in vitro propagation of apricot (prunus armeniaca l

35 Pages·2015·1.02 MB·English

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J.Agric.&Env.Sci.Dam.Univ.,Egypt Vol.11 (1)2012

IN VITO PROPAGATION OF APRICOT (Prunus
armeniaca L.) GROWING AT SAINT CATHERINE
VALLEYS IN SINAI PENINSULA, EGYPT.

KARIM M. FARAG1 , NEVEN M. N.NAGY1, HEMAID I. SOLIMAN2AND
MANAL E.E.AHMED2.
1 Department of Horticulture (Pomology), Faculty of Agriculture, University of Damanhour,
Egypt.
2Tissue Culture Unit, Desert Research Center, Mariut Research Station, Alexandria, Egypt.

ABSTRACT

Conserving rare germplasms of apricot is one of the
important means to achieve diversed fruits characteristics
with preserving desired parameters. This acquires an
establishment of a mass production system that overcomes
all the obstacles of propagating a woody plant such as
apricot. This study was conducted during the period 2009 to
2012, utilizing apricot germplasms known as (Allawzi) from
Saint Catherine Valleys, Sinai, Egypt. It could be concluded
from the sterilization experiments of stem node section with
apricot that NaOCl solution was very effective. In addition,
the survival percentage is controlled by the explant type.
Stem node sections needed higher concentrations and
durations of NaOCl application (1.5 % for 10 min and 1%
for 25 min, respectively) to give 90 and 70 %, respectively of
survived explants. This could be contributed to the large size
and age of stem node sections, which contain high
percentage of microbial contaminants compared to the
smaller and younger shoot tips. Concerning the
establishment and multiplication stage, it could be concluded
for Prunus armeniaca L. that MS medium containing 2 mg/L
BA + 0.5 mg/l 2ip and that containing 2 mg/l BA with 1 mg/l
Kn were the most suitable media for the establishment of
stem node sections. These media gave the highest percentage
of survival and axillary shoot formation and mean number
and length of axillary shoots per explant comparing to the
other used treatments. It was noticed that MS medium
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containing BA with 2ip gave higher mean number of short
axillary shoots per explant than that containing both BA and
TDZ or that containing BA alone, in contrast to the mean
length of axillary shoots, which was higher on the medium
containing 2ip or BA alone, the mean number of axillary
shoots /explant increased with each subculture until
reaching the forth subculture (it reached the maximum
value of 9.7 axillary shoots /explant) and decreased in the
fifth subculture. It could be concluded that the medium with
2 mg/L BA and 0.5 mg/l 2ip was the more responsible for the
multiplication of apricot plant. Regarding to the elongation
stage, it could be concluded that 2iP gave promising results
in enhancing elongation of axillary shoots of Prunus
armeniaca L., since the medium containing 0.5 mg/l 2iP in
addition to 0.5 mg/l BA was superior in its effect on shoot
elongation. It gave the highest mean length of axillary shoots
(5.05 and 2.49 cm) which was significantly the optimum
growth regulators combination comparing to the other
tested media. From the different treatments used to induce
rooting of apricot, IBA alone without NAA gave the highest
rooting percentages than IBA with NAA or NAA alone. For
Prunus armeniaca L. the highest rooting percentage (90%)
was obtained on liquid MS medium supplemented with 1.5
mg/l IBA after 30 days of incubation. This treatment also
gave the significantly highest mean root number (5.1) and
length (3.35 cm), in addition to the maximum shoot height of
5.67 cm. For Prunus armeniaca L. the highest rooting
percentage (80 %) was obtained on solidified MS medium
supplemented with 2 mg/l IBA after 30 days of incubation.
This treatment also gave the significantly highest mean root
number (3.8) and length (4.8 cm), in addition to the
maximum shoot height of 5.5 cm. Rooted plantlets of Prunus
armeniaca L., survived when were acclimated of inside the
growth room in soilrite for two weeks then transferred into
peatmoss: sand mixture (1:1). An average of 92–98% of the
acclimatized transplants survived in the green house.
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INTRODUCTION

Apricot is a member of the genus Prunus, which comprises
other economically important crops like peach, almond, cherry and
plum. Apricot (Prunus armeniaca L.) is an important stone fruit tree
growing in temperate climates. Apricots are mainly used to prepare
juices and for fresh consumption, with affair amount of the 2.7
millions of tons of fruit per year world production being destined to
comfitures, drying and canning. Woody plants raise frequent
propagation difficulties when using conventional techniques. Tissue
culture can overcome this problem since it has been reported that
plants may acquire higher rooting capabilities after continuous sub
culturing in vitro (Haward et al., 1989; Jones and Haddlow, 1989 and
Grant and Hammat, 1999). Apricot is one of the more difficult - to-
root fruit tree species and it has been propagated by grafting onto
heterogeneous seedling rootstocks. In vitro culture has been widely
used for the propagation and conservation of crop genetic resources in
both agriculture and horticulture crops (Barakat and El-lakany, 1992).
In addition, in vitro propagation has been used to produce large
quantities of desired genotype. Apricot is well suited for in vitro
propagation because cuttings are difficult to root and scions are
currently budded onto apricot seedlings (Reighard et al., 1990).
Therefore, growing apricot on their own roots appears to be logical
undertaking. The literature on in vitro culture of apricot, however, is
very important compared to that on other Prunus species (Marino et
al., 1993). Many economic and valuable germplasms are available in
Egypt especially in very remote or distant areas. These germplasms
have been threatened by distinction due to some stress full conditions
or pests, eventhough; they are characteristics with many desired traits.
Rare germplasms, in general represent a wealth or source of fortune to
the field of agriculture. It is very important to pay more attention to
the available germplasm especially those growing under arid
conditions. The valley of Saint Catherine is one of the rich sources of
fruit trees germplasm. The utilization of available techniques such as
encapsulation-dehydration would provide a valuable mean to preserve
such invaluable plants. To be able to achieve mass production of such
as rare germplasms while maintaining their traits, it is very important
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to establish an in vitro production system that could be adapted and
implemented on a commercial scale. Thus, the objectives of this
research were to propagate, in vitro, rare apricot plants grown at Saint
Catherine Valleys as a stepstone to do the other steps such as the
preservation of such germplasm by encapsulation- dehydration. The
second objective would be to introduce a new clone of apricot with
greatly desired fruit characteristics.

MATERIAL AND METHODS

Micropropagation Study.
1. Preparation of Explants:
Shoots of apricot (Prunus armeniaca L.) were cut into
approximately 1-2 nodal segments, and then washed under running tap
water overnight for assisting in surface sterilization. Cultures were
initiated using stem node segments. The explants were rinsed several
times with sterile anti-oxidant solution (150mg/L citric acid and 100
mg/l ascorbic acid) to avoid browning of the tissues. Surface
sterilization of the explants was carried out under complete aseptic
conditions in the Laminar air flow hood. The explants were subjected
to different sterilization treatments (1.5% NaOCl for 10 min) and
washed with sterilized distilled water for 3-4 times to remove all
traces of the disinfection. All steps of the sterilization had been done
under aseptic conditions. During sterilization experiment, using of an
antibiotic detergent has been taken.
2. The Basic Nutrient Medium:
Stem node sections (3-4 cm) long were dissected out of the
cuttings and planted vertically on solid basal medium of MS
(Murashige and Skoog, 1962), and vitamins supplemented with 100
mg/L myo-inositol and 30 g/L sucrose with 2.5 g/L phytagel (Table1).
Growth regulators such as Benzyl adenine (BA), 2-isopentenyladenine
(2ip), Zeatin, Thidiazuron (TDZ), -Naphthalene Acetic Acid (NAA),
kinetin (Kn), and 3-Indole Butyric Acid (IBA) were used
independently or in combination at different concentrations.
The pH value of the nutrient media was adjusted to 5.7 ± 0.1
with adding few drops of 0.1 N either HCl or NaOH prior to addition
of phytagel or agar to medium and autoclaving.
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The media were dispensed into 25 x 150 mm tissue culture
tubes where each tube contained 15 ml per tube or for multiplication
and rooting stages 30 ml volumes into jars. All jars were closed with
autoclavable polypropylene caps and autoclaved for 15 minutes at
121C and 1.1 kg/cm 2 pressure for sterilization, then left to cool in a
slant position at room temperature (22 ± 2 oC).
3. Culture Growth Conditions:
The sterilized explants were cultured on the media under
complete aseptic conditions in the Laminar Air Flow Hood. Tissue
culture tubes and jars were then placed in an incubation room at a
temperature of 26 ± 2C under 16 hours photoperiod of 3000 Lux
supplied with cool white fluorescent lamps.
4. Effect of Anti-oxidant Treatment:
Shoot tip and stem node section were excised from branch lets
of Prunus armeniaca L., surface sterilization of explants was done as
described previously (section 1-4 mm). The explants were subjected to
the anti-oxidant treatment as following:
1- Control; explants were immersed in sterilized distilled water for
two hours.
2-Anti-oxidant solution; the explants were immersed for 1 hour in a
solution consisted of 100 mg/L ascorbic acid and 150 mg/l citric
acid as a pre-treatment.
3-Polyvinylpyrlidine (PvP); 160 mg/l was add to cultured medium.
4-Activated Charcoal (A.C); 200 mg/l was added to the medium.
5- Combination of Anti-oxidant solution + PvP.
6- Combination of Anti-oxidant solution + A.C.
7- Combination of PvP + A.C.
8- Combination of A.C + PvP +Anti-oxidant solution.
5. Effect of Different Growth Regulator Combination Establishing
Stage:
The induction of shoot development from the stem node
segments was attempted with MS (Murashige and Skoog, 1962)
supplemented with 0.1 g/l myo-inositol, 30.0 g/l sucrose and 2.5 g/l
phytagel. The pH of the medium was adjusted to 5.7 and autoclaved at
1.2kg.cm-2 and 121 ºC for 20 minutes. Different growth regulator
combinations added to the MS medium supplemented with different
concentrations of BA alone (0.2, 0.4, 0.5, 1.0, 2.0, and 3.0 mg/l) or in
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combination with 2ip (0.5 mg/l), Kn (0.5-1.0 mg/l), TDZ (0.0- 0.5
mg/l), IAA (0.0-0.05 mg/l) of apricot (Prunus armeniaca L.), in
addition to the control (MS nutrient medium without plant growth
regulators).
Explants were cultured in tissue culture tubes or jars and each
treatment consisted of 10 replicates. Percentage of survived explants
(%), percentage of explants forming axillary shoots (%), mean number
of axillary shoots/explant and mean length of axillary shoots (cm)
were recorded after 4 weeks of culture.
5.1. Multiplication Stage:
Established shoots were subjected to be multiplied on MS
medium containing different concentration of BA (1.0, 1.5, and
2.0mg/l) with 2ip (0.2- 0.5 mg/l) and Kn (0.5-1.5 mg/l) of apricot
(Prunus armeniaca L.). For further multiplication, the explants were
subcultured five times on the best medium using large jars to obtain
stock materials to be used for the following experiments. Mean
number and length (cm) of axillary shoots/explant were recorded after
6 weeks of each subculture.
5.2. Elongation Stage:
The proliferated shoots were transferred to jars containing full
strength MS medium supplemented with various concentrations of
BA (0.5-1.0 mg /l) and 2ip (0.5-1.0 mg/l), to select the best
concentration improved shoot elongation. MS medium was
supplemented with 100 mg.l-1 myo-inositol, 3 % sucrose, pH was
adjusted at 5.7, then purified phytagel was added at 2.5 g/l. Shoot
length and number of leaves per shoot for each explant type were
recorded individually.
MS medium was supplemented with 100 mg/l myo-inositol, 3
% sucrose, pH was adjusted at 5.7, and then purified agar was added
at 2.5g/l phytagel. Then, media were autoclaved then cultured and
incubated under culture conditions for two weeks. Mean shoot length
and mean numbers of shoots for each explant type were recorded
5.3. Rooting Stage:
A factorial experiment between auxin type and activated
charcoal was conducted to investigate the effect of the auxin type
(IBA and NAA) and activated charcoal on root formation. Shoots (3-4
cm in length).The obtained explants from shoot elongation experiment
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were transferred to jars contained MS medium liquid or solid
supplemented with or without activated charcoal and with IBA or
NAA at levels 1.0, 1.5, 2.0 mg/l. The following measurements were
recorded after four weeks on rooting medium: rooting percentage,
number of roots/ plantlet and average of root length (cm).
5.4. Acclimatization Stage:
Apricot rooted shoots (5-8 cm long) were washed from
medium residues and treated with 1 mg/l Ridomil solution as a
fungicide, then transplanted into pots filled with a soil mixture of sand
and peat moss (1:1 v/v). Pots were covered with transparent
polyethylene bags and placed in a greenhouse. One week later, the
covers were gradually removed within one month. The percentage of
survived transplants (%) was recorded.
Statistical Analysis:
Variance analysis ANOVA was done using SAS software
program for statistical analysis (SAS, 2000). The differences among
means for all treatments were tested for significance at 5% level by
using Duncan, 1955 new multiple range tests. Means followed by the
same letter are not significantly different at p≤ 0.05.

RESULTS AND DISCUSSION

1. Explant Sterilization of Apricot (Prunus armeniaca L.).
Contamination by microorganisms has been considered a
bottle neck in tissue culture. Therefore, the work with tissue culture
should be carried out under effective aseptic conditions. The choice of
the sterilization method depends on the relative occurrence of
contamination and death. High concentrations and durations of the
sterilizing agent prevent contamination and cause higher death
percentage and vice versa.
Explants of the Prunus armeniaca L. were surface sterilized to
eliminate fungi and bacteria. In this respect, different concentrations
of Clorox containing 5.25% sodium hypochlorite (NaOCl), ethanol
and HgCl were used to prevent the contamination during growth.
From the various-examined sterilization agents and procedures, the
following results were obtained for both stem node sections.
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Stem Node Sections:
Stem node sections of apricot achieved the highest survival
percentage (90 %) by immersing the explants into 1.5 % NaOCl
solution for 10 min (Table 1, Fig.1 and photo 1). Increasing or
decreasing the duration reduced the survival percentage significantly
to 70 and 60 %, respectively. Lower concentrations of NaOCl solution
significantly resulted in a reduction in the survival percentage and
caused an increase in the contamination of explants even with longer
durations, up to 25 min.
Table 1: Effect of surface sterilization with different concentrations of
NaOCl, HgCl and Ethanol on the survival percentage of
2
Prunus armeniaca L. stem node section.
Treatments Duration Survival
(minute) (%)
70 % ethanol + 0.25 % NaOCl 1+15 20h*
70 % ethanol + 0.5 % NaOCl 1+15 20h
0.5 % NaOCl 20 25gh
0.75 % NaOCl 10 25gh
0.75 % NaOCl 20 40efg
0.75 % NaOCl 25 35fgh
70 % ethanol + 0.75 % NaOCl 2+15 70b
70 % ethanol + 1.0 % NaOCl 1+10 50cde
1.0 %NaOCl 15 60bcd
1.0 %NaOCl 25 70b
1.0 %NaOCl 20 45def
1.25 %NaOCl 10 65bc
1.25 %NaOCl 15 60bcd
1.5 % NaOCl 10 90a
1.5 % NaOCl 15 60bcd
70 % ethanol + 1.0 % HgCl 2+10 25gh
2

* Values, within a column, of similar letters were not significantly different
according to the least significant difference (LSD) at 0.05 levels.
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Photo 1: Sterilized stem node sections of
Prunus armeniaca L. using 1.5%
NaOCl solution for 10 min.

Fig.1: survival percentages of Prunus armeniaca L. stem node sections
after surface sterilization with different treatments of ethanol,
HgCl and NaOCl solution.
2
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These results were similar to those obtained by Rocha et al.,
2007 who found that sterilizing explants of prunus armeniaca L. was
done by immersing them in 70% alcohol for one minute and 1.5%
NaOCl for 15 minute.
It could be concluded from the sterilization experiments of
stem node sections of Prunus armeniaca L., that NaOCl solution was
very effective. In addition, the survival percentage was controlled by
the explant type. Stem node sections needed higher concentrations and
durations of NaOCl application (1.5 % for 10 min and 1 % for 15 min,
respectively) to give 90 % survival.
This could be attributed to the large size and age of stem node
sections which contain high percentage of microbial contaminants or
that tree grown in Saint Catherine valleys between rocks under various
environmental stresses.
2- Effect of Anti-oxidant Treatment:
Table 2 indicated that combination of (Anti + PvP + active
charcoal) significantly reduced browning (5%). On the other hand,
increased survival percentage (95%), followed by combination of
Anti+ active, then Active charcoal + PvP as compared with the other
anti-oxidants used and the control (Table 2, Fig.2 and Photo 2). These
results were similar to those obtained by Bayomy, 1998 who found
that the combination of anti-oxidant solutions as explant per treatment
+ PvP in the medium was effective in reducing phenolic compound in
Communis pear.
Table 2: Effect of different anti-oxidant treatments on the survival and
browning percentages of Prunus armeniaca L.
Treatments Survival (%) Browning (%)
Control 0.0c 100a*
Anti-oxidant 40b 60b
PvP 30b 70b
Active charcoal 30b 70b
Anti. + Activ, 90a 10c
Anti. + PvP 70a 30c
Activ. + PvP 60ab 40c
Anti. + PVP + Activ. 95a 5.0cd
* Values, within a column, of similar letters were not significantly different
according to the least significant difference (LSD) at 0.05 levels.