Rootstock Breeding in Grapes

Many nurseries now offer trees with a choice of several rootstocks. Rootstocks play an
essential role in determining orchard performance. They are responsible for water and mineral
uptake and provide anchorage for the tree. Rootstocks may also provide some degree of tolerance
to drought and flooding. However, disease resistance and quality improvement is the major
components of rootstock breeding programs (Roper, 2001).
Importance of Grape rootstock
It is generally believed that Vitis vinifera, the grape of antiquity, has the
genome of 2n=38, originated in the Middle East and its cultivation began during
the Neolithic era (6,0005,000 BC) along the eastern shores of the Black Sea
(Mullins et al., . 1992). In India, grape is grown on an area of 45,000 hectare with an
annual production of 1,20,000 tones in Maharashtra and in Karnataka it is grown on an area of
12,000 hectare with a production of 1,65,000 tones (Sheikh and Manjula, 2009).
Rootstocks offer grape producers a risk avoidance strategy (www.csiro.au) by acting as a
tool to manipulate vine performance. With reports of declining yield in the states of Maharashtra
and North Karnataka due to salinity of soil, water and severe drought, the farmers have realized
the importance of grape rootstock. Apart the drivers for rootstock adoption are wide ranging with
the more important being phylloxera, nematode and salt tolerance. Water-use efficiency and
drought tolerance are also increasingly important.
Rootstock breeding in grapes
Rootstock breeding has driven call for reduced vigour to counter the negative impacts of
high vigour on berry composition; for reduced potassium uptake to counter the impact of high
berry potassium on pH; and to reduce the need for pH adjustment during winemaking (Walker
and Clingeleffer, 2009). Different Vitis species are known for their ability to tolerate soil
infestations of phylloxera and nematodes. In breeding new rootstocks, the aim is to combine
resistance to these pests along with other key traits. At this point of view, it is important to test
hybrids for resistance to different races or biotypes of pests before they are selected for use as
rootstocks in evaluation trials.

Planchon (1887) divided the Vitis species into two sub-genera namely
Euvitis (bunch grapes) and Muscadinia (muscadine grapes). Muscadinia has a
genome of 2n =40, similar to the one of several other genera of Vitaceae
(Ampelopsis, Ampelocissus, Parthenocissus), while for Euvitis it is 2n =38.
Muscadinia is endemic to the southeastern states of the USA and among the
three species known, only Vitis rotundifolia Michx. is of commercial value
(Olien, 1990).
Great genetic diversity is found in grapevines and they are adapted to
different soils and climates. A number of American Vitis spp. were used in the
past and are still being used in breeding programmes, especially in
developing phylloxera resistant rootstocks, but also disease resistant wine
and table grape cultivars. Of these, the following are of highest importance:
V. labrusca, V. aestivalis and V. rupestris Vitis spp. native to tropical America,
like V. smalliana, V. caribaea and V. shuttleworthii are included in breeding
programmes to develop cultivars adapted to climatic conditions of the tropics
(Camargo, 2000). In the Eastern European and Asian countries, V. amurensis
is often included in breeding programmes to introduce cold hardiness and
disease resistance.
Objectives of rootstock breeding

Breeding rootstocks for tolerance to root pests of phylloxera, nematodes

Tolerance to adverse soil conditions salinity, high lime and pH, low nutrient
status, poor water availability

To possess favourably nursery and viticultural characteristics of high propagation

rate, graft compatibility, appropriate growth, yield and fruit quality.

History and results of grape rootstock breeding

In Europe, the need of using of grapevine rootstocks occurred after the phylloxera
invasion in the second half of the 19th century. The destruction of European viticulture took

place above all after 1860s when phylloxera was inadvertently introduced to Europe and
devastated the European grapevine (Vitis vinifera L.). The first vines began to deteriorate in
France and the problem spread rapidly across the European continent (Granett et al., 2001).
Several species of North American grapes viz V. berlandieri, V. champinii, V. rupestris and

V.

riparia were found to have certain characteristics like phylloxera resistance, nematode
resistance, special value as rootstocks in certain soils and drought tolerance.
After several experiments with a chemical liquidation of this pest the plant breeders and
selectionists established new nurseries and started to breed resistant rootstocks. In France, the
breeding of rootstocks was oriented either to genuine Vitis species Vitis rupestris Scheele, Vitis
riparia Michx. or to their interspecific hybrids and hybrids with the botanial species Vitis
berlandieri Planch. (Kocsis et al., 2002). As usual, roots of rootstocks selected on the base of
North American Vitis spp. are not at all (or only a little) infested by phylloxera. Rootstocks with
a partial share of Vitis vinifera L. in their pedigree can also be highly phylloxera-resistant but
such a resistance is not of permanent nature (Martinez-Peniche, 1999). In the former AustroHungarian Monarchy, former Czechoslovakia and present Czech Republic, the breeding work
was performed by the Hungarian breeder Zsigmond Teleki. His breeding and selection process
resulted in the creation of a number of rootstocks that are still being used in all wine-growing
countries of the worlds (Bakonyi et al., 1997). Telekis rootstocks are still an important part of
the breeding programme of resistant rootstocks.
Use of Euvitis Species in Resistance Breeding
The introduction of American species resistant to phylloxera (mainly V. riparia, V.
rupestris and V. berlandieri) and intensive breeding programmes saved grapevine production in
many regions of the world (Pouget, 1990). Among the resistant species, only V. berlandieri was
adapted to the highly calcareous soils, but could not be used in its pure form and hybridisation
was necessary to develop rootstocks resistant to lime-induced chlorosis. Rootstocks were also
breed for resistance against nematodes like Meloidogyne spp. (Cousins et al., 2003) and
Xiphinema spp. (Meredith et al., 1982). Although chemical sprays (sulphur and copper) were
rapidly effective against the fungal diseases, these are still difficult to control and costs are high.
Very early, numerous European breeders made crosses between V. vinifera and resistant
American Vitis spp. like V. rupestris, V. labrusca, V. riparia and V. aestivalis to combine their

resistance with the fruit quality of V. vinifera. Although hundreds of so-called direct producer
hybrids were introduced to the French wine industry during the first half of the twentieth
century, they are not of commercial importance today, but some of them (Villard blanc,
Chambourcin, Seyval) were intensively used in modern breeding programmes for disease
resistance (Eibach and Topfer, 2003). Resistance to powdery mildew was also found in the wild
Chinese species V. bryoniifolia, V. davidii and V. piasezkii (Wang et al., 1995) and to downy
mildew in the Asiatic species V. amurensis (Korbuly, 2000). Other fungal diseases apart from the
mildews, that breeders endeavour to develop resistance against, include anthracnose (Mortensen,
1981) and Botrytis, while resistance against bacterial diseases include Pierces Disease, of
importance in the USA (Krivanek et al., 2005).
In Euvitis, Boubals (1959) postulated resistance to downy mildew to be dependent on two
genic systems: a single gene for the hypersensitive reaction at the time of infection and several
genes for the inhibition of growth of the fungal mycelium. He also postulated resistance to
powdery mildew to be dependent on a polygenic system. Li (1993) also viewed resistance to
powdery mildew to be of a polygenic nature and found minor resistance genes in V. vinifera.
Eibach (2000) found different genes to be responsible for resistance to downy and powdery
mildew and that no marked linkage seemed to exist between those genes.
Use of Muscadinia in Resistance Breeding
Muscadinia is native to the southeastern United States and is a useful source for genes of
resistance to phylloxera, nematodes, PD and fungal diseases. Often, these genes have a high
degree of dominance (Olmo, 1986). This was confirmed by Bouquet (1983) who found a high
degree of resistance to phylloxera in Muscadinia. Bouquet (1981) found the muscadines resistant
to Xiphinema index the vector of grapevine fanleaf virus (GFLV), but not to the virus itself and
also confirmed a large degree of dominance of this resistance (Bouquet et al., 2000a). By backcrossing a resistant F1 hybrid to the rootstock cultivar 140 Ruggeri, a new rootstock resistant to
virus spread has been selected (Bouquet et al., 2004). In Muscadinia, the same author also
identified a dominant gene called Run 1 that confers resistance to powdery mildew and which he
introduced in advanced back-cross progenies with V. vinifera (Bouquet, 1986; Bouquet et al.,
2000b). It appeared that genotypes carrying the Run 1 gene also showed partial resistance to
downy mildew due to the Rpv 1 linked gene (Merdinoglu et al., 2003). Current strategies being

developed in European countries aim at combining genes of resistance from Muscadinia and
Euvitis (Kozma and Dula, 2003).

Table 1A. Characteristic features of certain major rootstocks of grapes

Rootstock
Carman

Parentage
V. lincecumii x

Dog Ridge

Triumph
A selection from

Schwarzmann

Amos

Characteristic features
- High vigour, cold hardiness

- resistant to Pierce disease, and root knot

nematode
Vitis champini
- Adopted to both light sandy soils and
calcareous soils
A selection from
- resistance to active limestone is lower
and does not exceed 10%
Vitis riparia
- suitable for light, sandy, less fertile soils
with a good heat accumulation capacity
- good affinity to all common varieties of
Vitis vinifera L.
Severnyj x (Vitis riparia x - resistance to the content of low active
limestone in soil
Vitis rupestris)
- suitable for cultivation in lighter soils
- good affinity to Vitis vinifera L.
- rootstock variety is phylloxera-tolerant

Table 1B. A detailed characterisation of rootstock clones selected in the Grapevine

Breeding Station Poleovice (Kivnek, 1989)

Procedure involved in grapevine breeding

Some wild grapevine species have unique nematode resistance mechanisms, and are thus
used as parents in rootstock breeding. The procedure involved are as follows:

Wild grapevines, and their hybrids, are dioecious; individual vines bear clusters of either
male or female flowers

Dioecious vines are relatively easy to cross as the flowers do not have to be emasculated;
to prevent unwanted crosses, enclose female flower clusters in paper bags prior to bloom

As bloom approaches, periodically tap the bag and listen for the rattle of detached petals
that signals the blossoms are ready to be pollinated

Properly prepared pollen, by grinding flowers, can be stored for a relatively long time,
facilitating crosses between vines

To pollinate, the bags are removed, the appropriate pollen is slathered on with paint brush
and the cluster is labeled, recovered with the bag

In the summer, extract the seeds from the fruit and sow them in polybags

Later, seedlings will then be screened for nematode resistance, and the best selections
would be evaluated alongside their parents.

Fig 1. Dusting of pollen to the grape flower

Fig 2.

Removal of bags after pollination

Pest tolerance
It would be a major risk to overlook phylloxera resistance and tolerance when selecting
rootstocks for future plantings, irrespective of location, because there is still no proven, long
term method of controlling phylloxera with insecticides (Buchanan and Godden, 1989). There
are multiple strains of phylloxera and ideally, rootstocks should be assessed against as wide a
range of strains as possible. For example, in a laboratory based excised root bioassay, the number
of insects of phylloxera strain G30 counted on Schwarzmann roots after eight weeks was 285,

compared with just two insects of strain G4. This compared with just one insect each of strain
G30 and strain G4 on roots of 1103 Paulsen and 612 and 725 insects, respectively of strain.
Resistance to nematodes and pierce disease
Root nematodes (Meloidogyne spp.) are another severe pest problem in Vitis. It results in
galled roots, weak vines, shortened vine life, and reduced establishment. Nematode resistant
Vitis species include V. champini, V. cinerea, and V. longii (Mullins et al., 1992.). Magoon and
Magness (1937) tested 42 rootstocks in South Mississippi and found only 6 of them exhibited
resistance to root nematodes, namely, Barnes (V. champini), Joly (V. champini), Monticola x
Rupestris, Ramsey (V rupestris x V. candicans), Riparia x berlandieri 161-49, and Rupestris
St. George. Some other rootstocks considered to be resistant to nematodes are Ramsey, Dog
Ridge, Harmony, 1613 C and SO4 (Mullins et al., 1992). Some novel sources of resistance
to aggressive root-knot nematodes, including Vitis mustangensis, V. cordifolia, V. biformis, V.
aestivalis, V. nesbittiana, V. rotundifolia (muscadine grape), and V. cinerea and its hybrids
(Cousins et al., 2007).
Pierces disease (PD) caused by gram-negative bacterium Xylella fastidiosa, is the
primary limiting factor of growing Euvitis grape. Grape rootstock trials in Mississippi showed a
large effect of rootstock trial on vine longevity in a region recognized for high Pierces disease
pressure (Loomis, 1965). When the Florida hybrid bunch grape cultivar Blanc du Bois was
grafted on to muscadine, which is relatively tolerant or resistant to the bunch grape pests and
diseases common in North America, the scion showed a reduction in both PD and anthracnose
symptoms and fruiting improved (Ren and Lu, 2003). Rootstocks demonstrating high survival
rates had lower PD scores, while the lower survival percentage rootstocks had higher PD ratings
(Lu and Cousins, 2003).

Fig 3. PD score effect on vine survival rate after three growing seasons
Resistance to Abiotic Stress
In breeding programmes for some wine grapes and rootstocks, tolerance to abiotic stress
factors is also important. Rootstocks of V. rupestris V. berlandieri ancestry were found the
most tolerant to drought in greenhouse conducted tests by Carbonneau (1985). Pouget (1980)
bred a new rootstock cultivar highly resistant to iron chlorosis by inter-crossing rootstocks of V.
vinifera V. berlandieri ancestry. Further work, using inter-crossing rootstocks of V. riparia, V.
rupestris and V. berlandieri ancestry led to another new cultivar well adapted to acid soils
(Pouget and Ottenwaelter, 1986). Cold tolerance is found in V. riparia, V. labrusca and V.
amurensis (Reisch and Pratt, 1996) and was largely used in breeding programmes for wine
grapes in the USA (Hemstad and Luby, 2000) and Eastern Europe (Korbuly, 2000). Some salt
tolerant rootstocks are also important as the grapevine is easily affected by salinity. Troncoso et
al. (1999) found in vitro techniques suitable for the selection of salt tolerance in various
rootstocks. However, Skene and Barlass (1988) stressed the need for verification under field
conditions. Some rootstocks are known to be susceptible to magnesium deficiency and Bouquet
et al. (1990) investigated the possibility of in vitro selection.
Rootstock Transformation

GFLV is transmitted by the nematode X. index. Dangerous chemicals are used for soil
disinfection and, therefore, many researchers put their efforts into producing transgenic plants
with coat protein mediated protection. Krastanova et al. (1995) obtained V. rupestris and 110
Richter plants transformed with the coat protein of GFLV, while Mauro et al. (1995) obtained
41B and SO4 plants also transformed with GFLV-CP. From 18 independent transgenic grapevine
lines established in a naturally infected vineyard, 3 did not show reaction to GFLV infection 3
years after planting (Vigne et al., 2003). Bouquet et al. (2003a) found that transgenes (nptII,
uidA and GFLV-CP), introduced into 110 Richter and V. rupestris rootstocks and transmitted by
hybridisation in X. index resistant rootstocks developed by conventional cross-breeding,
expressed normally with a mendelian segregation in offspring.

Fig 4. GFLV infected vines which are infested with Blister mite form white blisters
Le Gall et al. (1994) transformed 110 Richter with the coat protein of Grapevine Chrome
Mosaic Virus (GCMV-CP). Torregrosa and Bouquet (1997) co-inoculated in vitro grown
plantlets of the rootstock Gravesac with a mixture of wild A. rhizogenes and A. tumefaciens
carrying plasmids containing GCMV-CP genes. Transformed hairy root cultures were initiated
from excised root tips. Plant regeneration was not achieved, but the authors mentioned the
possibility to graft in vitro transgenic roots to non-transformed shoot systems. Sade et al. (2000)
cloned the gene encoding the coat protein of Grapevine Virus A (GVA-CP) and used this gene to
transform the rootstock 41B and tobacco. Martinelli et al. (2002) reported stable transformation
of V. rupestris with the movement protein of GVA.

Guillen et al. (1998) purified a NADPH-dependent aldehyde reductase from Vigna

radiata that converts eutypine, the toxin that is involved in eutypa dieback, into non-toxic
eutypinol. Grapevine (V. vinifera) cells transformed with the gene (Vr-ERE) encoding the
eutypine-reducing enzyme showed in vitro resistance to the toxin. Transformed plants were
established only for the rootstock cultivar 110R and were not affected by relatively high
concentrations of eutypine, whereas growth of untransformed plants were highly inhibited
(Legrand et al., 2003).
Scion Transformation
Mauro et al. (1995) reported the first transformed plants of a scion
cultivar (V. vinifera cv. Chardonnay) with a gene of agricultural value, namely
the GFLV-CP gene, while Gambino et al. (2005) reported transformation of cv.
Nebbiolo with the same gene. Scorza et al. (1996) reported that by
combining particle bombardment of somatic embryos with Agrobacterium
co-cultivation produced Thompson seedless plants transformed with the lytic
peptide Shiva-1 or the tomato ring spot virus (Tom RSV) coat protein genes.
Chitinase is one of the hydrolytic enzymes, which can degrade fungal
cell wall components. Because of this characteristic, hydrolytic enzyme
coding genes are very attractive for researchers in their efforts to improve
disease resistance. Neo Muscat (V. vinifera) plants transformed with a rice
chitinase gene showed enhanced disease resistance to powdery mildew and
anthracnose (Yamamoto et al., 2000). Aguero et al. (2005) obtained
Chardonnay and Thompson Seedless plants transformed with the pear
polygalacturonase inhibiting protein (pPGIP) gene. Plants were evaluated for
tolerance to PD and Botrytis and delay in the development of PD was
observed in some transgenic lines with increased pPGIP activity. Chardonnay
plants, transformed with an antimicrobial peptide gene by using biolistics,
were evaluated for resistance to crown gall and powdery mildew (Vidal et al.,
2006).

Rootstock and scion compatibility

There are potentially a range of factors affecting the level of compatibility between
rootstock and scion, the most obvious being the anatomy and morphology of the graft union
itself. A successful graft union is one where xylem and phloem integrity is maintained between
rootstock and scion with no adverse impacts on upward flow in the xylem of water, mineral
elements and root to shoot signaling compounds, or downward flow in phloem of carbohydrates
or other essential factors for root function. The level of deformity of the graft union, measured by
the ratio of stem girth above and below the graft union, is not necessarily indicative of vine
performance.
For example, in a rootstock trial involving the variety Sun Muscat as scion, the
scion/rootstock girth ratio measured above and below the graft union was 0:92 for 1103 Paulsen,
1:24 for 140 Ruggeri and 1:25 for Ramsey. Mean yield of Sun Muscat was 28.9kg/vine for 1103
Paulsen, 26.1kg/vine for 140 Ruggeri and 25.8kg/vine for Ramsey. However, Sun Muscat on
101-14 had a scion/rootstock girth ratio of 1:21, but a yield of only 18.7kg/vine, indicating that
scion/ rootstock girth ratio is not strongly related to yield performance (Clingeleffer and
Emanuelli, 2006).
Rootstock B2/56, resembling 110 R was found to have better salt exclusion as indicated
by lower sodium content in petiole of Thompson Seedless vines grafted on this rootstock. The
yield was also highest in vines grafted on this rootstock (NRC Grapes, 2010).
Conferred vigour, vine performance and wine quality
Performance of Thompson Seedless grafted on Dogridge B, Salt Creek, 1613 C and St.
George to single level of imposed soil moisture stress in an existing vineyard revealed that
Thompson Seedless on Dogridge B performed well in terms of growth, yield and quality both
under normal and stressed conditions (NRC grapes, 2010).
Ough et al. (1969) reported large and easily detectable differences in wine composition
and quality that were related to scion/rootstock combinations. Grapes from vines grown on the
relatively vigorous Ramsey rootstock had a higher pH and higher levels of titratable acidity,

malate and potassium and a lower level of soluble solids than grapes from vines grown on their
own roots (Hale and Brien, 1978). Wines made from these grapes also showed similar
differences in composition. These effects are not restricted to vines on Ramsey, as other
rootstocks induce similar effects (Cirami et al., 1984). Rootstock and trellis interactions also
influence wine quality. Excessive shading of berries, like that associated with the dense canopies
of scions on vigorous rootstocks, also result in higher pH and potassium content of musts (Smart
et al., 1985).
In a study involving seven scions (Gamay, Chasselas, Ehrenfelser, Richensteiner,
Egiodola, Perdea and Roussanne), five rootstocks (SO4, Schwarzmann, 1103 Paulsen, Ramsey
and Dog Ridge) and own-rooted vines of the scion varieties, there was a three-fold range in
conferred vigour based on overall mean weights of one year- old pruning wood for the range of
scion varieties. Strong positive correlations were obtained between the weight of one year- old
pruning wood and yield (r=0.74), grape juice K (r=0.55) and grape juice malic acid (r=0.57).
Strong positive correlations were also obtained between grape juice potassium and pH (r=0.77)
and malic acid (r=0.69). Strong negative correlations were obtained between yield and grape
juice tartaric acid (r=0.62) and between berry weight and tartaric acid (r=0.56) (Clingeleffer,
G.H. Kerridge and E.H. Rhl, unpublished data).
The prime importance of pH for wine quality is well-known. It plays a major role in
anthocyanins equilibria and in compositional changes during wine ageing. It is known to
influence taste, the composition of wine volatiles and susceptibility of the wine to oxidative and
biological spoilage. The excellent correlation between wine pH and potassium content confirms
that potassium content has a controlling influence on pH (Somers, 1975). Any increase in berry
potassium content that might be attributable to rootstock would be undesirable from a
consideration of the chemical and biological stability of wine. Accordingly, selection of
rootstocks with reduced potassium uptake (Rhl, 1989) became a focus of the Australian
rootstock breeding effort, eventually leading to selection of 60 promising rootstock genotypes for
evaluation as grafted vines.
In the study of 60 rootstock genotypes each grafted with Shiraz, there was a 10 fold range
in conferred vigour (assessed by the weight of one-year-old pruning wood), but there was no
relationship between conferred vigour and grape berry maturity assessed by total soluble solids

measurements. In this case, there were strong positive correlations between grape juice pH and
the weight of one-year-old pruning wood ( r=0.70) and berry weight (r=0.55); between grape
juice titratable acidity and the weight of one-year-old pruning wood (r=0.85), yield (r=0.66) and
berry weight (r=0.66); and between grape juice potassium and the weight of one-year-old
pruning wood (r=0.81), yield (r=0.80) and berry weight (r=0.61). There were strong negative
correlations between grape berry colour (anthocyanins) and the weight of one-year-old pruning
wood (r=0.76), yield (r=0.81) and berry weight (r=0.72); and between grape berry total phenolics
and the weight of one-year-old pruning wood (r=0.69), yield (r=0.71) and berry weight (0.62)
(Clingeleffer, 1996).
The total nitrogen and free amino acid content of wines is another factor that can be
influenced by the rootstock (Ough et al., 1968). In the study involving Shiraz with 60 rootstock
genotypes, higher berry nitrogen was associated with higher conferred vigour. Very high or very
low total nitrogen is associated with deterioration in wine quality. This appears to be related to
the nature and content of specific nitrogenous compounds in the must. The initial total nitrogen
content of the must, and relative amounts of individual nitrogenous constituents, will greatly
affect yeast growth, rate of fermentation, end-product formation and ultimately, the final
organoleptic quality of wine (Bell et al., 1979).
In any rootstock-breeding program the main aim was to select rootstock types, which
through their lower vigor, resulted in acceptable yields and berry weights, lower grape juice
potassium (and, therefore, lower grape juice pH), and higher grape berry anthocyanins relative to
that conferred by standard higher vigour rootstocks. With respect to the above statement, three
new rootstocks have been selected, named and released with Plant Breeders Rights in Australia.
They are Merbein 5489, Merbein 5512 and Merbein 6262. A comparison of their performance
with two standard rootstocks Ramsey and 1103 Paulsen is shown as follows.

Table 2. Conferred vigour, juice total soluble solids and pH, yield and wine spectral parameters of Shiraz
grafted to 1103 Paulsen, Ramsey and three Australian rootstock selections grown in the warm irrigated
Sunraysia region of north-west Victoria

LSD = least significant difference. Values are means of three seasons (2001-2003). Wine was pH adjusted to
3.5. Different superscript letters indicate significant differences (P < 0.05) between treatment means.

Pruning wood weights, grape juice pH at harvest, acid added during winemaking and
wine pH (data not shown) is lower than for the standard stocks. Yield is also lower, except for the
comparison between Merbein 5489, 1103 Paulsen and Ramsey, and between Merbein 6262,
Merbein 5512 and 1103 Paulsen. Wine colour density of Merbein 5489 and Merbein 6262 and
total phenolics of Merbein 5489, Merbein 5512 and Merbein 6262 are higher than for the
standard stocks.
Water use efficiency and drought tolerance
Increased emphasis is being placed on these characteristics and, accordingly, they are a
focus of current research. Water use efficiency can be measured in various ways. A common
measure is crop water use index which is yield /evapotranspiration or preferably yield/vine water
use. Rootstock transpiration efficiency, assessed using carbon isotope discrimination (Gibberd et
al. 2001), and drought tolerance, assessed by transpirational cooling under drought conditions,
has been the main approaches used to date. The work with ungrafted vines so far has shown a
wide variation (50%) in carbon isotope discrimination across 220 hybrids from 14 families,
indicating significant opportunity to select for increased transpiration efficiency.
Encouraging data with respect to water use efficiency has obtained using the low to
medium vigour rootstocks produced from the CSIRO rootstock breeding program. Specifically,
in a comparative study involving mature vines in the field of Shiraz grafted to Merbein 5489 and

1103 Paulsen, Shiraz on Merbein 5489 had a smaller canopy (measured by the weight of oneyear-old pruning wood), lower water use over a 75-day period (measured by sap flow) and
higher yield, resulting in a two-fold higher crop water use index for Merbein 5489 relative to
1103 Paulsen.
Table 3. Cumulative water use over a 75-day period from 14 October 2003 to 29 December 2003, yield, pruning
wood weight and crop water use index for Shiraz vines on 1103 Paulsen (n = 4) and Merbein 5489 (n = 3). Different
superscript letters indicate significant differences (P < 0.05) between treatment means.

Tolerance of salinity
Salt tolerance breeding in grapevines is hampered by the complex nature of the plant
response to a soil solution containing a high concentration of salt. Salt-tolerant rootstocks are
known to confer moderate to high vigour to scions and are good chloride and sodium excluders
(Walker et al. 2002 and 2004). All grapevine rootstocks appear to exclude sodium and chloride to
some extent. For example, comparison of chloride concentrations in xylem with concentrations
in the soil solution show that a good chloride excluder, 140 Ruggeri, excludes 99.4% of chloride
from xylem, whereas a poor chloride excluder K51-40, excludes 96.4% of chloride from xylem
(Tregeagle 2007). The higher concentration of chloride in the xylem of K51-40 over the course
of a season leads to excessive accumulation of chloride in grape juice and laminae (Tregeagle et
al. 2006). Initial evidence obtained using a fluorescent apoplast tracer to monitor uptake
pathways in grapevine roots (H. Gong and R. Walker, unpublished data) suggests that chloride
entry into xylem is principally regulated by membrane ion transport. Chloride uptake studies
involving rooted leaves of 140 Ruggeri and K51-40 and using 36chloride as a tracer suggest that
the better chloride exclusion of 140 Ruggeri is linked to reduced transport of chloride into the
xylem (Tregeagle 2007).
In a screening study of grape rootstocks for sulphate salinity, the results revealed that the
rootstocks H-516 and Dogridge recorded the lowest reduction in growth attributes and so, they

can be further used for grafting of seedless / wine grape varieties for their cultivation under
sulphate salinity soils (Deshmuki and Patil, 2010).
Current research activities in NRC, Grapes
Certain research work on rootstock breeding in grapes are undergoing at NRC, Grapes
under progress and their details are as follows.

Evaluation on the performance of Cabernet Sauvignon grapes raised on different

rootstocks under various salinity levels (Upadhyay et al., 2011)

Standardization of propagation techniques in grapes based upon the assessment of plant

sap extract effect on rooting of promising grape rootstock and commercial varieties
(Somkuwar et al., 2011)

Studies to assess the effect of grape rootstock in influencing the protein profile in
Thompson Seedless at different phenological stages (Sathisa et al., 2011)

Apart, several important rootstocks were introduced from Oenological Research Institute,
South Africa, during 2009-10 vis 135-5 EVEX (EC659392), 143 Mgt. (EC659393), 1045
Paulsen (EC659394) and 140 Ruggeri (EC659395).
Biotechnological approaches in rootstock breeding
The present generation within the wine industry may be too presumptuous to
believe that success of traditional cultivars, based on today's context, will continue forever on
such unchanged basis. In fact, there is a general industry perception that to be successful a new
wine cultivar must be very similar to, or if possible indistinguishable from the traditional cultivar
that it is designed to replace except for the addition of a desirable trait such as disease or pest
resistance. So, in such a case, biotechnology offers a means of inserting new characters into the
genome of traditional cultivars without changing any of their other characteristics, particularly
wine typicity and quality. The major biotechnological approaches under grape rootstock breeding
are as follows.

a) Proteomic analysis in grape rootstock

Proteome is the entire set of proteins expressed by a genome, cell, tissue or organ. It is
the set of expressed proteins in a given type of cell or an organism at a given time under defined
conditions.
As far as grapevine is considered, it contains a wide range of proteins, like chitinase,
thaumatin and some osmotins (Monteiro et al., 2001). The protein present in the grapes is
dictated by several factors such as cultivars, cultural practices, soil, climate, period of the year
and rootstocks (Baily and Berg, 1967).
Grapevine proteomic profiles have been characterized by proteomic studies (Vincent et
al., 2006). Though many methodologies have been developed for characterization of protein
profiles, two dimensional electrophoresis (2 DE) remain one of the most efficient strategies to
isolate proteins showing qualitative variation in response to a treatment (Gorg et al., 2004).
Hence, proteomic analysis helps to characterize the protein profile of Thompson Seedless grapes
grafted on different rootstocks at different phenological stages of vine growth and fruit
development.
b) Microsatellite markers
Microsatellite markers are one of the desired type of DNA marker for identification of
grapevine cultivars, and their properties enable a wide range of applications from cultivar
identification based on various parts of the grapevine plant to pedigree reconstruction and
genome mapping. 24 rootstocks and 44 other accessions were characterized with micro satellite
markers. The work on molecular database is initiated for storage and retrieval of molecular data.
The confusion between two morphologically different Dogridge rootstock plants, available with
grape growers as Dogridge-A (America) and Dogridge-B (Bangalore) was resolved based on
micro satellite marker analysis and ampelometric studies (NRC Grapes, 2010).
Rootstocks Dogridge-A resembled 110 Richter (110 R), which is a hybrid of V.
berlandieri V. rupestris, whereas Dogridge-B belongs to the species V. champini.

Fig. 5. Micro satellite analysis of grape accessions

c) Molecular characterization using SSR analysis
In usual, SSRs are best suited for genetic analyses, due to their reproducibility, simple
implementation and interpretation of allelic patterns, low cost and because results can be shared
among different laboratories, a key issue when working on fingerprinting (Jung, 2004).
In a research work, carried out at NRC grapes (2010) revealed that 134 grape accessions
were analyzed with 25 primers. 25 primers detected 405 alleles in 134 accessions with an
average of 16 alleles per primer. The number of alleles detected by each primer ranged between 9
to 26. With this analysis, so far, 188 genotypes have been characterized. These include all the
rootstocks available at the Centre, indigenous material collections and certain hybrids developed
from IIHR and IARI.
d) Antiporter gene expression
The expression of Na+/H+ antiporter gene was affected by different levels of salt and
moisture stress. DDRT-PCR of 110R RNA identified differentially expressed transcript specific
to salinity, moisture and combined stress.
In a pot study conducted at NRC Grapes, four rootstocks viz. Dogridge, 110-R, 1613 C
and Salt Creek were subjected to combined salinity and moisture stress. The analysis of
expression of Na+/H+ antiporter gene in rootstocks 110R and 1613C showed differential
response by different rootstock. Two levels of salt stress viz. 2 EC and 4 EC were used either at
100% or 50% field capacity. Different biochemical parameters viz. total protein, total sugar, total
phenols, proline and glycine betain were estimated. Total protein content varied significantly in
response to salinity as well as combined stress among different rootstocks. In 110R there was no

significant difference in proline accumulation under salinity stress, whereas proline content
increased in response to water and combined stress. Based on the sequence information available
in online grape gene index database, primers were designed to study the expression of Na+/H+
antiporter gene, a gene known to be involved in salinity tolerance. The real time PCR was used
for relative quantification of gene expression in different treatments. The expression of Na+/H+
antiporter gene was found to be upregulated in 110R in response to salinity, moisture as well as
combined stress (Fig. 6). Within seven days, the expression increased 4.5 and 7.5 fold under
saline stress (4EC) and moisture stress (50% field capacity) respectively. However in 1613 no
effect on gene expression levels of this gene was observed in response to salinity. Increased
expression was observed in response to moisture and combined stress at 21 days (Fig. 7).

Fig 6. Expression of Na+/H+ antiporter gene in 110R

Fig 7. Expression of Na+/H+ antiporter gene in 1613 C

e) Genetic transformation of grape rootstocks

The development and use of a genetic transformation platform has become of much
importance for various activities in grape breeding and biotechnology. More than 2,500
transformed lines have been produced, with approximately 750 lines already released to a bio
safety field.
Gene encoding the iron binding protein, ferritin, cloned from Medicago sativa, support
and response oxidative stress. Therefore, it seems that the overexpression of ferritin in grapevine
plants makes them tolerant to oxidative damage and pathogen attack. Somatic embryos (with size
of 1-2 mm) of Richter 110 were treated with Agrobacterium tumefaciens EHA101 (pRok2: Ferr).
The germinating embryos were transferred on light three months ago and the embryos developed
to green plantlets (Olh, 2004). Other applications of genetic transformation focused on the

testing of a series of candidate genes derived from functional genomics experiments from Vitis or
other species or by crossing the information coming from linkage mapping and QTL
identification plus the mapping of candidate genes by using Single Nucleotide Polymorphisms
(SNPs) markers.
f) Genes of interest in grape rootstock breeding
Viral resistance was obtained by introducing the grape chrome mosaic virus (GCMV) CP
gene into the rootstock 110 R (Le Gall et al., 1994). This success was concomitant with the
transformation of the rootstocks 110 R and V. rupestris cv. du Lot (Krastanova et al., 1995) with
the grape fan leaf virus (GFLV) CP gene. The same plasmid p1660, bearing CP, uidA, and nptII
genes, was used to obtain transgenic plants from the rootstocks 41 B, SO4, and V. vinifera cv.
Chardonnay (Mauro et al., 1995). The GFLV replicase, proteinase, and movement protein genes
were inserted in the rootstocks 110 R and 41 B (Valat et al., 2006). With regard to other
nepoviruses, the tomato ring spot virus (TomRSV) CP gene was inserted into V. vinifera cv.
Thompson seedless (Scorza et al., 1996). The gene coding for the movement protein of GVA was
inserted in sense and antisense orientation into the table grape cultivar Superior seedless and the
rootstock V. rupestris cv. du Lot (Martinelli et al., 2002).
CONCLUSION
Key rootstock characteristics for selection includes phylloxera, nematode and salt
tolerance, and good viticultural characteristics. Water-use efficiency and drought tolerance have
increasing priority. Conferred vigour by the rootstock to the scion has important consequences
for yield and berry composition. Rootstock vigour is linked to salt tolerance and there is evidence
that higher vigour rootstocks with penetrating root systems perform better under water deficit
conditions. Until now, there has been a demand for rootstocks with reduced vigour to counter the
negative impacts of high conferred vigour on berry composition. Accordingly, the capacity to
select for rootstocks with both low-medium and medium-high vigour will be important to retain
flexibility in capacity to address future needs. However, biotechnological approaches paved a
new way for developing elite rootstocks and further studies have to be made in this line in future.

Table 4. Rootstocks of grapes obtained as Transgenic forms with Vitis species

Cultivar

Selectable

Promoter

Gene product

References

marker
V. riparia,

NPTII

NOS, 35S

3309 Couderc,

Translatable, antisense,
untranslatable coat protein (GFLV,

101-14,

Krastanova et
al., 2000

GLRaV), GUS

110 Richter
110 Richter

NPTII

NOS, 35S

GFLV replicase, GUS

Barbier et al.,
2000

V. rupestris

NPTII

NOS, 35S

ArMV coat protein

Spielmann et al.,
2000

110 Richter

NPTII

NOS, 35S

Eutypine-reducing enzyme

Legrand et al.,
2003

110 Richter

NPTII

NOS, 35S

Ferritin (iron binding protein)

Olah et al., 2004

41 B

NPTII

NOS, 35S

GFLV coat protein and movement

Valat et al., 2006

protein
Source: Bouquet et al. (2009)