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In vitro propagation and acclimatization of pepino (Solanum muricatum)

Article  in  Journal of Food Agriculture and Environment · January 2013

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Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment Vol.11 (1): 410-415. 2013 www.world-food.net
Helsinki, Finland
e-mail: info@world-food.net

In vitro propagation and acclimatization of pepino (Solanum muricatum)

Aysun Cavusoglu * and Melekber Sulusoglu

Kocaeli University, Arslanbey Agricultural Vocational School, TR-41285, Kocaeli, Turkey.
*e-mail: cavusoglu@kocaeli.edu.tr, aycavusoglu@hotmail.com

Received 12 September 2012, accepted 18 January 2013.

Abstract
A method for the micropropagation of pepino (Solanum muricatum Aiton) from nodal segments was developed. Detailed surface disinfection
methods of leaves and nodes were tested. Via establishment of initial culture using nodal segments, effect of Murashige and Skoog Basal Medium
(MS) with N6-benzylaminopurine (BAP) (1, 2, 3, 4, 5, 6 and 7 mg/l) or MS with α-naphthalene acetic acid (NAA) (2, 4, 6 and 8 mg/l) on some
morphologic and vital parameters were noted. To obtain the best shoot multiplication medium from shoot tips of in vitro growth plants; MS with
BAP (1, 2, 3, 4, 5 and 6 mg/l) and control medium without any plant growth regulators were tested. In both of the two stages, proliferation rate (%),
number of shoots (shoot number/explant), length of shoots (cm), callusing rate (%) and callus size (cm) were determined. In addition, at initial culture,
rooting rate (%), number of roots (root number/explant) and length of roots (cm) were also evaluated. Fully expanded shoots were transferred rooting
medium MS with or without indole-butyric acid (IBA) (0, 0.5, 1, 2 and 3 mg/l) and rooting rate (%), classification of root quality, callusing rate (%),
callus size (cm), and length of shoots (cm) were determined. Initiation time of rooting according to treatments were also observed in every day and
fully in vitro rooted plants transferred two different acclimatization media under two different climatic conditions step by step and for each step
survival rate (%) were evaluated.

Key words: Solanum muricatum, pepino, micropropagation, node culture, acclimatization.

Introduction
Pepino (Solanum muricatum Aiton) is a subtropical fruit originating
in the Andes in South America. The fruit of pepino, a whitish-
yellow skin with clear purple stripes and flavour similar to a melon,
has been appreciated not only as a food but for its appearance as
well 1. The plant is cultivated very recently in areas other than
South America. Breeding programme in non-native countries has
to be established for developing cultivars with adaptation to new
growing conditions 2 or with greater resistance to diseases 3.
Pepino is highly diverse, and by using appropriate breeding
strategies, it has been possible to develop new improved materials
for such traits 4. Although most pepino cultivars are sexually fertile
and produce viable seeds, they cannot breed truely because of
their high level of heterozygosis 5. Pepino tissue culture
regeneration systems can achieve a rapid propagation for true to
name of type of seedlings from vegetative tissue 6, 7, 11, for gene
transformation of identified genes 8 and regeneration of intergeneric
somatic hybrid 1.
The aim of this study was to establish an efficient and reliable
protocol for in vitro micropropagation of a pepino cultivar
currently grown in Turkey, which presumedly will lead to mass
multiplication of seedlings.
Materials and Methods
Plant material, culture media and incubation condition: The
original stock plant material of Solanum muricatum was obtained
from one of the main commercial tropical and subtropical seedling
and sapling suppliers. Pepino seedlings were kept under culture
room conditions and used as a source for explants throughout
the experiments. Fresh branches were handpicked from the mother
plants and leaves were separated from the main branches with a
blade before sterilization experiments and procedures. The in vitro
experiments reported here, used MS media supplemented with 30
g/l (w/v) sucrose and solidified with 7 g/l (w/v) agar and pH was
adjusted 5.7 before autoclaving. All established in vitro cultures
were incubated in a growth chamber at 25±2°C with a 16 h light/8
h dark photoperiod provided by cool white fluorescent light at 50
µmol s-2m-1 at 70% humidity.
Sterilization experiments: For surface sterilization, fully grown
leaves and bud parts were cut in 10 cm long washed for 5 min with
tap water and then sterilized by ethanol (MerckTM- C2H5OH-
ethanol-96% at 5-70% (v/v) for 5-30 min), Na-hypochlorite
(Commercial bleach-ACETM at 20-70% (v/v) for 1-45 min), tween-
20 (MerckTM-polyoxyethylene sorbitan monolaurate-Tween® 20
as drop for additive), H2O2 (MerckTM-hydrogen peroxide-35% at a
ratio of 4-8% (v/v) for 5-10 min). These were followed by rinsing
three times with distilled sterile water. The sterilized and washed
leaves and buds were blot-dried with sterile paper and cut into 1
cm2 leaf and 1 cm lateral bud including nodiums and internodiums
before cultured in vitro in 9 cm diameter Petri dishes containing
10 ml Murashige and Skoog’s Medium (MS) 9 without any plant
growth regulators.

410 Journal of Food, Agriculture & Environment, Vol.11 (1), January 2013
Initial shoot induction: Axillary branches of the seedlings under
culture room condition of pepino were separated from main branch
and cut in 10 mm long stem segments including 3 to 5 nodiums.
These were surface sterilized with ethanol (30% v/v) for 5 min,
Na-hypochlorite (20% v/v) for 5 min and H2O2 (4% v/v) for 5 min
and followed by rinsing three times with distilled-sterile water and
blot-dried. Every nodium was excised 1 cm long parts with
internodes and placed laterally in plastic disposable Petri dishes
(9 cm in diameter) containing 10 ml of basal MS medium
supplemented with N6-benzylaminopurine (BAP) (1, 2, 3, 4, 5, 6
and 7 mg/l) or α-naphthalene acetic acid (NAA) (2, 4, 6 or 8 mg/l).
For continuation of freshness and healthy growth, the explants
were transferred to same fresh medium in Erlenmayer flasks after
15 days. The proliferation rate (%), number of shoots (shoot
number/explant), length of shoots (cm), rooting rate (%), number
of roots (root number/explant), length of roots (cm), callusing rate
(%) and callus size (cm) were counted, measured and recorded
after 4 weeks, and treatment means were calculated .
Shoot multiplication: Formed shoot tips 0.5-1 cm long, were
transferred from in vitro initial culture medium to shoot
multiplication medium, MS, supplemented with BAP (1, 2, 3, 4, 5
and 6 mg/l) or MS without any BAP as control treatment in
Erlenmayer flasks. In the medium proliferation rate (%), number of
shoots (shoot number/explant), length of shoots (cm), callusing
rate (%) and callus size (cm) were recorded after 4 weeks, and
treatment means were calculated .
Rooting: Experiments on in vitro rooting of in vitro grown shoots
were attempted 1 cm long shoots produced during shoot
multiplication. Shoots were cultured in glass tube 1 cm in diameter
on MS medium supplemented with indole-3-butyric acid (IBA)
(0.5, 1, 2, 3 mg/l) or MS without any IBA as control treatment. At
the end of the fourth week rooting rate (%), classification of root
quality (%) [categorized as 1 (the worst), 2 (not good),3 (good)
and 4 (the best)], callusing rate (%), callus size (cm), length of
shoots (cm) and daily root initiation rate (%) were recorded, and
treatment means were calculated .
Acclimatization: In vitro rooted plants were removed from rooting
medium and gently washed with sterile distilled water supplemented
with a fungicide in a proper dose to remove rooting gel medium
and to avoid from any damping-off contamination and then
transplanted holed plastic pots containing autoclaved mixture of
peat and perlite in 3:1 ratio and all plant body covered with
transparent plastic to maintain humidity. The plastics were
punctured once a day to reduce the humidity and allowing
adaptation to the ambient environment gradually. These were kept
moist with sterile water as per requirement. At the end of 3rd week,
the plants were irrigated with Hoagland’s nutrient solution 10 10
ml per pot. At the end of 4th week the transparent plastics were
completely removed. The plants were kept under room condition
25±2°C with photoperiod (16 h in light/8 h in darkness) for 5 weeks.
At the end of the 5th week, acclimatization data were recorded
according to initial rooting medium and transferred to controlled
greenhouse condition. Each seedling was transplanted to plastic
pots (10 cm x 15 cm) containing sifted field soil and peat at 1:1 ratio
and placed on shaded tables. The plantlets were irrigated with tap
water as per requirement and growth was recorded.
Journal of Food, Agriculture & Environment, Vol.11 (1), January 2013
Experimental design and statistical analysis: All experiments
were triplicated so that in sterilization and initial culture experiments
each repeat consisted of 3 plants in shoot multiplication
experiments of 5 plants in rooting and acclimatization experiments
in 7 plants. The data was analysed using analysis of variance
(ANOVA) of completely randomized design and the groups that
showed variance were then subjected to Duncan’s Multiple Range
Test with a significance value at P<0.05. The percentage data was
transformed using angular transformation (Arc Sin √%) before
carrying out ANOVA.
Results and Discussion
Sterilization experiments: Although, several studies included in
vitro regeneration of pepino 1, 6- 8, 11, 12, there are still in difficulties
for sterilization procedures of pepino according to our experience.
In the step, leaves and buds with nodium were treated with some
chemicals as shown in Tables 1 and 2. Although the leaf culture
was not established, sterilization experiment was done because of
the idea of the leaf culture may be done by us or another scientists.
As can be seen from the tables when dose of the chemicals and
duration increased, a decrease of survival rate occurred, for both
explant types (leaves and buds) treatment with 30% ethanol for 5
min; 20% Na-hypochlorite for 5 min and 4% H2O2 for 5 min without
any pretreatment, was the best sterilization procedure. As a step
of a study, shoot fragments with terminal buds were taken from
the pepino plants, treated and surface disinfected with 70% ethanol,
10% solution of Domestos (Na-hypochlorite) and rinsed with
sterile distilled water 3. In another study, internodes, nodes, apical
parts and lamina of pepino were treated with 3% commercial Na-
hypochlorite 6. In the two studies, duration was not mentioned
but it can be obvious that the chemicals were common in use.
Initial shoot induction: Cultured nodium explants showed signs
of proliferation and callus growth (as regards used media) within
7-10 days. Morphogenic response varied with respect to used
plant growth regulators (Tables 3 and 4). MS media with different
BAP doses added did not show any significant differences except
callusing rate. MS media with different NAA doses added did not
show any significant differences except length of roots among
tested features. As can be seen from the Table 3, with the increase
of the BAP dose, callusing rate also increased at MS with 5 mg/l
BAP. The other high BAP doses caused a sharp decrease on
callusing. According to Table 4, MS supplemented with different
NAA doses only showed significant differences in root length.
The least NAA dose (2 mg/l) caused longest root, but high NAA
dose of 8 mg/l never caused proliferation. The frequency of
proliferation rate, number of shoots, length of shoots and number
of roots increased with decreasing concentration of NAA.
Increasing concentrations of NAA caused callusing (Fig. 1B).
According to general observation for initial nodium culture for
shoot induction, MS with 1 mg/l BAP (Table 3, Fig. 1A) and 2
mg/l NAA (Table 4, Fig. 1C) gave the best results with higher
proliferation rate and shoot number per explant with the least
callusing rate. Moreover, the data with the doses on the
proliferated shoots appeared strong and healty looking.
Although there are not many studies on pepino in vitro culture,
nodes have been used as explants 7 and MS media as basal
medium1, 3, 8. Mostly leaves or meristem tips were used as explants
so supplemented plant growth regulators could not be compared.
411
 Table 1. Sterilization procedures of pepino leaves. Shoot multiplication: When cultured shoot tip explants
Sterilization treatments and period of time Infection rate Survival rate from in vitro proliferated plants for shoot multiplication,
(%) (%) new proliferation rate varied between 46.67 and 93.33%
Pretreatment : Tap water with 5% ethanolĺ30 min. on MS accordingly doses of BAP used (0 (control), 1, 2,
Main treatment: 50% ethanolĺ1 min. 33.33 0 b*
70% Na-hypochloriteĺ1 min. 3, 4, 5, 6 mg/l) (Fig. 1D), but only length of shoots showed
Pretreatment :- statistical significance. Shoot length was longest with
Main treatment: 70% ethanolĺ5 min.
0 0b 7.67 cm/shoot in the control medium. The control medium
30% Na-hypochloriteĺ15 min. gave no response on callus growth at the same time.
8% H2O2ĺ10 min.
Pretreatment :- The other vital and morphologic features, except length
Main treatment: 30% ethanolĺ5 min. of shoots (Table 5), showed some numerical differences.
11.11 100 a
20% Na-hypochloriteĺ5 min. Jordan et al. 11 stated that shoot-tip cultures were very
4% H2O2ĺ5 min. productive since a 14-fold multiplication rate through
Pretreatment :-
Main treatment: 20% Na-hypochloriteĺ20 min.
16.67 88.89 a axillary branching could be achieved using 1.6 µM NAA,
Pretreatment :- 0.4 µM BA and 0.03 µM GA3. In one of the steps of
Main treatment: 25% ethanolĺ12 min. 0 0b another study, meristem tip explants with primordial
25% Na-hypochloriteĺ12 min. leaves were cultured in MS with 0.1 mg/l BA and after
Pretreatment :-
Main treatment: 25% ethanolĺ5 min. 100 - four weeks the explants were transferred to MS medium
25% Na-hypochloriteĺ5 min. without plant growth regulators 3. Because of the
* Means within the column indicate that the treatment means for each treatment having a different letter were significantly treatment differences, the mentioned studies and our
different at P<0.05.
study could not be comparative. When we considered
the observation for shoot multiplication; MS with 1 or 2

Table 2. Sterilization procedures of pepino lateral buds.

Sterilization treatments and period of time Infection rate Survival rate
(%) (%)
Pretreatment : Tap water with 5% ethanolĺ30 min.
Main treatment: 50% ethanolĺ1 min. 55.56 0 c*
70% Na-hypochloriteĺ1 min.
Pretreatment :-
Main treatment: 30% ethanolĺ5 min.
11.11 100 a
20% Na-hypochloriteĺ5 min.
4% H2O2ĺ5 min.
Pretreatment :-
44.45 83.33 ab
Main treatment:20% Na-hypochloriteĺ35 min.
Pretreatment : Tap water with 5% commercial blench with
1% Tween-20ĺ60 min. 0 88.89 ab
Main treatment: 20% Na-hypochlorite with 1% Tween-20ĺ45 min.
Pretreatment :-
Main treatment: 25% ethanolĺ12 min. 0 0c
25% Na-hypochloriteĺ12 min.
Pretreatment :-
Main treatment: 70% ethanolĺ5 min.
0 88.89 ab
30% Na-hypochloriteĺ15 min.
7% H2O2ĺ10 min.
Pretreatment : Tap water with 1% Tween-20ĺ20 min.
Main treatment: 40% ethanolĺ10 min.
19.44 75 b
40% Na-hypochloriteĺ10 min.
7% H2O2ĺ10 min.
Pretreatment : Tap water with 1% Tween-20ĺ35 min.
Main treatment: 30% ethanolĺ10 min.
0 91.30 ab
30% Na-hypochloriteĺ10 min.
7% H2O2ĺ8 min.
* Means within the column indicate that the treatment means for each treatment having a different letter were significantly different at P<0.05.

Table 3. Effect of BAP on some vital and morphologic features from nodiums at initial culture stage.
MS Proliferation Number of Length Rooting Number of roots Length Callusing Callus
medium rate shoots (Shoot of shoots rate (Root number of roots rate size
with BAP (%) number/exp.) (cm) (%) /explant) (cm) (%) (cm)
1 mg/l 88.89 1.67 0.75 0 - - 0 b* -
2 mg/l 80.56 1.08 1.09 16.67 1 0.75 0b -
3 mg/l 55.56 1.00 2.25 0 - - 0b -
4 mg/l 71.25 1.29 1.25 5.56 2 0.63 0b -
5 mg/l 70.37 1.31 1.44 0 - - 14.81 a 0.5
6 mg/l 81.95 1.37 0.93 11.11 1.5 0.79 2.78 b 1
7 mg/l 72.23 1.67 1.71 16.67 2 2.5 5.56 b 0.5
* Means within the column indicate that the treatment means for each BAP having a different letter were significantly different at P<0.05.

412 Journal of Food, Agriculture & Environment, Vol.11 (1), January 2013
Table 4. Effect of NAA on some vital and morphologic features from nodiums at initial culture stage.
MS Proliferation Number of Length Rooting Number of Length Callusing Callus
medium rate (%) shoots (Shoot of shoots rate roots (Root of roots rate (%) size
with NAA number/exp.) (cm) (%) number/exp.) (cm) (cm)
2 mg/l 58.33 1.33 0.78 27.77 3.67 0.94 a* 94.44 0.85
4 mg/l 19.44 1 0.75 5.55 0.75 0.50 b 100 0.68
6 mg/l 11.11 1 0.50 33.33 1.75 0.50 b 100 0.87
8 mg/l 0 - - 0 - - 100 0.83
*Means within the column indicate that the media means for each NAA doses having a different letter were significantly different at P<0.05.

Table 5. Effect of BAP on some vital and morphologic features at shoot multiplication
culture from shoot tips.
MS medium Proliferation Number of shoots Length of Callusing Callus
with BAP rate (%) (Shoot number/exp.) shoots (cm) rate (%) size (cm)
0 mg/l (Control) 86.67 1.27 7.67 a* 0 -
1 mg/l 73.33 3.33 1.49 b 40 0.46
2 mg/l 86.67 4.58 1.21 b 53.33 0.71
3 mg/l 86.67 4.35 0.88 b 53.33 0.68
4 mg/l 93.33 2.1 2.07 b 33.33 0.62
5 mg/l 93.33 4 0.95 b 66.67 1.23
6 mg/l 46.67 1 1 b 0 -
* Means within the column indicate that the media means for each parameter having a different letter were significantly different at P<0.05.

Table 6. Effect of IBA concentration on rooting and root classification rate with some morphological data.
MS medium Rooting Classification rate of rooting quality (%) Callusing Callus Length
with IBA rate (%) 1 2 3 4 rate (%) size of shoots
(the worst) (not good) (good) (the best) (cm) (cm)
0 mg/l (Control) 95.26 45.24 54.76 0 0 14.29 c* 0.5 6.46
0.5 mg/l 100 47.62 9.53 4.76 38.1 47.62 c 0.55 7.09
1 mg/l 90.42 41.9 9.52 6.67 41.91 100 a 0.78 5.53
2 mg/l 95.25 26.19 10.32 48.41 15.08 80.95 b 0.77 4.86
3 mg/l 90.48 22.86 9.53 67.62 0 80.95 b 0.73 5.54
* Means within the column indicate that the media means for each parameter having a different letter were significantly different at P<0.05.

mg/l BAP gave healthy and resistant looking multiple shoots with
relatively lesser callusing rate.

Rooting: MS with IBA doses (0; 0.5; 1; 2; 3 mg/l) showed high

rooting rate (90.42-100%). MS with 1 mg/l IBA gave namely “the
best” rooting group (41.91%), but as can be seen in Table 6, control
and 3 mg/l IBA with MS showed no response in “the best” rooting
group with 0%. MS with 0.5 mg/l IBA is the best for root quality
A B
with relatively high rate in the best rooting group (38.1%) and
relatively the secondly least callusing rate (47.62%). Observations
on root initiation rate day by day with % are in Table 7. MS without
IBA (control) treatment was firstly rooted media and MS with 1
mg/l IBA and MS with 2 mg/l IBA showed late rooting. Rooting
occurred mostly in two weeks (Fig. 2A-D).

Acclimatization: As it is seen in Table 8, if in vitro seedlings

C D
Figure 1. Initial proliferation and multiplication culture of pepino from
nodal segments; A: Proliferation from nodal segments on MS medium
containing 1 mg/l BAP, after 2 weeks. B: Proliferation and callus
development from nodal segments on MS medium containing 6 mg/l
NAA after 4 weeks. C: Proliferation from nodal segments on MS medium
supplemented with 2 mg/l NAA after 4 weeks. D: Shoot multiplication
culture from in vitro tips on MS medium supplemented with or without
BAP (0-Control; 1, 2, 3, 4, 5, 6 mg/l from left to right).
come from control medium, acclimatization to the greenhouse
condition is relatively least, but generally, acclimatization to room
100% for all quality of rooting in vitro medium (Fig. 3A). In the
second step, adaptations to greenhouse (Fig. 3B-C) were very
high, varied between 80-100%, which depended upon the in vitro
rooting medium and rooting quality.
Conclusions
Although there were not many studies on in vitro pepino culture,
when we searched at in terms of Solanaceae family, there are some
correlative examples of shoot tip culture for Capsicum chinense 13
and apikal meristem of Capsicum annuum 14 , effect of BAP and/
or NAA with MS medium for Withania somnifera 15 and Solanum

Journal of Food, Agriculture & Environment, Vol.11 (1), January 2013 413
414
Table 7. In vitro root initiation rate day by day (%).
Days for MS without MS with MS with MS with MS with
root initiation IBA (Control) 0.5 mg/l IBA 1 mg/l IBA 2 mg/l IBA 3 mg/l IBA
2. 4.76 - - - -
3. 14.29 - - - -
4 14.29 - 4.76 4.76 4.76
5. 9.53 - 4.76 - 4.76
6. 28.58 23.81 9.52 9.53 14.29
7. - 19.05 23.81 28.57 28.57
8. 4.76 9.53 9.53 19.05 4.76
9. 14.29 19.05 - 4.76 4.76
10. - 4.76 4.76 9.53 4.76
11. - - 4.76 - -
12. - 9.52 - - -
13. 4.76 - 9.53 - 9.53
14. - 4.76 - 4.76 9.53 A B
15. - - - - -
16. - - - - -
17. - - - - -
18. - - - - -
19. - - - - -
20. and after than…… - 9.52 19.05 14.29 4.76
Total Rooting (%)* 95.26 100 90.42 95.25 90.48

Table 8. Survival rate at acclimatization in the room and the

greenhouse condition.
In vitro Root quality The survival rate of acclimatization step
rooting Classification Room condition Greenhouse
C D
medium (%)* Condition (%)** Figure 2. Rooting media of pepino shoots; A: 1. quality (the worst)
Control 1 100 87.5 rooting on MS supplemented with 0.5 mg/l IBA (left) and control (right).
2 100 85.71
B: 4. quality (the best) rooting on MS supplemented with 1 mg/l IBA. C
3 - -
4 - - and D: From 1. quality (the worst) to 4. quality (the best) for classification
MS+0.5 mg/l 1 100 100 of root quality from left to right.
IBA 2 100 100
3 100 100
4 100 100
MS+1 mg/l 1 100 83,3
IBA 2 100 100
3 100 100
4 100 100
MS+2 mg/l 1 100 100
IBA 2 100 100
3 100 80
4 100 100
MS+3 mg/l 1 100 100
IBA 2 100 100 A B C
3 100 80 Figure 3. Acclimatization of pepino in vitro regenerated plants; A:
4 - -
* Survival rate at the acclimatization to the room condition at the end of the 5th week according to the initial
Acclimatized plantlets after 5 weeks under culture room conditions. B:
in vitro rooting medium and classification of root quality. ** Survival rate at transferring to the soil mixure Transferring to the soil mixure at shaded greenhouse in the transferring
at the end of the 8th week of transferring according to the initial in vitro rooting medium and classification

Journal of Food, Agriculture & Environment, Vol.11 (1), January 2013

of root quality.
day. C: Healty plantlets transferring to greenhouse after 8 weeks.
laciniatum 16 and effect of IBA on rooting of Capsicum annuum17 in Capsicum annuum L. from nodal segments. Biol. Plantarum
and acclimatization of in vitro seedlings of Withania somnifera 18. 50(4):701-704.
According to our search on the topics, there were nearly no study
18
Kulkarni, A. A., Thengane, S. R. and Krishnamurthy, K. V. 2000. Direct
of detailed acclimatization of in vitro pepino plantlets. Since the shoot regeneration from node, internode, hypocotyl and embryo
explants of Withania somnifera. Plant Cell Tiss. Org. Culture 62:203-
succes rate of seedling derived from seed can show heterozygosis
209.
at high level 5 , the above protocol can be used for large-scale
clonal propagation of a true to name phenotype.

Acknowledgements
This research was a part of the project (Project Code:KOU-BAP-
2004/25) supported by Kocaeli University, Scientific Research
Projects Coordination Office, Turkey.

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