The effect of nutrient media in micropropagation and in vitro conservation of wild population of mahaleb cherry (Prunus mahaleb L.).

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THE EFFECT OF NUTRIENT MEDIA IN MICROPROPAGATION AND IN VITRO CONSERVATION OF WILD
POPULATION OF MAHALEB CHERRY (PRUNUS MAHALEB L.)
Valbona Sota*1, Efigjeni Kongjika2
Address(es):
1Department of Biotechnology, Faculty of Natural Sciences, University of Tirana, Bul. Zog I, Nr. 25/1, Tel. +355 4 2229590, Albania.
2Academy of Sciences of Albania, Section of of Natural and Technical Sciences, Tel. +35542250368, Albania.
*Corresponding author: bona_sota@yahoo.com
ABSTRACT
Keywords: In vitro culture, micropropagation, mid-term storage, MS, LP, WPM media, Prunus mahaleb L
INTRODUCTION
Prunus mahaleb (syn. Cerasus mahaleb L., St. Lucie cherry, also occasionally
Rock cherry or Mahaleb cherry) trees occur in thickets and open woodland on
dry slopes; in Central Europe at altitudes up to 1700 m a.s.l. and in highlands at
1200-2000 m a.s.l. in Southern Europe (Rushforth, 1999). It is drought resistant
and can grow in very poor and calcareous soils, in full sun or partial shade
(Guitian, 1994).
The plant is cultivated for a spice, which is fragrant and has the taste of bitter
almonds. It is used in small quantities to sharpen sweet foods (El-Dakhakhny,
2006). The wood is hard, and is used in cabinet-making and for carving
ornamental objects such as pipes and walking sticks. The bark, wood and seeds
contain coumarin. They have anti-inflammatory, sedative and vasodilation effects
(Vedel et al., 1960). Away from its native range, the species is grown as an
ornamental tree for its strongly fragrant flowers, throughout temperate regions of
the world.
The most common use is as rootstock for sweet or sour cherry cultivars. A
number of cultivars have been selected (Bean, 1976). In the first stage of the
cherry rootstock breeding, the activity is focused on the collection of native
mahaleb cherry (Prunus mahaleb L.) varieties (Hrotkó, 2004; Hrotkó et al.,
2004). The common Prunus mahaleb L. rootstocks used for cherry production
are becoming more unsatisfactory. Trees from grafted varieties are too large, fruit
set is too late and harvest costs are too high (Edin et al., 1996).
Micropropagation is a suitable method for obtaining a large quantity of
genetically homogeneous and healthy plant material which can be used for
planting (Kongjika et al., 2002; Damiano et al., 2008). The rapid in vitro
multiplication of cloned plants is desirable to shorten crossing programs in fruit
tree breeding (Daorden et al., 2004). In vitro culture is an effective method for
ex situ conservation of plant genetic diversity, allowing rapid multiplication from
very little plant material and with little impact on wild populations. For safe
preservation, the in vitro slow growth storage method was developed and is
considered an alternate solution for medium term storage of fruit germplasm
(Neveen et al., 2008). The aim of medium term storage is to increase the interval
period between subcultures by reducing growth. This might be achieved by the
use of modified environmental conditions, modified culture medium, growth
retardants, osmotic regulators and/or reduction of oxygen concentration
(Kameswara, 2004). Slow growth storage via In vitro cultures has been reported
in many species (Maqsood et al., 2010).
The aim of this study is to determine the optimal nutrient media for
micropropagation and to develop a suitable protocol for mid-term storage of
Prunus mahaleb L. germplasm.
MATERIAL AND METHODS
Plant material: collection and disinfection
Cultures of P. mahaleb L. isolated from wild populations of Zejmen (Lezhe),
were established from apical and lateral buds removed from adult field-grown
trees. The plants were collected during February and March. Most often shoot
tips and meristems are the explants of choice due to their genetic stability. Plant
material used to introduce P. mahaleb L. cultivars is obtained by collecting the
active explants between January and March, when buds were starting to swell
from shoots in dormancy.
Active shoots were cut in two- or three-node sections. Two types of disinfection
reagents were used: HgCl2 and NaOCl. The stem sections were washed carefully
with water and than were shaken for 5 min. in 70% ethanol, followed by 20 min.
treatment with HgCl2 0.01% or NaOCl 0.3% and two drops of Tween 20. Finally
stem sections were rinsed three times with sterile distilled water. Explants size is
not as important for micropropagation as purposes as for obtaining disease-free
plants. The buds were dissected up to 3 mm by removing the outer scales and
showed no sign of contamination after over one year of continued culture.
Shoot tips of Prunus mahaleb L. isolated from wild populations of Zejmen (Lezhe), promising as rootstocks for sweet cherry cultivars,
were submitted to in vitro culture to test if micropropagation could be used for their rapid production. This study was carried out to
determine the optimal nutrient media for micropropagation and to develop a suitable protocol for mid-term storage of Prunus mahaleb
L. germplasm. For micropropagation were tested three different basal media MS, WPM and LP, all the three combined with 0.3 mg l-1
BAP, 0.1 mg l-1 IBA, 0.3 mg l-1 GA3. The highest shoot length (5.53 cm) was observed on explants cultured on MS media, whereas this
parameter was reduced on explants cultured on WPM and LP media (4.63 and 2.10 respectively). During subculture stage, MS and
WPM media didn’t show statistical differences regarding to shoots number/explants and leaves number/explants. The rooting
percentages of plantlets ranged from 10 to 90%, depending on NAA concentration in the rooting media. In order to find out a medium-
term in vitro preservation protocol effect of reduced sucrose and MS salts concentrations and elimination of PGRs from nutrient media
on a collection of 30 days old of in vitro wild mahaleb cherry nodal segments have been examined for different periods. The highest
survival and regeneration percentage (respectively 93.36 % and 83.72 %) were found in cultures stored at ½ MS media without sucrose
for the period of 3 months. The maximal time of conservation without subculture on reduced sucrose and MS salt (1/2MS)
concentrations is up to 5 months and in basal MS media without PGRs is up to 3 months. Hence the shoot tips of Prunus mahaleb L. can
be successfully stored in vitro for medium terms at ½ MS media without sucrose.
ARTICLE INFO
Received 13. 3. 2014
Revised 6. 4. 2014
Accepted 30. 4. 2014
Published 1. 6. 2014
Regular article

J Microbiol Biotech Food Sci / Sota and Kongjika 2014 : 3 (6) 453-456
454
Media composition for in vitro cultivation
Proliferation and subculture media: Three nutrient media were tested MS
medium (Murashige and Skoog, 1962), LP medium (Quoirin and Lepoivre,
1977) and WPM medium (Lloyd and McCown, 1980), all the three combined
with 0.3 mg.L-1 BAP; 0.1 mg.L-1 IBA; 0.3 mg.L-1 GA3 and supplemented with
3% sucrose and solidified with 0.55% agar. The pH of the media was adjusted
5.7 – 5.8 before autoclaving. After a month, the developed buds were transferred
to fresh media for further multiplication. Measurements of proliferation (%),
length of the shoots and leaves number were taken.
Rooting media: When the explants derived from MS medium during proliferation
and subculture stage reached 2 – 4 cm in length, they were transferred to rooting
media. Three variants of rooting medium were analyzed:
- Rooting media I: ½ MS macronutrients, MS micronutrients, MS
vitamins containing 0.1 mg.L-1 NAA;
- Rooting media II: ½ MS macronutrients, ½ MS micronutrients, MS
vitamins with 0.1 mg.L-1 NAA;
- Rooting media III: MS macronutrients, ½ MS micronutrients, MS
vitamins with 2 mg.L-1 NAA.
Rooting response was evaluated after 4 – 5 weeks of culture.
Plantlets acclimatization: Rooted plantlets were transferred after 3 – 4 weeks on
rooting media to a mixture of soil, peat and perlite (2:1:1) (v/v), in 7 cm diameter
plastic pots and placed in a controlled growth chamber at 20oC. The plantlets
were covered with plastic bags in order to maintain high humidity by removing
them periodically for some minutes day after day. The bags were removed
completely after about four weeks.
In vitro chamber conditions
The culture in the proliferation stage was grown in the growth chamber at
temperature of 25  2 C in a 16 h/8 h light/dark regime with cool, white
fluorescent light of intensity 43.4 mol m-2 s-1.
For in vitro conservation, two different methods of minimal growth are
tested:
- Effect of reduced sucrose and MS salts concentrations: The cultures are
transferred onto ½ MS media without sucrose and supplemented with the same
rate of plant regulators and agar as in the multiplication media. The incubation
conditions are the same as in the multiplication stage.
- Absence of phytohormones or growth regulators in the growth media: The
cultures are transferred onto MS media without growth regulators or
phytohormones and supplemented with the same rate of other components as in
the multiplication media. The incubation conditions are the same as in the
multiplication stage.
The cultures are stored in these conditions for different periods (3, 4, 5 months)
for each method tested. For each method are at least 15 shoots in each replication.
Survival of the cultures is assessed on the basis of criteria as suggested by Reed
(1992) as dead and brown shoots are considered as unsurvived while those with
vigorous growth and having healthy leaves are considered survived.
Statistical analysis
All experiments were repeated at least twice. Data collections in experiment were
subjected to analyses of variance and evaluated by computer using the statistical
evaluation program JMP 7.0.
RESULTS
In vitro cultivation
Explants disinfection: The explants isolated from developing buds, after the
surface sterilization with HgCl2 0.01% for 20 min resulted in the highest
percentage of developed explants (85%). Contamination rates were about 15%
for primary explants and less than 2% for subcultures.
Sodium hypochlorite in the concentration 0.3% was not effective in disinfecting
explants derived from field-grown adult trees. The contamination rates in this
case were about 80%. Must be noted that the explants are isolated from the field
trees with high contamination and it is required the disinfection with the most
powerful reagents such as mercury chloride.
Proliferation and subculture stage: The shoots number per explants (SN) was
affected by the type of media (Tab. 1, Fig. 1a, b). Explants cultured on MS and
WPM media showed the highest shoot number/explants (3.00 and 2.80
respectively) compared to the explants cultured on LP media (1.50), after 4
weeks of culture. The results showed that the highest leaves number/explants
(LN) (9.50) was recorded on explants cultured on MS media, value that wasn’t
statistically different from those cultured on WPM media (7.80), whereas leaves
number/explants was smaller (4.20) on explants cultured on LP media. The mean
shoot length (SL) was also affected by the type of media. The highest shoot
length (5.53 cm) was observed on explants cultured on MS media, whereas this
parameter was reduced on explants cultured on WPM and LP media (4.63 and
2.10 respectively).
Even in this stage, shoots number/explants, shoots length/explants and leaves
number/explants were affected by the type of media. SN and LN values weren’t
statistically different between the explants cultured on MS and WPM media,
whereas SL values were statistically different (Tab. 1).
Table 1 Shoot number per explants (SN), shoot length (SL) and leaves number per explants (LN) during proliferation and subculture
stage of explants cultured onto MS, WPM and LP media, and rooting percentage on three different rooting media
Culture stage
Nutrient media
Shoot number (SN)
Shoot length (cm) (SL)
Leaf number (LN)
Proliferation
MS
3.00 ± 0.55 A
5.53 ± 0.16 A
9.50 ± 1.00 A
WPM
2.80 ± 0.25 A
4.63 ± 0.12 B
7.80 ± 0.51 A
LP
1.50 ± 0.22 B
2.10 ± 0.25 C
4.20 ± 0.53 B
Subculture
MS
3.80 ± 0.49 A
5.80 ± 0.31 A
13.90 ± 1.23 A
WPM
4.00 ± 0.33 A
4.94 ± 0.15 B
11.50 ± 0.98 A
LP
2.10 ± 0.27 B
4.30 ± 0.24 B
7.60 ± 0.56 B
Rooting media I (%)
Rooting media II (%)
Rooting media III (%)
Rooting
90 ± 2.33
10 ± 1.92
30 ± 2.91
Note: Values represent mean + standard error. Means followed by the same letter within the column do not differ significantly (P ≤
0.05) according to a Tukey’s Honestly Significant Difference test
Rooting and acclimatization stage: Rooting induction appears very difficult,
especially regarding to trees species. For this reason, three nutrient rooting media
containing different concentrations of auxin, α-naphthalene acetic acid, NAA and
macro- and micronutrients, presented in the universal medium MS were
compared for the explants derived from MS medium during proliferation and
subculture stage. After 3 weeks of the culture on rooting medium, rhizogenesis
was observed. The explants reacted differently in three types of rooting media
(Fig. 1c, d, e). The mineral and NAA concentration of the culture media affect
rooting percentage and roots characteristics.
Best results were observed in explants cultured on I rooting media where the
percentage of rooting appeared to be too high (90% ± 2.33). The two other
media, II rooting media and III rooting media showed lower rooting percentage,
respectively 10% ± 1.92 and 30% ± 2.91 (Tab. 1). At the ultimate case (rooting
media III), higher concentrations of NAA tended to induce callus formation on
the proximal end of shoots. In this case, the number of roots was high, but those
had an abnormal look being two short and thick (Fig. 1e).
Acclimatization was affected directly by rooting conditions. Survival was best
when plantlets were transferred to pots after a short period of root emergence on
rooting media (Fig. 1f). Maintenance on rooting media gave longer roots but
resulted in poor survival. Acclimated plantlets are able to pass in the permanent
place in the green-house.

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Figure 1 Micropropagation of wild mahaleb cherry shoot tips a, b) Development
of explants during proliferation and subculture stage c, d, e) Rooted explants in
three different rooting media, respectively I rooting media, II rooting media, III
rooting media f) Acclimated plant
Germplasm mid-term storage
In Table 2 and Graphics 1, 2 are presented and analyzed the results of survival
percentage and regeneration percentage for both methods of conservation tested
in three different periods (3, 4 and 5 months). The regeneration percentage is
calculated after transferring the survived shoots in the nutrient media used for
micropropagation purposes.
From the obtained data result that the highest survival and regeneration
percentage is found in cultures stored in ½ MS media without sucrose
(respectively 93.36% and 83.72%) for the period of 3 months. The maximal time
of conservation without subculture on reduced sucrose and MS salt (1/2MS)
concentrations is up to 5 months and in basal MS media without PGRs is up to 3
months.
With increase in storage period, survival rate as well as regeneration is reduced
significantly. Also are observed significant statistical differences in survival and
regeneration rates of shoots for each method tested (Graph. 1).
Table 2 Survival and regeneration percentage of wild mahaleb cherry explants
conservated with different methods of minimal growth, for different periods
Prunus mahaleb L.
Survival percentage
3 months
4 months
5 months
½ MS media without
sucrose
93.36+ 2.14
74.10 + 0.73
29.6 + 0.77
Basal MS media
(without PGRs)
65.00 + 3.00
32.00 + 2.08
0
Regeneration percentage
3 months
4 months
5 months
½ MS media without
sucrose
83.72 + 1.51
63.31 + 1.87
17.80 + 0.86
Basal MS media
(without PGRs)
79.65 + 1.76
30.02 + 2.64
0
Note: Values represent mean + standard error.
Graphic 1 Oneway analysis of survival and regeneration percentage by storage
period
Graphic 1 - continue Oneway analysis of survival and regeneration percentage
by storage period
Graphic 2 Survival and regeneration percentage of wild mahaleb cherry explants
conservated with different methods of minimal growth
DISCUSSION
Better results obtained in in vitro culture of Prunus mahaleb explants in MS
medium comparing to LP and WPM ones could be related to different
composition of these media. The major differences in macronutrients among
these three basal media are in ammonium and nitrate ion concentrations and total
ion concentration. Full-strength MS medium has higher values of ammonium and
nitrate, while WPM and LP are low ammonium mediums. MS medium has even
potassium nitrate as a major nitrogen source, while LP and WPM medium have
calcium nitrate. Beside this, some micronutrients are present only in the MS
medium. Effectiveness of MS media in micropropagation of Prunus sp. is also
reported from other authors (Sedlák et al., 2008; Shatnawi et al., 2007; Ružić et
al., 2008; Gurel et al., 1998; Namli et al., 2011; Marino et al., 1989; Muna et
al., 1999). Meanwhile, from other studies resulted that LP basal media gives
better results (Lamrioui et al., 2009; Hasan et al., 2010) and others report WPM
basal media more effective (Yao et al., 2011; Harada et al., 1996; Liu et al.
2010).
Positive effect of lower doses of one of the auxin, “inductor” of rhizogenesis, α-
naphthaleneacetic acid, NAA, is reported in the studies of some authors on the in
vitro rooting of apple plantlets (Nemeth, 1981; Monter, 1992). Inside a species
in the level of different varieties is demonstrated that the use of higher
concentration of auxins (2-3 mg l-1) favors the development of callus and limits
root formation. As result, the use of the lower doses than 0.5 mg l-1 is
recommended.
Effectiveness for minimal growth conservation reducing MS salt concentration in
basal media without sucrose is also reported from other authors during
conservation of Vitis sp. (George, 1996) Pyrus sp. (Ahmed et al., 2009;
Moriguchi et al., 1989), Coffea sp. (Desbrunais et al., 1992) etc.
Plant growth could be reduced even if PGRs concentration in nutrient media is
under optimal levels (Gunning et al., 1985). Reducing PGRs concentration
resulted effective in conservation of Fragaria sp. (Jungnickel, 1988), meantime
eleminating them from nutrient media is reported as an optimal storage method
for this specie (Reed et al., 1995). This storage method is also reported for
conservation of Ramonda sp. (Kongjika et al., 1998).
CONCLUSION
The most optimal nutrient medium is considered Murashige & Skoog
medium (MS) supplemented with MS vitamins and combined with 0.3 mg l-1
BAP; 0.1 mg l-1 IBA; 0.3 mg l-1 GA3, which favors the buds development in the
first stage of in vitro culture.
Comparing the data of the response of the explants in three different proliferation
media is observed the difference in shoots number, leaves number and shoot
-10
0
10
20
30
40
50
60
70
80
90
100
Survival
percentage
3 months 1/2 MS basal
media without sucrose
3 months MS basal
media without PGRs
4 months 1/2 MS basal
media without sucrose
4 months MS basal
media without PGRs
5 months 1/2 MS basal
media without sucrose
5 months MS basal
media without PGRs
Storage Period
Each Pair
Student's t
0.05
All Pairs
Tukey-Kramer
0.05
-10
0
10
20
30
40
50
60
70
80
90
Rigeneration
percentage
3 months 1/2 MS basal
media without sucrose
3 months MS basal
media without PGRs
4 months 1/2 MS basal
media without sucrose
4 months MS basal
media without PGRs
5 months 1/2 MS basal
media without sucrose
5 months MS basal
media without PGRs
Storage Period
Each Pair
Student's t
0.05
All Pairs
Tukey-Kramer
0.05

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length parameter. During subcultures was observed not only the production of a
considerable number of plantlets, but even increase in length of secondary and
tertiary adventitious shoots in the explants multiplicated on MS medium.
Best results on rooting percentage were observed in explants cultured on I rooting
medium containing ½ MS macronutrients, MS micronutrients, MS vitamins
supplemented with 0.1 mg l-1 NAA.
Conservation via reduction of MS salt concentration and sucrose elimination
from nutrient media resulted effective for mid-term storage periods up to 5
months. With increase in storage period, survival rate as well as regeneration is
reduced significantly.
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