3 3 3 45.0

Garnacha 20 7 1 1 5 35.0

Tempranillo 15 7 2 2 3 46.7

Muscat Hamburg 10 8 1 1 6 80.0

Sultanina 20 9 6 3 0 45.0

Verdejo 15 10 2 2 6 66.7

125 60 16 14 30 48.0

a Some established lines were lost and discarded due to contaminations or browning of the embryogenic masses during the 2 years of cultureb Embryogenesis potential as determined by visual observation of positive TCC lines was recorded on three-category scale: (?) low, (??)

medium and (???) high

Table 4 Number of embryos emerged per Petri plate from embryogenic callus and from cell suspension

Embryogenic linea Number of embryos per Petri plate No. of embryos

selected and

germinated

Percentage (%)

of plant

regenerationper gram of embryogenic callus per ml of cell suspension

Albarino 18 1,350 ± 378 4,683 ± 905 110 61.8

Garnacha 9** 435 ± 59 6,767 ± 546 86 48.8

Tempranillo 7* 322 ± 11 1,800 ± 404 69 71.0

Sultanina 16** 789 ± 163 7,920 ± 586 100 69.0

365 62.5

The percentage of plant regeneration from selected embryos of four embryogenic lines is showna Significant difference (average ± standard error) in the number of embryos emerged between gram of callus and milliliter of cell suspension

(* P \ 0.05; ** P \ 0.001)

Plant Cell Tiss Organ Cult (2009) 96:85–94 91

123

Sultanina (2,149 ± 307)]. Several repetitions for each

cultivar were performed; although different cell suspen-

sions were used, similar results were obtained. To mention

that results for Garnacha and Sultanina are from bom-

bardments with a first version of Biolistic PDS unit,

however results for Albarino and Tempranillo are from

bombardments with a new version of Biolistic PDS unit.

These results confirm the competence of the somatic

embryogenic cell suspensions obtained to approach genetic

transformation projects.

Discussion

Most reports on initiation of somatic embryogenic cultures

of grapevine are mainly focused on few elite cultivars.

Embryogenic induction has been so far difficult or

impossible for certain genotypes. Nowadays, researchers of

traditional and new winegrowing countries, are interested

in initiating somatic embryogenesis of other elite cultivars

for genetic transformation projects. Therefore, it is neces-

sary to have available protocols to initiate somatic

embryogenesis of grapevine with success within a year. We

report an experimental design based on four parameters

[induction medium, floral tissue, developmental stage, and

year] to initiate embryogenic cultures. Embryogenesis was

achieved every year with at least one parameter combina-

tion for all genotypes assayed. Moreover, established

embryogenic cultures were competent for plant regenera-

tion and transformation. Here, new and improved results

are reported.

Medium MS with 2,4-D and BAP has been widely used

for anther culture of Vitis sp. with variable success on

establishment of embryogenic cultures (Gray 1995; Perrin

et al. 2004 [0.0–42.5%]). In our trials, the frequency of

embryogenesis for anthers was less than 7% with all

genotypes tested except for Garnacha (up to 14%) and

Sultanina (up to 27%). In contrast, embryogenesis from

ovaries on MS-derived medium was higher than from

anthers (Table 1). Medium NN with 2,4-D, NOA, 4-CPPU

and glutamine was successfully used for anther and ovary

culture but embryogenesis percentage (up to 20%) was

genotype dependent (Kikkert et al. 2005). Sources of

reduced nitrogen such as glutamine have been reported to

aid embryogenesis in Vitis sp. (Mauro et al. 1986; Marti-

nelli et al. 1993). In our experiment, the highest

percentages of embryogenesis (up to 54%) were obtained

with NN-derived medium, especially when ovaries were

used as explants (Table 1). Previously, somatic embryo-

genesis of grapevine anthers on NN medium using the

same PGRs has been reported (Iocco et al. 2001; Franks

et al. 2002). However, in these reports glutamine was not

include and the success of embryogenesis was lower,

confirming the beneficial effect of glutamine in NN med-

ium for embryogenesis induction from anthers and ovaries

(Kikkert et al. 2005).

In our research, the frequency of embryogenesis from

ovaries was generally better than from anthers. The fre-

quency from anthers during early phase A was generally

higher than at phase B and C for all cultivars, in agreement

with a previous report on cultivars Chardonnay and Bar-

bera (Gribaudo et al. 2004). However, embryogenesis from

ovaries was favored by later phases (B and C). The

advantages of ovaries compared to anthers are the larger

size and the easier handling. Anthers are really small and

difficult to handle compared to ovaries during early phys-

iological phases such as stage II, which benefits the choice

of ovaries. In the present work, embryogenesis frequency

was generally higher from ovaries than anthers for all

developmental stages considered (Table 2). Whole flowers

are also a suitable material; however, contamination from

flower explants could be a pitfall (Gambino et al. 2007). To

the best of our knowledge, this is the first report on ovary-

derived embryogenesis at different developmental stages.

Somatic embryogenesis was obtained from all geno-

types tested every year on at least one medium formulation

and one type of plant tissue (Table 1). Differences between

consecutive years for a cultivar may be due to the physi-

ological status of the vines, slight differences in floral

developmental stage or the person who extracted and pla-

ted the explants (Perrin et al. 2004; Kikkert et al. 2005).

The percentage of embryogenesis was strongly influenced

by the induction medium as well as the floral explant and

developmental stage. Somatic embryogenesis has been

widely reported in Chardonnay and Cabernet Sauvignon

using several media (Gray 1995; Perrin et al. 2004; Kikkert

et al. 2005). Here, embryogenic cultures from Chardonnay

anthers (1.3–7.7%) and ovaries (4.4–17.9%) were pro-

duced. In contrast, Cabernet Sauvignon was recalcitrant in

agreement with previous reports (Perrin et al. 2004) and

required the culture of many explants to establish

embryogenic cultures from anthers (0.2–1.6%) and ovaries

(0–0.6%). Embryogenesis in Sultanina was previously

obtained from anthers, but frequency was not reported

(Scorza et al. 1996; Franks et al. 1998). Here, Sultanina

yielded the highest rates of embryogenesis from anthers

(11.7–27.1%) and ovaries (10.7–54.2%) on both MS and

NN media. This is the first report on ovary-derived

embryogenesis for Sultanina. Embryogenesis of Muscat

Hamburg has not been previously reported. The frequency

of embryogenesis with Muscat was low in both years

ranging from 0–4.6% with anthers to 0.7–5.9% with ova-

ries. Embryogenesis from Pinot Meunier was previously

obtained from anthers on NN-derived medium without

glutamine although percentage was not reported (Franks

et al. 2002), and also on MS-derived medium at low (0.2%)

92 Plant Cell Tiss Organ Cult (2009) 96:85–94

123

efficiency (Perrin et al. 2004). Here, we obtained a rela-

tively high percentage of embryogenesis only from anthers

(14.1% in stage II) on NN-derived medium including

glutamine.

The embryogenesis efficiency reported for Garnacha,

always from anthers, ranged from 2.4 to 14.1% (Perrin

et al. 2004) while for Tempranillo, efficiency was not

reported (Ben Amar et al. 2007). In our experiment, the

frequency of embryogenesis for Garnacha ranged from

3.9–14.5% with anthers to 7.5–21.1% with ovaries. In

contrast, the percentage of embryogenesis for Tempranillo

with anthers (0–0.25%) and ovaries (0.7–5.2%) was sub-

stantially low, therefore, it could be considered a

recalcitrant cultivar for somatic embryogenesis. To our

knowledge, somatic embryogenesis from Albarino and

Verdejo has not been previously reported. Here, we

obtained a low percentage of embryogenesis from Verdejo

anthers (0–3.6%) and ovaries (0.6–12.6%). Most of Verd-

ejo explants rapidly died on both induction media and half

of ovaries developed a non-embryogenic callus. However,

the percentage of embryogenesis from Albarino anthers

(1.3–2.4%) and ovaries (6.3–15.7%) was slightly higher.

Anthers and ovaries formed high quality embryogenic

cultures characterized by fine, granular, friable PEM with

rapid growth. The frequency of embryogenesis was taken

6 months after the start of embryogenesis induction.

Comparing frequency of embryogenesis for a particular

genotype among reports is complicated because of differ-

ences in manipulation, induction media and developmental

stage of the explants. Kikkert et al. (2005) reported that

somatic embryos continued arising over a 12-month period.

Here, PEM of Albarino, Garnacha, Tempranillo and Sul-

tanina that had fine, granular whitish aspect and gave

positive viability by TTC test were assayed for starting

suspension cultures and evaluating plant regeneration.

Because cell suspensions aged after a year of culture, to

reduce the risk of somaclonal variation and declining

regenerative capacity, cell suspensions should be re-initi-

ated periodically (Kikkert et al. 2005). For this important

goal, the addition of conditioned media or arabinogalactan

proteins for initiation of embryogenic suspensions sug-

gested by Ben Amar et al. (2007), could greatly benefit

long-term transformation programs. Embryo development

from cell suspensions was significantly higher than from

callus masses, as expected, which justifies the selection of

suspensions for genetic transformation. The efficiency of

regeneration for Garnacha (48.8%) and Tempranillo

(71.0%) was higher than in previous reports 15.5 and

11.7%, respectively, (Perrin et al. 2001; Ben Amar et al.

2007). Plant regeneration from embryogenic cultures of

Albarino (61.8%) and Sultanina (69.0%) has not been

previously reported. Preliminary bombardments of cell

suspensions from those cultivars gave up to 6,387 blue

spots per plate as determined by the transient GUS assay.

Thus, embryogenic cell suspensions were suitable for bi-

olistic transformation in agreement with previous reports

(Hebert et al. 1993). However, it should be noted that

following particle bombardment there is an important

decrease from transient to stable transformation (up to

99.4%) as reported elsewhere (Vidal et al. 2003).

The strategy described here based on 12 combinations of

three parameters per genotype and year [induction medium

(2), floral explant (2), developmental phase (3)], allowed the

reliable production of embryogenic cultures suitable for

grapevine transformation. Two years after establishment of

the first embryogenic cultures, approx. 50% of the

embryogenic lines were viable for cell suspension initiation.

Our experience suggests focusing in ovaries cultured on

NN-derived medium to initiate embryogenic cultures of

grapevine genotypes. This is the first report of embryogen-

esis from Albarino, Verdejo and Muscat Hamburg as well as

transient transformation of Albarino and Tempranillo.

Acknowledgments We gratefully acknowledge financial support

from a Marie Curie International Re-integration Grant (MIRG-2005-

28341) of the European Commission and an I ? D ? i Grant (AGL-

2006-5856) of the Spanish Ministry of Education and Science (MEC).

JR Vidal was fully supported by a Ramon y Cajal contract from MEC.

Thanks are also extended to F. Cabello for allowing us to collect

flower buds from vineyards at IMIDRA (Comunidad de Madrid) and

to MJ Carmona for the use of her laboratory facilities.

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