regeneration and micropropagation of grapevine - BioIT ...

International Journal of Advanced Biotechnology and Research ISSN 0976-2612,
Vol 2, Issue 4, 2011, pp 484-491
http://www.bipublication.com
REGENERATION AND MICROPROPAGATION OF
GRAPEVINE (VITIS VINIFERA L.) THROUGH SHOOT TIPS
AND AXILLARY BUDS
Ayman A. Diab 1 * S. M. Khalil 2 , Roba M. Ismail 2
1 Molecular Markers and Genome Mapping Department - Agricultural Genetic Engineering Research
Institute (AGERI), Agricultural Research Center (ARC), Giza, 12619-Egypt
2 Plant Genetic Transformation Department - Agricultural Genetic Engineering Research Institute
(AGERI), Agricultural Research Center (ARC), Giza, 12619-Egypt
* Corresponding author: Ayman Diab. Email: aymanalidiab@gmail.com
Abstract
Efficient regeneration methods are a priority for the successful application of genetic engineering to
vegetatively propagated plants such as grape (Vitis vinifera L.). An efficient in vitro shoot
micropropagation protocol leading to the regeneration from shoot tip explants was successfully
developed for mass production. Callus was induced from shoot tips and axillary buds on C 2 D medium
supplemented with 4 mg/l 6-benzyladenine (BA). Bud differentiation from callus was possible on C 2 D
medium supplemented with 1 mg/l gibberellic acid and 0.1 mg/l kinetin. Bud initials were elongated by
sub-culturing on elongation medium containing C 2 D salts supplemented with 0.1 mg/l BA and
resulting shoots could be mass micropropagated on C 2 D medium supplemented with 1 mg/l BA. Shoots
that were regenerated via organogenesis or micropropagation were rooted on C 2 D medium
supplemented with 1 mg/l indole-3-butyric acid. In this study, regeneration and micropropagation of
grape cv. ‘Spero’ was successfully achieved through shoot tip and axillary bud culture. Such a protocol
would be useful for future use in the strategic improvement of grape using genetic engineering.
Key words: Regeneration, shoot tip, micropropagation, genetic engineering
Introduction
Grapevine (Vitis vinifera L.) is one of the
most widely grown fruit crops in the world
[1]. Grape, as an ancient food in the life and
history of humankind over several millennia,
has increased production because the fresh
fruit is nutritious for humans. Grapes are
widely used in the production of juice and
wine and an increase in industrial raw
materials for developing new food products is
required. Sustained efforts to ensure high,
reliable grapevine yields are usually
threatened with damage to production caused
by numerous diseases Martelli (1993) [2].
Micropropagation is a tool used to propagate
genotypes by applying in vitro culture
methods. Depending on the plant species and
culture conditions, tissue culture may enable
the mass production of genetically
homogeneous populations from elite (e.g.,
high-yielding or disease resistant) individuals.
Micropropagation of selected Vitis genotypes
can be carried out, among others, by the
culture of total or fragmented shoot apical
meristems, axillary bud microcuttings or
through adventitious bud development [3-6].
Somaclonal variation is undesirable in
applications pointing to reproduce selected
elite genotypes, e.g., ancient and rare clones,
pathogen-resistant or drought/salinity-tolerant
genotypes and it is caused by high levels of
plant growth regulators (PGRs), mainly
cytokinins, usually useful to promote shoot
multiplication and thus increase yield (Gray et
al. 2005) [7]. Micropropagation is an
efficient method of plant regeneration and
rapid propagation through organogenesis and
embryogenesis of any valuable genotype
obtained by non-conventional methods
(Stamp et al. 1990) [8] and therefore
regeneration of whole plants by somatic
embryogenesis and organogenesis has been
intensively studied [9,10]. Conventional
methods of propagation of nearly all grape

REGENERATION AND MICROPROPAGATION OF GRAPEVINE (VITIS VINIFERA L.)
varieties are stem cuttings, layering and
grafting. Genetic improvement of classic
cultivars in order to obtain high quality wine
and table grape varieties through conventional
hybridization methods does not appear to be
practical and therefore unconventional
approaches have been proposed [8,9,1]. A
prerequisite to develop such approaches for
grapevine is the ability to establish efficient in
vitro regeneration techniques[1]. Thus, the
objective of this study was to establish a
regeneration and micropropagation protocol
for Speryo grape.
MATERIALS AND METHODS
Preparation and sterilization of plant
material
Speryo, a cultivar of grapevine of highly local
importance in Egypt, was used in this
investigation. Grapevine seedlings of 5-10
years old vines were kindly provided from El-
Karma Farm, Monofeya Government. Shoot
tips (0.3-0.5 cm) were used as explants for
micropropagation and/or regeneration through
indirect organogenesis, i.e. via callus
induction. Shoot tips explants were washed
thoroughly for 15 min under running tap
water. For surface sterilization, explants were
dipped in 75% (v/v) ethanol for 1 min,
followed by immersion in 25% (v/v)
commercial bleach (final concentration of
sodium hypochlorite = 1.5% ) containing
0.1% Tween-20 for 30 min and rinsed three
times in sterile, distilled water.
Media composition and preparation
Nutritional requirements for optimal growth
of a tissue in vitro may vary with the species.
Even tissues from different parts of the plant
may have different requirements for
satisfactory growth. There is no single
medium that is suitable for all types of plant
tissues and organs.
Therefore, three media that represent high,
medium and low salt concentrations were
selected for the present investigation.
Murashige and Skoog (MS; Murashige and
Skoog, 1962 [11]), C 2 D Vitis basal medium
[12], and WPM medium [13] were used. The
composition of three different media is
presented in Table (1).
Table 1 The composition of different media used in Vitis
micropropagation
Compound
Macronutrient
MS
(mg/l)
C2D (mg/l)
NH 4NO 3 1650 1650 400
KNO3 1900 1900 --
MgSO4.7H2O 370 370 180.7
KH2PO4 170 170 170
CaCl2. 2H2O 440 -- 72.5
Ca(NO3).4H2O -- 709 386
FeSO4.7H2O 28 27.8 27.5
Na2.EDTA 37 37.3 37.3
Micronutrient
KI 0.83 -- --
MnSO4.4H2O 22.3 0.845 22.3
H3BO3 6.2 6.2 6.2
ZnSO4.7H2O 8.6 8.6 8.6
Na2MoO4.2H2O 0.25 0.25 0.25
CuSO4.5H2O 0.025 0.025 0.25
CoCl2.6H2O 0.025 0.025 --
K2so4 --- --- 999
WPM (mg/l)
The principal components of most tissue
culture media can be categorized into
inorganic nutrients (macro and micro),
organic nutrients (vitamins and amino acids),
a carbon source, PGRs and a gelling agent.
Other undefined organic supplements such as
CH and CM were also added to the medium
when required (PhytoTechnology
Laboratories, Inc. www.Phytotechlap.com).
PGRs were purchased from Sigma-Aldrich
(St. Louis, MI, USA). Bacteriological grade
agar was used as the gelling agent throughout
the study.
Different cytokinin (BA, TDZ or kin) with
different concentrations was added to each of
these media, aimed to study the effect of it on
micropropagation and regeneration of grape.
While different concentration of BA were
used aimed to have no vitrified plantlets.
All media were supplemented with 3% (w/v)
sucrose and 0.6% agar (w/v). The cultures
were incubated at 25°C under a 16/8 h
day/night photoperiod (1000-Lux).
Shoot induction and micropropagation
media
Ayman A. Diab, et al.
485

REGENERATION AND MICROPROPAGATION OF GRAPEVINE (VITIS VINIFERA L.)
Sterilized explants were transferred to one of
three shoot induction medium, i.e., C 2 D, MS
and WPM. The C 2 D medium contained salt
with vitamin, 3% (w/v) sucrose, 0.6% (w/v)
agar in addition to BA, or Kin in three
concentrations (1, 2 and 4 mg/l for each), or
TDZ in three concentrations (0.1, 0.2 and 1
mg/L). The two other media (MS and WPM)
were supplemented with BA only at three
concentrations (1, 2 and 4 mg/l). Four weeks
later and after selecting the best basal
medium, the selected medium was
supplemented with different concentrations of
auxins for elongation shoots. The shoot
primordia was transferred to medium
supplemented with (BA, GA and Kin) at three
different concentrations (0.1, 0.5 or 1 mg/l for
each hormone), each plate contained 20
explants. The plates were sealed with
parafilm and incubated at 25°C under a 16-h
photoperiod (1000-Lux).
Grape regeneration
Thirty two calluses from 60 explants were
induced on C 2 D and WPM media
supplemented with 4 mg/l BA or 4mg/l kin.
These calluses were examined on C 2 D basal
medium supplemented with 30 g/l sucrose, 6
g/l agar and with two PGRs: NAA and BA.
After two weeks from planting on this
medium, calluses were transferred to C 2 D
medium supplemented with 1 mg/l gibrillic
acid GA3 and 0.1 mg/l Kin for differentiate.
After 2-4 weeks, differentiated calluses were
transferred to shoot elongation medium.
Organogenesis shoot proliferation cultures
were subjected to light with photoperiod of 16
h a day. After another four weeks, the shoots
were transferred to rooting medium
supplemented with (IAA, NAA and IBA) at a
concentration of 1mg/l for each; roots were
grown after 6 weeks on the rooting media.
The pH of all media was adjusted at 5.8 and
autoclaved at 121°C with 15 psi pressure for
15 min.
Acclimatization
For acclimatization, “In vitro” derived rooted
plantlets were acclimatized under controlled
environment. The pots were covered with a
plastic bag inside greenhouse. The problem of
phenolic exudation from explants was tackled
by repeated Sub-culturing on fresh media.
The damaged tissues on both explants sides
were aseptically cut off and the nodal
segments were immersed base down into
solid culture medium.
Results and Discussion
Grape-vine (Vitis vinifera L.) is a perennial
deciduous woody vine which is cultivated all
over the world. The conventional method of
grape vine propagation is time consuming and
allows diseases transmission. A planted
grapevine needs four to five years to produce
propagation materials by cuttings. By using
the tissue culture technique, a mass
production of genetically homogeneous
populations and healthy plants occurs.
Therefore, is a very important technique for
grape vine culture program [14].
Explants sterilization and oxidative
browning
Shoot tips explants sterilization is considered
to be a serious problem in all woody plants as
explants were derived from field-grown
grapevines. The sterilization protocol used in
this investigation was according to Banilas
and Koraks (2007) [15], proved to be quite
successful, since only less than 10 % of the
explants contaminated. Nevertheless, the time
period of explants collection was a critical
factor for efficient sterilization of explants,
since in earlier experiments conducted at the
end of the growing season the frequency of
infected explants was much higher.
Gray and Benton (1991) [16]reported that
meristems taken from 10 cm long shoots had
less contamination (3%) and a higher survival
rate (94%) than those from shorter or longer
shoots. The problem of contamination in vitis
vinifera L. CV. Perlette was solved by Jaskani
et al. (2008) [17], using 10% Clorox
treatment for 5, 10 and 15 min, treatments for
10 and 15 min showed non-significant
differences and successfully controlled
contamination.
Like other woody plants the oxidative
browning was also a problem in grapes. This
Ayman A. Diab, et al.
486

REGENERATION AND MICROPROPAGATION OF GRAPEVINE (VITIS VINIFERA L.)
problem was solved by transferring the
explants on fresh medium every 2-3 weeks;
subculture was repeated for about 2-3 times.
Oxidative browning problem was solved by
Jaskani et al. (2008) [17]by continuous
subculture on fresh media up to 3 subcultures.
Similarly Dalal et al. (1991) [18] reported
oxidative browning control with modification
in MS macro salts.
Effect of media composition on
regeneration and micropropagation of
grapevine
In medium study, three different media, i.e.,
C 2 D, MS and WPM were compared for their
effect on shoot production. Table (2)
illustrates that WPM medium is not suitable
for micropropagation as most of shoots
produced on this medium became vitrified;
while C 2 D medium gave the best results for
vitis micropropagation followed by MS
medium. Using C 2 D medium, 16 and 10
normal shoots were showed using 1 mg/l and
2 mg/l BA, respectively, while the vitrified
shoots obtained were only 3 and 5 shoots out
of 20 using the same concentration from BA.
The development of shoot tip Speryo explants
on C 2 D medium supplemented with 1 mg/l
BA after two week of culture, was illustrated
in figure (1 A). Gray and Benton (1990) [19]
established micropropagation in Muscdine
grape, they showed that shoot produced on
WPM medium have been vitrified and
suffered from leaf abscission. On media tested
by Gray and Benton (1991) [16], MS, 1/2
MS, and C 2 D resulted in equivalent shoot
proliferation rates, whereas, WPM produced
stunted shoots.
Table (2): Effect of the three different media with BAP
hormone on shoot formation
BA 1 mg/l 2 mg/l 4 mg/l
N V N V N V
MS 15 2 8 3 6 -
C2D 16 3 10 5 4* -
WPM 8 10 7 11 3* -
The total number of explants is 20 in each treatment, N:
Normal, V: Vitrified, *explants form callus in this
concentration
Effect of cytokinin on shoot development
The effect of three cytokinins (BA, TDZ, and
Kin) on shoot development of
micropropagation of Speryo grape vine
cultivar was studied. using the number of
shoot formed (Table 3).
Table (3): Effect of cytokinin on micropropagation of
grapevine. The total number of explants is 20 in each
treatment.
BA
(mg/l)
No. of shoot
induction
TDZ
(mg/l)
No. of shoot
induction
1 16 0.1 10
2 10 0.2 12
4 5* 1 15
*explants form callus in this concentration
Results showed that BA and TDZ generally
produce shoots in all concentrations used
except BA in concentration 4 mg/l which
produced callus, C 2 D medium was used in
this investigation. However, shoots produced
on media containing TDZ were stunted and
distorted compared to media supplemented
with BA . Similarly, Gray and Benton (1991)
[16] study the effect of BA and TDZ on
micropropagation of muscadine grape
cultivars (Vitis rotundifolia), they found that
BA (5, 10 and 20 µM) and TDZ (5 µM)
produced the highest average number of
shoots per cultured apex. However, shoots
produced with TDZ were stunted and did not
root well. In this investigation, Kinetin was
ineffective, being comparable to other media
as no shoots were induced using 1mg/l or 2
mg/l kin, while 4 mg/l kin formed callus only.
Similar results were shown by Gray and
Benton (1990) [19], they illustrated that BA
gave the best result in comparison to Kin and
TDZ.
Lee and Wetzstein, (1990) [20] and Heloir et
al. , (1997) [3] showed that BA is the most
effective among other cytokinin for inducing
shoot development in Vitis. Banilas and
Korkas (2007) [15] demonstrated that lateral
buds into shoots were also efficient at
medium lacking BA. Only the main shoot
developed while the growth of axillary buds
was inhibited. The presence of BA, even at
relatively low levels, enhanced bud
multiplication. At relatively high BA
concentrations axillary bud proliferation was
Ayman A. Diab, et al.
487

REGENERATION AND MICROPROPAGATION OF GRAPEVINE (VITIS VINIFERA L.)
apparent, while at the same time the growth
of the main shoot was suppressed.
For shoot multiplication, three concentration
of BA, GA3, and Kin (0.1, 0.5 or 1 mg/l for
each hormone) were studied using C 2 D
medium; we notice that 0.1 mg/l BA was the
best for shoot multiplication as shown in
Table (4), figure (1B).
Table (4): Effect of BA, GA3, and Kin on shoot multiplication
on C2D medium. The total number of explants is 20 in each
treatment
Hormone
Concentration
(mg/l)
BA 0.1
0.5
1
GA 0.1
o.5
1
Kin 0.1
o.5
1
No. of explants
gave shoot
18
14
9
10
12
11
14
12
7
Alizadeh et al. , (2010) [21] studied the effect
of different plant growth regulators
supplemented to MS medium on culture
establishment and time to bud sprouting, they
found that culture establishment was
enhanced using BAP alone or with NAA in
combination as compared to KIN but among
the different combinations tried, 2.0 mg/l BA
with 0.2 mg/l NAA showed better response.
BA has been the most commonly used
cytokinin in grape tissue culture media
[12,22-27]. Reisch (1986) [23] reported a
small but significant negative response with
regard to shoot bio-mass by increasing the
kinetin levels in ‘Concord’ variety but shoot
proliferation rate did not vary significantly to
the treatments. Martinez and Tizio (1989)
[24] found that combination of 1 mg/l BAP +
0.5 mg/l GA3 was most useful for in vitro
culture ability of seven different grape
varieties. Poudel et al. (2005) [28] reported
the effectiveness of KIN on culture
establishment of two wild grapes.
induction. Media having 1 mg/l IBA proved
the best for root formation in micro shoots
followed by IAA (Table 5, figure 1C). The
percentage of rooting was 80% using C 2 D
medium supplemented with 1 mg/l IBA as 16
shoots were rooted out of 20 shoots used in
this experiment. While NAA was not suitable
for rooting as the percentage of rooting was
35% only. Similarly, in a previous study on in
vitro rooting of cv. `Pinot noir' microshoots
Helior et al. (1997) showed that IBA is a
suitable auxin, while other types of auxins
(e.g., NAA) may lead to callus formation.
Jaskani et al. , (2008) [17] reported that media
having 10 µM IBA proved the best for root
formation in micro shoots while in the absent
of IBA shoots showed complete failure in
root formation. Hicks & Dorey (1998) [29]
also reported roots at high frequency on MS
plus IBA but level of IBA was different than
our treatment which may be due to different
varietal response.
Table (5): Effect of IAA, NAA, and IBA on rooting. The total
number of explants is 20 in each treatment
Hormone Concentration (mg/l) No. of explants gave root
IAA 1 11
NAA 1 7
IBA 1 16
Root formation and acclimatization
In this study, Micro shoots were transferred
on C 2 D medium supplemented with IAA,
NAA, or IBA (1 mg/l for each) for root
Ayman A. Diab, et al.
488

REGENERATION AND MICROPROPAGATION OF GRAPEVINE (VITIS VINIFERA L.)
Figure 1: Stages of micropropagation through
auxiliary buds culture of Grapevine cv.
Speryo. (A) Shoot tip explants development
on C 2 D Medium supplemented with 1mg/l
BA, after two weeks in culture. (B) Grapevine
shoots multiplication after four weeks on C 2 D
medium supplemented with 0.1 mg/l BA. (C)
Elongation of shoot after four weeks. (D)
Rooting of grapevine shoot in vitro on C 2 D
medium supplemented with 1mg/l BA.
Grape regeneration
The use of genetic engineering for plant
development permits the introduction of
useful agronomic traits without altering the
features of the cultivar, demanding the
development of in vitro systems for the
genetic transformation and plant regeneration
[30]. Until our time, the regeneration of grape
plants has been obtained by both
organogenesis and embryogenesis. Shoot
regeneration from fragmented shoot apices
has been successfully applied to several grape
species and hybrids [4]. In this investigation,
regeneration system was established for grape
vain cv. Speryo, using shoot apices as
explants.
Callus induction and differentiation
As we indicated previously in material and
methods, thirty two calluses from 60 explants
were induced on C 2 D and WPM media
supplemented with 4 mg/l BA or 4mg/l kin.
(Table 2 and 3). Kinetin leads to callus
formation when it was used in concentration
(4mg/l) with all media. Shoot tips explants
induced 22.5% callus on C 2 D media and 15%
callus on WPM media. C 2 D medium was used
for grape vain regeneration. Calluses were
examined on C 2 D basal medium
supplemented with NAA and BAP, Figure (2,
A). Wang et al. , (1985) [31] reported callus
development on different concentrations of
BA and 2,4-D. Jaskani et al. , (2008) [17]
reported that nodal segments induced 80%
callus on media having 5 µM BA and that
only the 50% stem segments induced callus
on media with 10 µM BA whereas the
cultures on MS media without BA did not
respond.
Figure 2 A. Callus production from shoot tip after
subculture explants on C 2 D medium supplemented
with NAA and BA
For callus differentiation, calluses were
transferred to C 2 D medium supplemented
with 1 mg/l gibrillic acid GA3 and 0.1 mg/l
kinetin, after 8-10 weeks, 24 shoots were
differentiate out of 32 calluses used in this
experiment, in a percentage of 75%. Figure
(2, B) illustrates callus differentiation stage.
Figure (2, B) Differentiation of callus to shoots buds
For shoot elongation, 24 Shoots were
elongated on C 2 D media containing 0.1 mg/l
BA (figure 2,C), 16 of them were succeeded
in rooting on MS media containing 1mg/l
Ayman A. Diab, et al.
489

REGENERATION AND MICROPROPAGATION OF GRAPEVINE (VITIS VINIFERA L.)
IBA (figure2, D), and 6 plants were
acclimatized (figure 2, E).
Figure (2, D) Shoot production via organogenesis
on media containing 0.1 mg/l BA.
Figure (2, E) Plantlet of grape vine via
organogenesis on rooting media consist 1mg/l IBA
Park et al. , (2001) [32] studied the effect of
cytokinin on the shoot induction of Grape
(Vitis labruscana cv. Kyoho) regeneration
using shoot tip as explants, they test the effect
of different concentrations of kinetin and BA
on shoot micropropagation. They found that
the multiple shoots produced on the kinetincontaining
medium were stunted compared
with those from media containing BA.
In V. vinifera cv. Perlette Barreto and
Nookaraju (2007) [33], Butiuc-Keul et al. ,
(2009) [34], and Lewandowski, (1991) [35]
found that up to 95% rooting of microcuttings
were obtained on MS medium
supplemented with IBA and NAA .
References
1. Mederos-Molina, S. 2007. Culture medium
requirements for micropropagation of Vitis
vinifera L. cv. Listan Blanco. Acta Hort., 754
(1): 265-271
2. Martelli, G.P. 1993. Graft- transmissible
diseases of Grapevine. Hand book for
detection and diagnoses. FAO. Part 1. pp 9-
18.
3. Heloir, M.C., Fournioux, J.C., Oziol, L. and
Bessis, R. 1997. An improved procedure for
the propagation in vitro of grapevine (Vitis
vinifera cv. Pinot noir) using axillarybud
microcuttings Plant Cell Tiss. Org. Cult., 49:
223-225.
4. Barlass, M., and Skene, K.G.M. 1978. In vitro
propagation of grapevine (Vitis vinifera L.)
from fragmented shoot apices. Vitis, 17: 335-
340.
5. Gray, D.J. and Fisher, L.C. 1985. In vitro
shoot propagation of grape species, hybrids
and cultivars. Proc. Fla. State Hort. Soc., 98:
172-174
6. Monette, P.I. 1988. Grapevine (Vitis vinifera
L.), Biotechnology in Agriculture and
forestry, vol.6, Crops Bajaj Y.P.S. (ed.) 6: 3-
37. Springer Verlag Berlin Heidelberg,
Germany.
7. Gray, D.J., Jayasankar, S. and Li, Z.T. 2005.
Vitis spp. Grape. In: Litz RE (ed)
Biotechnology of fruit and nut crops, pp. 672–
706.
8. Stamp, J.A., Colby, S.M., and Meredith, C.P.
1990. Direct shoot organogenesis and plant
regeneration from leaves of grape (Vitis spp.).
Plant Cell Tiss. Org. Cult., 22: 127-133.
9. Torregrosa, L., and Bouquet, A. 1996.
Adventitious bud formation and shoot
development from in vitro leaves of
Vitis×Muscadinia hybrids. Plant Cell Tiss.
Org. Cult., 45: 245-252.
10. Mei-Chun, L. 2005. Micropropagation of Vitis
thunbergii Sieb. Et Zucc., a medicinal herb,
through high frequency shoot tip culture. Sci.
Hort., 107 (1): 64-69.
11. Murashige, T. and Skoog, F. 1962. A revised
medium for rapid growth and bioassays with
Ayman A. Diab, et al.
490

REGENERATION AND MICROPROPAGATION OF GRAPEVINE (VITIS VINIFERA L.)
tobcco tissue cultures. Phsiol. Plant., 15: 473-
497.
12. Chee, R., and Pool, R.M. 1982. The effects of
growth substances and photoperiod on the
development of shoot apices of Vitis cultured
in vitro. Sci. Hort., 16: 17-27.
13. Lloyd, G., McCOWN, B. 1981.
Commercially feasible micropropagation of
montain laurel, Kalmia latifolia, by use of
shoot tip culture. Com. Proc. Int. Plant Prop.
Soc., 30: 421-327.
14. Kinfe, B. 2010. Multiple shoot regeneration
of grape vine (Vitis vinifera L.): Optimum
BAP concentration for shoot initiation,
survival and multiplication optimum IAA
concentration for rooting. LAP Lampert
Acad. Publ. 64.
15. Banilas, G. and Korkas, E. 2007. Rapid
micropropagation of grapevine CV.
Agiorgttiko through lateral bud development.
In: e-journal of science and technology (e-
JST)., 2: 31-39
16. Gray, D.J. and Benton, C.M. 1991. In vitro
micropropagation and plant establishment of
muscadine grape cultivars (Vitis rotundifolia).
Plant cell, tissue and organ culture., 27: 7-14.
17. Jaskani, M.J., Haider, A., Sultana, R., Khan,
M.M., Qasim, M., and Iqrar, A.K. 2008.
Effect of growth hormones on
micropropagation. Of vitis vinifera L. CV.
Perlette. Pak. J. Bot., 40(1): 105-109.
18. Dalal, M.A., Sharma, B.B., and Sharma. H.C.
1991. Effect of macro mineral salts
modification in MS culture medium on
oxidative browining in vitro culture of grape.
Indian J. Hort., 48:187-191.
19. Gray, D.J. and Benton, C.M. 1990.
Micropropagation and plant establishment of
Muscadine grape. In: Proc. Fla. State Hort.
Soc., 103: 300-302.
20. Lee, N., Wetzstein, H.Y. 1990. In vitro
propagation of muscadine grape by axillary
shoot proliferation. J. Amer. Soc. Hort. Sci.,
115: 324–329
21. Alizadeh, M., Singh, S.K., and Patel, V.B.
2010. Comparative performance of in vitro
multiplication in four grape (Vitis spp.)
rootstock genotypes. International Journal of
Plant Production 4 (1): 178-186
22. Harris, R.E. and Stevenson, J.H. 1982. In
vitro propagation of Vitis. Vitis., 21: 22-32.
23. Reisch, B.I. 1986. Influence of genotype and
cytokinins on in vitro shoot proliferation of
grapes. J. Amer. Soc. Hort. Sci., 111: 138-
141.
24. Martinez, E.A., Tizio, R., 1989. Grapevine
micropropagation through shoot tips and
minicuttings from in vitro cultured one-node
cuttings. Hort. Sci., 24: 3. 513
25. Thies, K.L., and Graves, C.H., 1992.
Meristem micropropagation protocols for
Vitis rotundifolia Michx. HortSci., 27: 447-
449.
26. Mhatre, M., Salunkhe, C.K., Rao, P.S., 2000.
Micropropagation of Vitis vinifera L. Towards
an improved protocol. Sci. Hort., 84: 357-63.
27. Singh, S.K., Khawale, R.N., Singh, S.P.,
2004. Techniques for rapid in vitro
multiplication of Vitis vinifera L. cultivars. J.
Hort. Sci. Biotech., 19: 267-272.
28. Poudel, P.R., Kataoka, I., Mochioka, R.,
2005. Effect of plant growth regulators on in
vitro propagation of Vitis ficifolia var. ganebu
and its interspecific hybrid grape. Asian J.
Plant Sci., 4: 466-471.
29. Hicks, G.S., and Dorey, M. 1998. Shoot
multiplication growth and adventitious
rooting in 3 cultivars of Vitis, in vitro. Proc.
Nova Scotian Inst. Sci., 38: 83-89.
30. Mezzetti, B., Tiziana, P. , Oriano, N.,
and Lucia, L. 2002. Genetic transformation
of Vitis vinifera via organogenesis. BMC
Biotechnology 2: 18-26
31. Wang, Y.Z., Ge, K.L., Zou, G.Z., Yang, J.S.,
and Ye, M.M. 1985. Callus induction and
plantlet regeneration in grapevines. Acta
Botanica Sinica, 27: 661-664.
32. Park, H.J., Lee, H.R., Pyee, J. , and Cha, H.C.
2001. Regeneration of Grape (V' s labruscana
cv. Kyoho) by Shoot-Tip Culture. Journal of
Plant Biology, 44(4): 185-192 .
33. Barreto, M.S., and Nookaraju, A. 2007. Effect
of auxin types on in vitro and ex vitro rooting
and acclimatization of grapevine as
influenced by substrates. India J. Hort., 64
(1): 11-17.
34. Butiuc-Keul, A.L., Coste, A., Halmagyi, A.,
Farago, M., Iliescu, M., and Luoras. R. 2009.
In vitro micropropagation of several
grapevine cultivars from Romania. Acta
Hort., 812 (1): 133-138.
35. Lewandowski, V.T. 1991. Rooting and
acclimatization of micropropagated Vitis
labrusca Delaware. HortScience, 26: 586-589
.
Ayman A. Diab, et al.
491