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Prof. Dr. Ibrahimov Ecoforest
AZERBEYCAN
CHANDLER/
walnun in vitro
Doku kültürü
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ALPARSLAN ŞEVKET ACAR
MUZAFFER YILDIRIM İSİMLİ ÇİFTÇİNİN DENİZLİDEKİ YÖRESEL CESİTLERİNDEN URETİLMİŞ.
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https://hal.archives-ouvertes.fr/hal-00882523/document
Walnut (Juglans regia L.) micropropagation
M.A. Revilla, J. Majada R. Rodriguez
Lab. Fisiologia Vegetal, Universidad de Oviedo, Espana
Introduction
One of the main problems involved in walnut
micropropagation by tissue culture
techniques is the low rate of multiplication.
This can be due to the slow growth of the
regenerated shoots leading to extended
culture periods and thus resulting in the
appearance of latent contamination in the
culture (Somers et al., 1982; Driver and
Kuniyuki, 1984; McGranahan ef al., 1986).
To overcome this problem, we have tried
to culture nodal segments from embryonic
and juvenile material in a double-phase
system, which has been shown to increase
production of axillary shoots
(Viseur, 1985), and to include in the culture
medium antibiotic mixtures to prevent
bacterial contamination (Phillips et al.,
1981; Young et al., 1984). This research is
being conducted to optimize the micropropagation
techniques for walnut (Juglans
regia L.).
Materials and Methods
Experiments have been made with embryonic
and juvenile nodal segments of walnut.
Embryonic axes were excised from seeds previously
imbibed for 24 h in water and disinfected
for 5 min in 0.5% NACIO solution followed by 5 min in 75% ethanol and 3 rinses in sterile
distilled water. Embryonic axes were cultured
for 8 wk before excising the nodal segments.
Juvenile material was taken from 2-3 mo old
plantlets germinated under greenhouse conditions
that had been sprayed every 15 d with a
solution of 0.04 g/l kasugamicin (Lainco), 0.97 g/l zineb (Agrocros) and 0.38 g/l cupric
oxychloride (Agrocros). Before taking the
explants from the juvenile material, the plantlets were sprayed 2 or 3 times, every 5 d, with a
solution of 100 mg/i BAP (benzylaminopurine)
and 50 mg/i GA3 (gibberellin) (McGranahan et
al., 1988) to induce vigorous growth.
The medium used was MS (Murashige and
Skoog, 1962), supplemented with 30% sucrose,
0.7% agar and different combinations of BAP
(1-5 mg/1), IBA (indole butyric acid, 0.1 mg/1), IAA (indole acetic acid, 0.05 mg/1) and GA3
(0.1-1 mg/1). The culture conditions were 16 h
photoperiod and 25°C. To decrease explant
exudation, they were transferred onto fresh
medium 1, 3, 5 and 8 d after culture (Driver and
Kuniyuki, 1984; McGranahan et al., 1988). For
double-phase culture, sterile liquid medium was
poured over explants after their transfer onto
the solidified medium. The liquid phase always
covered the solid medium surface.
Rooting was initiated by dipping the shoot
base into the liquid medium containing IBA
(2 mg/1) for 24 h and transferring it onto solidified
medium containing 1 °!° activated charcoal.
The addition of some antibiotic mixtures to
the culture medium allowed the recovery of a
high percentage of contaminated explants. The
levels of antibiotics tested were as follows:
cefotaxim 25-75 mg/l, tetracycline 25 mg/l,
rifampicin 6 mg/l, streptomycin 1-2 mg/I, ampicillin
25 mg/I. Antibiotic solutions were filter sterilized.
Results and Discussion
Micropropagation
The best growth regulator combinations
for micropropagation of J. regia L. from
nodal segments of embryonic or juvenile
material were 1.0 mg/i BAP and 0.1 mg/l
IBA. The same results were shown for
Juglans nigra (Somers et aL, 1982) and
Paradox (Driver and Kuniyuki, 1984).
Higher BAP concentrations, for short culture
periods, produced morphological
modifications in leaves and shoots, and
finally induced vitrification. The application
of 0.1 mg/I GA3 induced greater elongation
in the embryonary shoots but had no
effect on the juvenile shoots.
The establishment of the explants in
double-phase cultures increased the
micropropagation rate for both types of
plant material (Table I). Similar results
were observed for juvenile material when
the plantlets in the greenhouse were stimulated
with growth regulator solutions.
Presumably, the use of both treatments,
double-phase and plant growth stimulation,
will improve the proliferation rate in
walnut.
Sixty percent of the shoots regenerated
from embryonic material produced roots.
Similar rooting conditions were used by
Meynier (1985) for hybrid walnut.
Effect of antibiotics on shoot cultures
The pretreatments given to the plant
material together with the surface sterilization
of the explants allowed the recovery,
after 15 d of culture, of 85% of the
explants. However, latent endogenous
contamination appeared after 1 or 2 mo of
culture resuming in only 5% final recovery
of the explants.
In an attempt to solve this problem, antibiotic
mixtures were added to the initial
culture medium or to the fresh medium
used for transfers.
In the first case, the addition of cefotaxim
(25 mg/I), tetracycline (25 mg/I), rifampicin
(6 mg/I) and streptomycin (2 mg/I)
(mixture A) allowed the recovery, after 9
wk of culture, of 50% of the explants. The
use of cefotaxim (25 mg/I), tetracycline
(25 mg/I) and ampicillin (25 mg/I) (mixture
B) reduced the recovery to 38%. Higher
concentrations of cefotaxim induced
necrosis and death of all the explants.
Explants transferred after 12 wk of culture
to a medium without antibiotics showed
new growth of bacterial contamination.
Therefore, the bacteriostatic effect predominated
over the bactericidal one. No
morphological differences were observed
between control and antibiotic-treated
explants.
Experiments made with contaminated
explants, already established in a medium
without antibiotics, showed that, when
these were subcultured in fresh medium
with antibiotics (mixture A), 60% of the
explants could be recovered. In walnut, as
in other woody species, such as apple,
rhododendron and Douglas fir, the introduction
of antibiotics into the medium prevents
bacterial contamination (Young et
al., 1984).
From this research, it can be concluded
that the conditions under which the source
plant is grown and the treatments given to
the explant in culture are important for
success in walnut micropropagation. We
expect that the combined actions of the
phytosanitary treatment of the plants in
the greenhouse or in the field, the application
of growth regulator solutions, the culture
of explants in a double-phase system
and the addition of antibiotics, will greatly
aid the establishment of an efficient micropropagation
method from selected mature
walnut trees.
References
Driver J.A. & Kuniyuki A.H. (1984) In vitro propagation
of Paradox walnut Juglans hindsii x
Juglans regia rootstock. HortScience 19, 507-
509
McGranahan G.H., Leslie C.A. & Driver J.A.
(1988) In vitro propagation of mature Persian
walnut cultivars. HortScience 23, 220
McGranahan G.H., Tuleke W., Arulsekar S. &
Hansen J.J. (1986) Intergeneric hybridization in
the Juglandaceae: Pterocarya sp x Juglans
regia. J. Am. Soc. Hortic. Sci. 111, 627-630
Meynier V. (1985) Mise en culture in vitro de
m6rist6mes de noyers hybrides. C.R. Acad.
Sci. Paris Ser. /// 301, 261-264
Murashige T. & Skoog F. (1962) A revised
medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol. Plant. 15, 473-
497
Phillips R., Arnott S.M. & Kaplan S.E. (1981)
Antibiotics in plant tissue culture: rifampicin
effectively controls bacterial contaminations
without affecting the growth of short term
explant cultures of Helianthus tuberosus. Plant
Sci. Lett. 21, 235-240
Somers P.W., Van Sambeek J.W., Preece J.E.,
Gaffney G. & Myers O. (1982) In vitro
micropropagation of black walnut. Proc. 7th
North Am. For. Biol. University of Kentucky
Press, Lexington, pp. 224-230
Viseur J. (1985) Micropropagation of pear,
Pyrus communis L., in a double-phase culture
medium. Acta Hortic. 212, 117-124
Young P.M., Hutchins A.S. & Canfield M.L.
(1984) Use of antibiotics to control bacteria in
shoot cultures of woody plants. Plant. Sci. Lett.
34, 203-209
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http://www.davidpublishing.com/davidpublishing/upfile/7/22/2012/2012072284681417.pdf
Journal of Agricultural Science and Technology B 2 (2012) 665-670
Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250
In vitro Propagation of Walnut (Juglans regia) by Nodal
Explants
Layla Shaaban Mohammed Al-Mizory and Azad Ahmed Mayi

Received: February 27, 2012 / Published: June 20, 2012.
Abstract: Micropropagation techniques were set up for walnut. Many plantlets were obtained by culturing node explants in MS
medium,with 30 g/L sucrose, 7 g/L agar, and different concentrations of BA or Kin alone and different concentrations of BA + NAA
or BA + Kin. BA at concentration (2 mg L-1 BA) gave the best results in percentage response and number of shoots/plant (100%,
2.84) respectively. While, Kin at 3 mg L-1 gave the highest average shoot length (2.81 cm). However, MS medium supplemented
with 2.5 mg L-1 + 0.5 mg L-1 Kin gave the best results compared with all treatment shoot/explants and the highest number of
leaves/explants (3.7, 8.2) respectively. While the highest length of shoot (4.7 cm) in MS medium supplemented with 3 mg L-1 Kin.
and a high rooting percentage up to 60% at MS medium supplemented with 1 mg L-1IBA (Indole-3-butyric acid) was obtained .
Key words: Micropropagation, walnut, single-node explants, cytokinins.
1. Introduction
Juglans regia (Common walnut, Persian walnut, or
English walnut), is the original walnut tree of the Old
World. It is native in a region stretching from the
Balkans eastward to the Himalayas and southwest
China. The largest forests are in Kyrgyzstan, where
trees occur in extensive, nearly pure walnut forests at
1,000-2,000 m altitude [1-4].
Persian walnut is commercially cultivated in many
regions around the world for its timber, as well as for
its nuts. European production of Persian walnut still
largely depends on trees originated from seedlings.
During the last 20 years, important work on seedling
selection has been carried out in local populations of
walnut throughout Europe. Also, the characteristics of
wild walnut trees have been described in Bulgaria,
Germany, Greece, Hungary, Italy, Poland, Portugal,
Romania, Slovenia, Spain and the Ukraine. A valuable

Corresponding author: Layla Shaaban Mohammed
AL-Mizory, M.Sc., research fields: plant tissue culture,
horticulture and landscape. E-mail:
veenmizory@yahoo.com.sg.
edible nuts produced by walnut trees are well
appreciated because they are enriched with
unsaturated fat (linoleic, oleic acid). They also contain
other beneficial components like plant protein (e.g.
arginine, leucine), carbohydrates (e.g. dietary fiber),
vitamins (e.g. vitamin A, E), pectic substances,
minerals (magnesium, potassium, phosphorus, sulphur,
copper, iron), plant sterols, phytochemicals (phenolic
acids, flavonoids, etc.) [1, 5, 6]. Especially pellicle ea
thin cover that surrounds kernel, was found as the
most important source of walnut phenolics, although it
only represents 5% of the fruit weight [4, 7, 8].
A wide range of walnut cultivars grown in Duhok
governorate is mainly a consequence of a long-term
project called “Introduction and selection of fruit
plants” [4].
Walnut Can be propagated by seeds, cuttings, and
grafting. Seed propagation often results in variation
while other methods of walnuts propagation are slow
and time consuming. So, there is a need to introduce
efficient methods for faster propagation of walnuts.
Plant tissue culture technique has many advantages
DDAVID PUBLISHING
In vitro Propagation of Walnut (Juglans regia) by Nodal Explants

666
over conventional vegetative propagation and the most
important one is the propagation of a great number of
pathogen-free plants in a short time with high
uniformity. In vitro cultures are now being used as
tools for the study of various basic problems in plant
sciences. It is now possible to propagate all plants of
economic importance in large numbers through tissue
culture. Walnut
The aims of this study were to propagate walnuts
using tissue culture technique to investigate the effect
of different types and concentrations of growth
regulators. Moreover, the most important point is that
micropropagation of walnuts is a key step (transition
stage) to have adequate materials in order to be
extracted and have the medical component available
to be used in further studies.
2. Materials and Methods
This study was carried out at plant tissue culture
laboratory in the department of Horticulture College
of Agriculture, Duhok University during March
2008-July 2009. Actively growing shoots were
excised from field grown plants. Initially expanded
leaves were removed and explants (10 cm) were
dipped for 30 sec. in ethanol (70% v/v) and
disinfested by immersion in the solution containing
0.05% mercuric chloride (HgCl2) for 10 min. These
explants materials were washed three times with
sterile distilled water. The length of whole final of
explants (single nod 1 cm) were cut off and segment
was placed on initiation medium containing major and
minor salts as in [9] MS supplemented with 2 mg L-1
BA, 3% sucrose and 7 g L-1 agar-agar 2-3 weeks after
initiation, the shoots were transferred to proliferation
medium but enriched with different contents of BA (0,
1, 2, 3 mg L-1) and Kin (0, 1, 2, 3 mg L-1) and ascorbic
acid (100 mg L-1).
Shoots longer than 1 cm were used as micro
cuttings and were transfered to MS medium
containing four different concentrations (0, 0.5, 1, 1.5,
2, 2.5, 3 mg L-1) BA in combination with (0, 0.5, 1,
1.5, 2, 2.5, 3 mg L-1) NAA or BA in combination with
(0, 0.5, 1, 1.5, 2, 2.5, 3 mg L-1) Kin. After 4-6 weeks,
the following data were recorded; the percentage of
response, number of shoot/ explants, number of
leaves/ explants and average of shoots length (cm).
The pH of all media was adjusted to 5.7 ± 0.1 before
the media were autoclaved at 121 °C for 20 min at 1.4
kg cm2
. Cultures were routinely transferred every 2
weeks into fresh medium. Plantlets were maintained at
24 ± 2 °C with a 16 h photoperiod. The irradiance at
the level of the cultures was 1,000 Lux. After 4 weeks,
shoots were transferred to 1/2 strength salt of MS
medium with different concentrations (0. 0.25, 0.5, 1)
mg L-1 of IAA and IBA alone for in vitro rooting.
After 4 weeks, the following data were recorded:
percentage of root/culture, number of roots/ culture
and average of roots length (cm).
Statistical Analysis: Experiments were arranged out
using Complete Randomized Design (CRD) using 10
replicates. Significant differences among mean values
were separated using Duncan multiple range tests at P
≤ 0.05 [10].
3. Results and Discussion
3.1. Establishment of In vitro Culture and Shoot
Multiplication
Almost all 100% walnuts node explants placed on
culture initiation medium containing 2 mg L-1 BA
were established aseptic culture and transferred to
proliferation medium 3 weeks later. Plant hormones
are among the most important physiological factors
affecting the regeneration of plants in vitro and the
role of BA as an effective cytokinins in shoot
multiplication has been established in many plants,
which may occur naturally nodal explants showed
their first response by enlarging and bursting of
axillary buds within 1-3 weeks of culture (Fig. 1 a, b,
c). New shoot development from node explants was
observed within three weeks of culture and more
shoots were found to develop during subcultures.
In vitro Propagation of Walnut (Juglans regia) by Nodal Explants

667
Fig. 1 In vitro propagation of walnut. a: Culture single nodal explants on MS + 2 mg L-1 BA after 2 weeks. b: Initial nodal
explants healthy growth on MS + 2 mg L-1 BA after 2 weeks. c: Shoot formation and leaf primordial from nodal explants on
MS + 2 mg L-1 BA after 3 weeks.
3.2. Effect of Cytokinins alone on Shoot Multiplication
Results presented in Table 1 showed that increasing
the concentration of BA caused an increased in the
length of proliferated shoots and number of developed
shoots. The number of shoots per explants was found
in the range of 1.22 to 2.84 under different
concentrations of BA and Kin when used singly and
2.0 mg L-1 BA yielded maximum shoots. The in vitro
experiments of the present study initially involved the
establishment of nodal explants in aseptic cultures,
which resulted in the induction of multiple shoot
formation, elongation of shoots, development of roots
for plantlet formation and finally the establishment of
plantlets under ex vitro condition. The node explants
showed different responses when they were cultured
on MS with different concentrations of cytokinins. Of
the two cytokinins (BA and Kin) used, BA was found
to be comparatively better for shoot proliferation
(Table 1). Similarly, 100% the nodal segments
produced shoots on MS medium with 2 mg L-1 BA. In
this medium, the highest number of shoots per culture
was 2.84 and the average length of shoots per culture
was 2.28 cm. The nodal explants initially produced
about two to three shoots within three-four weeks after
inoculation on BA supplemented medium. On the
other hand, the highest of 60% cultures of nodal
explants produced shoots on the medium with 3 mg
L-1 Kin, where the number of multiple shoots was 1.66
and average length of shoots was 2.81 cm. With
respect to both shoot induction and increased shoot
Table 1 Effect of BA and Kin on in vitro shoot
proliferation from shoot tip and nodal explants of walnut at
establishment stage after 4 weeks of culture.
Growth regulator
mg L-1
% of
survival of
plantlets
No. of
shoots/explant
Average length
of shoots (cm)
Control 0 0f* 0c 0e
Kin
1 40e 1.22b 1.75cd
2 70c 1.32b 2.2cb
3 60d 1.66b 2.81a
BA
1 80b 2.67a 2.23bc
2 100a 2.84a 2.28b
3 60d 2.43a 1.63d
*Means followed by the same letter within each character
(column) do not differ significantly (P ≤ 0.05) according to
Duncan’s Multiple Range Test.
number, in BA proved better than Kin and it was
found to be as the most effective cytokinin for shoot
induction as well as shoot proliferation in walnuts.
The findings of the present study are in conformity
with those of many others where BA performed better
than Kin. As a synthetic cytokinin BA has the
advantage over other cytokinins in inducing in vitro
shoot production in woody plant like Ficus religiosa
[11].
3.3. Effect of Cytokinin and Auxin Combinations on
Shoot Multiplication from Nodal Explants
It is shown in Table 2 that increasing the
concentration of BA in combinations with NAA caused
an increased in the length of proliferated shoots and
number of developed shoots. The MS medium
supplemented with BA at 2.5 mg L-1 + 0.5 mg L-1 NAA
In vitro Propagation of Walnut (Juglans regia) by Nodal Explants

668
Table 2 Effect of BA + NAA on in vitro shoot proliferation
from shoot tip and nodal explants of walnuts on MS
medium at multiplication stage after 4 weeks of culture.
Growth regulator
mg L-1
No. of
shoots/expl
ant
No. of
leaves/explant
Average
length of
shoots (cm)
Control 0.0 2.2b* 2.3d 1.42b
BA+
NAA
3 + 0 3.2ab 2.6d 1.65b
2.5 + 0.5 3.6a 5.1a 2.21b
2 + 1 3ab 4.7ab 2.25b
1.5 + 1.5 2.8ab 4.3abc 3.16a
1 + 2 2.6b 3.8bc 3.29a
0.5 + 2.5 2.4b 3.3cd 2.18b
0 + 3 2.3b 3.1d 1.94b
*Means followed by the same letter within each character
(column) do not differ significantly according to Duncan's
Multiple Range Test (P ≤ 0.05).
gave the highest number of proliferated shoots
/explant (3.6) within six weeks of culture. Gill et al.
[12, 13] reported that a combination of BA and NAA
(5:1/10:1) respectively, was suitable for shoot
multiplication in woody plants. Shoot initiation was
observed in each subculture and the number of shoots
increased gradually with the age of the culture (Fig. 2).
Dealing with the effect of BA and NAA on the shoot
length, the tallest shoot as 3.29 cm and 3.16 cm were
produced from treating with BA at 1 mg L-1 + 2 mg
L-1 NAA and 1.5 mg L-1 BA + 1.5 mg L-1 NAA
respectively compared with all treatments. On the
other hand, the highest number of leaves/culture (5.1
leaves/explant) compared with some of the
transactions were produced form treatment with 2.5
mg L-1 BA + 0.5 mg L-1 NAA. However, the length of
proliferated shoots and number of leaves was
decreased by increasing the concentrations of BA and
NAA in the culture medium (Fig. 2). Results of this
study indicate that a large scale propagation of
Juglans sp. is feasible by tissue culture methods and
hundreds of plants can be regenerated from nodal
explant within fourth subculture. However, after
fourth subculture the cultured explants reach a stage
where the shoot multiplication rate of subcultured
material remained constant. The combined effects of
BA + NAA on multiple shoot induction were reported
earlier in different plants. Similar results were
reported by Atta-Alla et al. [14] who obtained the
highest number of leaves per explants by culturing the
explants of Bombax malabricum and Callistemon
lanceolatus on MS medium containing different
concentrations of BA and NAA.
3.4 Effect of Cytokinin Combination on Shoot
Multiplication from Nodal Explants
It has shown in Table 3 that the high frequency
regeneration of multiple shoots, a range of cytokinins
either individually or in combination with other
cytokinins were used. Growth becomes feasible only
on the addition of one or more of these classes of
hormones to the medium [15]. So, nodal segments
were cultured on MS medium supplemented with
various concentrations and combinations of cytokinins
BA and Kin. The response of nodal segments with
combinations was comparatively better than single
cytokinin. Nodal explants cultured on MS medium
containing BA at 2.5 mg L-1 + Kin at 0.5 mg L-1
produced 4.2 shoots/ explants. Whereas BA 2 mg L-1
+ Kin 1 mg L-1 produced 3.8 shoots/explant.
Maximum shoot length of 4.7 cm was obtained on
medium containing Kin, 3 mg L-1. Similar response
was observed with Rosa indica L [16]. The least
number were produced with MS medium without
growth regulators (control) and MS medium
containing 3 mg L-1 Kin in tissue cultures (as well as
in intact plants and plant organs), cytokinins appear to
be necessary for plant cell division. Cytokinins are
very effective in promoting direct or indirect shoot
initiation. To encourage the growth of auxiliary buds,
and reduce apical dominance in shoot cultures, one or
more cytokinins are usually incorporated into the
medium at proliferation stage [17]. Shoot proliferation
in this trial is comparable with those reported by
Bobrowski et al. [18]. They reported that the best
medium for shoot proliferation was MS medium
containing 1 or 2 mg L-1 BA, but Kin was not
effective on multiplication rate. While Villa et al. [19]
In vitro Propagation of Walnut (Juglans regia) by Nodal Explants

669
Fig. 2 In vitro propagation of walnut. a: Development shoots on MS medium with 2.5 mg L-1 BA + 0.5 mg L-1 NAA. b:
Development shoots on MS medium with 2.5 mg L-1 BA + 0.5 mg L-1 Kin. c: leaves formation on MS medium with 2 mg L-1
BA + 1 mg L-1 Kin. Only a limited number of shoots are observed in this medium.
Table 3 Effect of BA + Kin on in vitro shoot proliferation
from nodal explants of walnuts on MS medium at
multiplication stage after 4 weeks of culture.
Growth regulator
mg L-1
No. of
shoots/
explant
No. of
leaves/explant
average
Length of
Shoots (cm)
control 0.0 2.4b* 6.6bc 1.86d
BA+
Kin
3 + 0 3.6ab 7.3ab 2.46cd
2.5 + 0.5 4.2a 7.8ab 2.88c
2 + 1 3.8ab 8.4a 3.31bc
1.5 + 1.5 3.1ab 5.1cd 3.95ab
1 + 2 2.8ab 4.4d 4.24ab
0.5 + 2.5 2.5b 4.6d 4.61a
0 + 3 2.4b 3.8d 4.76a
*Means followed by the same letter within each character
(column) do not differ significantly according to Duncan's
Multiple Range Test Duncan (P ≤ 0.05).
reported that the greater numbers of shoots were
produced with 1 mg L-1 BA in WPM basal medium. In
our experiment the best result was obtained with 2 mg
L-1 BA in MS basal medium. The average number of
leaves per explants was recorded after 6 weeks of
culture. However, the average number of leaves per
explants on MS medium supplemented with BA at 2
mg L-1 + Kin at 1 mg L-1 was maximum as compared
to some treatments.
3.5. Effect of IBA and IAA on Rooting
The data presented in Table 4 were recorded after 4
weeks from culturing shootof walnuts. Results
indicated that, the highest number of roots (8.1) was
recorded when shoots were cultured on 1/2 strength
MS salt medium supplemented with IBA at 1 mg L-1,
while the lowest number of roots (2.9) was recorded
Table 4 Effect of different auxins and their concentrations
on adventitious root formation from the in vitro grown
micro-cutting walnuts cultured on half salt MS medium
after 4 week of culture.
Growth regulator
mg L-1
Percentage of
root %
No. of root /
explant
Length of
root (cm)
Control 0.0 0g* 0d 0c
IBA
0.25 50b 4.3b 2.12b
0.5 60a 4.7b 3.19a
1 30d 8.1a 2.13b
IAA
0.25 20e 5b 1.72b
0.5 40c 3.8bc 1.98b
1 10f 2.9c 1.66b
*Means followed by the same letter within each character
(column) do not differ significantly according to Duncan's
Multiple Range Test (P ≤ 0.05).
when growing on ½ strength MS medium
supplemented with IAA at 1 mg L-1, but there is no
root was recorded when culturing on ½ strength MS
medium supplemented without plant growth
regulators.
The highest root length (3.19 cm) was recorded
after 4 weeks when using MS medium at ½ salt
strength supplemented with 0.5 mg L-1 IBA, the
average root length of shootlets grown on MS medium
supplemented with 0.5 mg L-1 IBA was significantly
higher than those grown on MS media supplemented
with different concentration of IAA, but the shootlets
grown on MS medium hormone-free not produced the
roots. In conclusion, the highest percentage of root
and the best root length was recorded when culturing
shootlets on half strength MS medium supplemented
with 0.5 mg L-1 IBA and the highest number of roots
was recorded when culturing shootlets on half strength
In vitro Propagation of Walnut (Juglans regia) by Nodal Explants

670
MS medium supplemented with 1 mg L-1 IBA. This
agrees with the study of James et al. [20, 21] for
rooting. James et al. [20] reported that IBA stimulate
rooting of red raspberry explants. In contrast of our
result, Donnelly et al. [22] reported that MS medium
supplemented with 0.5-0.7 mg L-1 IBA was the best
medium for rooting of Rubus sp. shoots.
4. Conclusions
This study led to develop an effecvtive method for
micropropagation of Juglans regia, which in turn may
be applied to mature tissues of Juglans regia under in
vitro conditions.
References
[1] R.B.N. Prasad, Walnuts and pecans, in: B. Caballero, L.C.
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[10] D.B. Duncan, Multiple range and multiple F. teses, Biom.
11 (1955) 1-42.
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Akhteruzzaman, Large scale plant regeneration in Ficus
religiosa L., 4th Intl. Conf., 1-3 Nov, Dhaka, Plant Tissue
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[12] S.S. Gill, R.I.S. Gill, S.S. Gosal, Rapid propagation of
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[13] R. Islam, M. Hossain, O.I. Joarder, A.T.M.
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[14] H.K. Atta-Alla, E.I. Moghazy, A.K. Waly, S. Mohammed,
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[15] N. Mathur, K.G. Ramawat, K.C. Sonie, Plantlet
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[16] A. Shabbir, N. Hameed, A. Ali, R. Bajwa, Effect of
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Tissue Culture, Springer Netherlands Publishers, 2007.
[18] V.L. Bobrowski, C. Mello-Farias Paulo, A. Peters Jose,
Micropropagation of blackberries (Rubus sp.) cultivars,
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[19] F. Villa, A.G. Araujo, L.A. Salles Pio, M. Pasqual, In
vitro multiplication of blackberry (Rubus sp.) Ebano in
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agrotec, Lavras 29 (3) (2005) 582-589.
[20] D.J. James, V.H. Knight, I.J. Thurbon, Micropropagation
of Red Raspberry and the influence of phloroglucinol,
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