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

  • Albanian Academy of Sciences, Tirana, Albania
Valbona Sota*1, Efigjeni Kongjika2
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:
Keywords: In vitro culture, micropropagation, mid-term storage, MS, LP, WPM media, Prunus mahaleb L
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.
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.
J Microbiol Biotech Food Sci / Sota and Kongjika 2014 : 3 (6) 453-456
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
- 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.
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)
3.00 ± 0.55 A
5.53 ± 0.16 A
9.50 ± 1.00 A
2.80 ± 0.25 A
4.63 ± 0.12 B
7.80 ± 0.51 A
1.50 ± 0.22 B
2.10 ± 0.25 C
4.20 ± 0.53 B
3.80 ± 0.49 A
5.80 ± 0.31 A
13.90 ± 1.23 A
4.00 ± 0.33 A
4.94 ± 0.15 B
11.50 ± 0.98 A
2.10 ± 0.27 B
4.30 ± 0.24 B
7.60 ± 0.56 B
Rooting media I (%)
Rooting media II (%)
Rooting media III (%)
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.
J Microbiol Biotech Food Sci / Sota and Kongjika 2014 : 3 (6) 453-456
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
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
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
Regeneration percentage
3 months
4 months
5 months
½ MS media without
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
Note: Values represent mean + standard error.
Graphic 1 Oneway analysis of survival and regeneration percentage by storage
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
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.
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
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).
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
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
All Pairs
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
All Pairs
J Microbiol Biotech Food Sci / Sota and Kongjika 2014 : 3 (6) 453-456
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supplemented with 0.1 mg l-1 NAA.
Conservation via reduction of MS salt concentration and sucrose elimination
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... Other storage temperatures are also reported, such as Vitis spp. stored at 10 • C [33] and 15 • C [35] for 1 year, Arbutus unedo and Ceratonia sativa at 18 • C for 6 months [15], Citrus jambhiri at 22 • C for 1 year [17] and Prunus mahaleb at 25 • C for 4 months [26]. ...
... The same authors investigated the effect of reduced sucrose and MS salts concentrations without PGRs for the in vitro preservation protocol of Prunus mahaleb. In 1 2 MS media without sucrose, the shoots survival was 93.4% after only 3 months of conservation and 74.1% after 4 months [26]. The media composition in Prunus domestica and Prunus cerasifera did not affect the survival percentage during the cold preservation, even if several concentrations of sucrose, BAP and IBA were tested in MS-based media [27]. ...
Full-text available
Plant genetic resources conservation may be a potential option for the improvement of agricultural crops through modern biotechnologies, and in vitro conservation is a tool available to safeguard plant biodiversity. Ex situ conservation of plant genetic resources using the in vitro procedures is in progress in many countries. The slow growth storage (SGS) technique is a valid in vitro approach to preserve several vegetatively propagated species by controlling the growth and development of plantlets, economizing storage space and labor and reducing costs. Moreover, SGS prolongs the timing between subcultures, lowers the risk of losing germplasm through handling errors, such as contamination problems, and decreases the risk of genetic instability due to the reduction in the number of subcultures. SGS is applied by considering different factors: temperature, light or darkness conditions, medium composition, including mineral or sucrose concentrations, and the presence/absence of plant growth regulators, osmotic agents and growth inhibitors. SGS protocols for some fruit species have been well defined, others require additional research. The present review focuses on the effect of several factors that influence the SGS of in vitro shoots derived from temperate and tropical fruit species during the last ten years.
... Micropropagation, especially via meristem culture, is an in vitro technology suitable for maintaining endangered birch genotypes through the rapid proliferation of clone planting material. In vitro methods are advantageous as opposed to conven-tional cuttings, especially in old plants where the formation of new shoots and roots can be more easily controlled and stimulated by the influence of substances added to the nutrient medium (De Diego et al. 2010, Sota & Kongjika 2014. Micropropagation protocols have developed in recent decades for a large number of farm and ornamental tree species. ...
In vitro techniques could be applied for plant conservation through minimal growth. Growth reduction is generally attained by modifying the culture medium and/or the environmental condition. Conservation of St. John’s wort (Hypericum perforatum L.) in vitro through dilution of basic medium was conducted at the Tissue Culture Laboratory of Indonesian Spice and Medicinal Crops Research Institute (ISMCRI) in 2018. Sterile shoots were cultured into Murashige and Skoog (MS) medium by reducing macronutrients. The treatments used were full-MS + 0.1 mg L-1 N6 - benzyladenine (BA) as control; ¾ MS + 0.1 mg L-1 BA; ½ MS + 0.1 mgL-1 BA and ¼ MS + 0.1 mg L-1 BA. Observation of the culture growth was conducted three months after the treatments. The experiment was arranged in Completely Randomized Design with ten replications. The result showed that the use of a dilution of basic medium affected the growth of St. John’s wort. The use of ¼ MS + 0.1 mg L-1 BA composition could suppress culture growth (number of shoots, shoots length and number of leaves), without showing necrotic symptom until three months of conservation. This treatment could be used as an alternative to minimize the culture of St. John’s wort for in vitro conservation.
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Two apple cultivars, Golden delicious and Starking grown in Devoll, Albania, were analyzed to verify the rate of infection by ApMV (Apple Mosaic Virus), ASGV (Apple Stem Growing Virus), ACLSV (Apple Chlorotic Stem Virus) and ASPV (Apple Stem Pitting Virus) using in vitro propagated plantlets, and results taken from different categories of explants and among collections were compared based in the fact that template concentrations were equal. Samples, in total 450 trees, were buds taken from branches collected in early spring from symptomless trees, and seeds taken from fruits at three collection orchards during consecutive years 2013, 2014, 2015. Total RNA was extracted from plantlets grown in vitro from buds and seeds, which were used as template for One-step RT-PCR detection of the viruses. Results showed that viruses are present in both cultivars from the three collection orchards. Concentrations of amplicons varied among cultivars, explants categories and from collection to collection, which suggested different infection rates. The presence of mixed latent viral infection was discussed in terms of a possible mechanism of cross-protection among viral strains already amplified, and concentration of amplicons considered proportionate to the infection rate, was seen as a tool to monitor the situation in collections even in the absence of field symptoms.
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The present study aims to develop a reliable method for in vitro micropropagation and long-term storage of shoot tips in Prunus avium using vitrification and encapsulation dehydration methods. For micropropagation, maximum number of shoots per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L BA (benzyl amino purine). Several pretreatments prior to cryogenic storage were conducted to enhance shoot tips recovery after cryostorage. Using vitrification method, two different preculture treatments were tested to enhance regrowth after both dehydration and cryopreservation. Further accumulation of sucrose and glycerol was tested. The highest regrowth using this method was (up to 77.8%) when shoot tips were exposed to 60 min PVS2 at 0°C. Growth recovery of cryopreserved shoot tips using encapsulation dehydration was (up to 76.3%) obtained when encapsulated shoot tips were pregrown for 3 d in liquid medium containing 0.75 M sucrose, desiccated to 20-22% moisture content and immersed in liquid nitrogen. Recovery of whole plantlets from cryopreserved shoot tips took place directly without transitory callus formation. Both methods are promising techniques for in vitro propagation and germplasm preservation of P. avium shoot tips.
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Determination of the most optimal types and concentrations of plant growth regulators as medium constituents is one of the most important aspects of successful micropropagation, among other in vitro factors. With the aim of optimization of in vitro multiplication of sweet cherry cv. Lapins the effect of following cytokinins has been studied: benzyladenine (BA), isopcntenyl adenine (2iP), kinetin (KIN) and thidiazuron (TDZ) at concentrations of 1, 2, 5, 10 and 15 M, combined with auxine, indole-3-butyric acid (IBA) at concentrations of 0, 0.5, 2.5 and 5 mu M. MURASHIGE and SKOOG (1962) was the basic medium used in all the combinations. The following multiplication parameters were monitored: multiplication index, length of axial and lateral shoots. Fresh and dry shoot weight (callus, stem and leaves - S + L were determined. Some specific issues, such as colour, leaf and callus size, leaf roll, incidence of chlorosis or necrosis along with occurrence of rhizogenesis, i.e. roots unusual for this phase of micropropagation, were also monitored. The highest multiplication index as well as length of axial and lateral shoots was obtained on media with BA. Very poor multiplication, with large sized shoots and big leaves, was achieved on media with 2iP, TDZ and KIN, whereas in many combinations with 2iP, and particularly in those with KIN, rhizogenesis was induced. Obtained results suo, est that the choice of cytokinins for the phase of multiplication of sweet cherry is limited to BA. For more rapid micropropagation, through joining rooting and multiplication phases, KIN and 2iP may be applied. The latter two may be also used to obtain sturdy shoots (elongation phase, prior to rooting).
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The objective of this study was to investigate the possibility of optimizing routine tissue Culture methods to proliferate two sweet cherry cultivars Karesova and Rivan. Shoot tips of two genotypes were Successfully established in vitro. Six proliferation MS media containing 1, 2 and 4 mg/l BAP (6-benzylaminopurine), 0.5 and 1 mg/l TDZ (thidiazuron) or 10 mg/l 2iP (6-(gamma,gamma-dimethylallylamino)purine) were tested. The highest proliferation rate (3.0 +/- 0.1) was obtained for Rivan on MS medium containing 2 mg/l BAR In the case of cultivar Karesova, any of the cytokinins tested did not promote satisfactory proliferation. The highest proliferation rate (1.6) achieved on MS medium with 2 mg/l 2iP is not sufficient for a larger scale in vitro shoot production. It was proved that different genotypes of sweet cherry do not respond in the same way during proliferation in vitro. Future research and testing of other media and plant growth regulators will be carried out.
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Shoot cultures of 9 pear genotypes collected from different areas of Azad Jammu and Kashmir (northern Pakistan) were maintained on MS medium supplemented with 30 g L-1 of sucrose + 7 g L-1 of agar + 1 mg L-1of BAP. For in vitro preservation shoot tips were excised from these cultures and transferred onto different media, i.e. full strength, 1/2 strength, 1/2 strength MS medium, and full strength MS medium containing 2.5% or 3.5% (w/v) mannitol. The cultures were assessed for their survival after storage periods of 3, 6, 9, and 12 months. To test the regenerability of the cultures they were transferred onto fresh culture medium. The genotypes differed significantly in their survival; however, they were statistically similar in terms of regenerability when transferred onto fresh medium. Shoots of the Khurolli and Bagugosha genotypes remained quite healthy, with relatively high survival rates (53.25% and 50.50%, respectively), whereas those of the Desi nakh genotype had the lowest survival rate (41.02%). Full strength MS medium containing 2.5% (w/v) mannitol gave the highest survival and regenerability rates (55.82% and 52.31%, respectively) in cultured shoots. Preservation for the shortest period (3 months) resulted in the highest survival (63.41%) and regeneration (58.81%). Genotypes and storage period interaction showed maximum shoot survival in Kashmiri nakh stored for 3 months, while Raj btung cultures stored for 12 months responded poorly, with the minimum survival rate. Addition of mannitol at the lower concentration of 2.5% (w/v) was an effective technique to lengthen subculture duration, possibly because of a gradual increase in osmotic pressure in the medium, which resulted in reduced availability of water to the growing cultures.
Strawberries pioneered large-scale application of in vitro techniques in pomology. Consequently, the scientific literature reflects all the ethusiasm and disillusionment, successes and failures, advantages and disadvantages, or finally, uses and misuses, which accompany the introduction of a fundamental new method into practice. However, specific mistakes, which could just as well have been made under axenic conditions as in the reestablishment procedure, or in experimental fields or in nurseries, gave rise to doubts, and numerous growers became worried about the “general” genetic instability of in vitro plants, regardless of the special method applied.
In this study the possibilities of in vitro vegatitive propagation of almond (Amygdalus communis L.) cv. Texas and cv. Nonpareil by shoot-tip culture were investigated. Different levels of IBA (0.0, 0.1 and 0.5 mg/l) and BAP (0.0, 0.5, 1.0, 2.0 and 3.0 mg/l) were tested by observing shoot development and growth during three successive stages, namely initiation, transplantation and multiplication. During the initiation stage, hormone-free medium or medium with low IBA (0.1 mg/l) only seemed favourable for shoot growth. During both the transplantation and multiplication stages, the combination of 0.1 mg/l IBA and 1.0 mg/l BAP was found to be the most effective in terms of new shoot production and shoot growth rate. In general, BAP was shown to be essential for shoot development during the last two stages but high levels (2.0 or 3.0 mg/l BAP) caused vitrification and callus formation which subsequently reduced the viability of the shoots.
Studies on the in vitro propagation ability of three species grown in the Mediterranean region (myrtle, pomegranate and mulberry) have been carried out in order to find an efficient propagation protocol. In particular the aptitude to growth under in vitro conditions, the micropropagation rate, the rooting percentage and the acclimatization phase were analysed. The myrtle (Myrtus communis L.) is an interesting plant with multipurpose use. As ornamental plant it is used for the production of green cut branches for indoor decoration and for pot plant for gardening. Pomegranate (Punica granatum L.) and mulberry (Morus alba L.) are also attractive ornamental plants. Moreover these species are interesting for their pharmaceutical properties as well as for their alimentary use. Axillary shoots have been successfully sterilised using a combination of NaOCl and Na merthiolate for 20 min.; the percentage of sterile explants of 65% was obtained. The shoots of the pomegranate were multiplied onto a basal medium QL with BA (0.4 mg l-1) and IBA (0.05 mg l -1); MS medium with BA (0.6 mg l-1), NAA (0.01 mg l -1) and GA3 (0.1 mg l-1) was used for myrtle; MS added with BA (1.0 mg l-1) for mulberry. Rooting was induced in all the species using IAA or IBA (0.75 or 1.0 or 2.0 mg l-1). All the species showed a good aptitude for multiplication and rooting.