World Journal of Agricultural Sciences 11 (2): 84-88, 2015

ISSN 1817-3047
IDOSI Publications, 2015
DOI: 10.5829/idosi.wjas.2015.11.2.12619

In vitro Propagation of Papaya (Carica papaya L.)

1
Adigo Setargie, 2Firew Mekbib and 3Eyassu Abraha

Mekelle University Biology Department, Ethiopia

1

2
Haramaya University School of Plant Sciences, Ethiopia
3
Mekelle Agricultural Research Center, Ethiopia

Abstract: This study was conducted to develop a protocol for in vitro propagation of hermaphroditic papaya
(Carica papaya L.) from shoot buds. In this study, MS media modified with varying concentration of auxin and
cytokinin were used to evaluate the effect of cytokinin and auxin for establishment of the appropriate medium
in regeneration of carica papaya under in vitro condition. Best initiated and proliferated shoots were obtained
on MS medium with 1mg/l BAP and 0.5 mg/l NAA. The highest mean number of shoots (16), highest shoot
length (1.7 cm) and mean number of leaves (21) were recorded on MS medium with 1.0 mg/l BAP and 0.5 mg/l
of NAA. Likewise, the minimum and maximum number of days for shoot emergence was counted on BAP at
0.5 mg/l +0.5mg/l NAA and BAP at 2.0 mg/l + 0.5 mg/l NAA respectively. The lowest number of shoots, number
of leaves and shoot length was recorded as BAP was increased from 0.5 mg/l to 2.0 mg/l and at 0.5 mg/l BAP.
On the other hand, the maximum number of roots (16.25) and root length (3.92 cm) were measured on shoots
pretreated on MS media supplemented with 1.5 mg/l IBA. Unlike this, minimum root length and number were
obtained from 3mg/l of IBA. Lowest survival capacity (40%) of seedlings were recorded during acclimatization
on mixture of garden soil, sand and cow dung at the rate of 2:1:1. Overall, the study showed that BAP and NAA
combination at medium level concentrations for shoot initiation and multiplication; and IBA at lower and
medium level concentrations for rooting were the best basal medium for in vitro propagation of papaya taken
from shoot buds; Nevertheless, further investigations are needed to minimize low survival rate of the seedlings
for mass and commercial propagation of the in vitro developed protocol.

Key words: BAP IBA NAA Root Induction Shoot Bud Shoot Initiation Carica Papaya

INTRODUCTION Southeast Asian region [4]. Now a days it is also

The papaya (Carica papaya L.) belongs to the small
family Caricaceae, which includes 35 species placed in
six genera. Among all species, 32 are dioecious, two
trioecious and one monoecious [1]. The papaya is the
only species of the genus Carica, also being the best
known and most economically important within the
family [2]. Botanically, papaya plant has three different
sex types: staminate (male plant) producing staminate
flower, female plant producing pistillate flower (female
plants) and hermaphrodite plants producing bisexual
flower [3]. It is an important fruit crop grown in the
tropical and sub-tropical regions of the world. It is now
widely cultivated throughout the tropical world and in the
warmest parts of the subtropics and elsewhere in the
popularly cultivated in Ethiopia as a good source of iron,
calcium, vitamins A, B, C [5-6] and the latex is also a good
source of proteolytic enzymes, papain and chymopapain.
It can be propagated sexually by using seeds and
asexual through grafting and rooted cuttings also exists.
However, the setback of propagating by seeds is the
production of non-true-to-type planting materials due to
the segregation of off springs at the second filial
generation; the inherent heterozygosity and dioecious
nature of the plant; and the seeds of open-pollinated
flowers exhibit considerable variation in shape, size and
flavour and susceptibility to diseases[7]. Similarly asexual
reproductions are also often tedious and impractical when
carried out on large scale. Therefore to minimize these
problems, efficient micro propagation of papaya has

Corresponding Author: Adigo Setargie, Mekelle University Biology Department, Ethiopia.

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 World J. Agric. Sci., 11 (2): 84-88, 2015
become critical for the multiplication of specific sex types
of papaya [8] and in the application of genetic
transformation technologies. Hence, breeding and
cultivation of papaya is limited to conventional
techniques in Ethiopia and no work has been conducted
on in vitro propagation of papaya, this study was carried
out to fill this gap by developing an efficient and
reproducible propagation protocol by using shoot bud
culture of papaya (Carica papaya L).
MATERIALS AND METHODS
The study was conducted at the Tissue Culture
Research Laboratory of Plant Biotechnology Division
in Mekelle Agricultural Research Center (MARC),
Northern Ethiopia during the period of 2012/13.
Preparation of Plant Material: The explants were shoot
buds obtained from three month old greenhouse grown
hermaphroditic Carica papaya L. cv. Maradol seedlings.
The shoot buds of papaya were washed thoroughly under
running tap water and then treated with 3 drops of
tween-80 for about 7 min with constant shaking.
Subsequently the explants were transferred to laminar
airflow cabinet and placed into sterilized jar. Then the
explants were immersed in 0.1% HgCl2 and shaked gently
for further sterilization for 5min. The explants were then
washed with sterile distilled water 5 times to remove every
trace of sterilants. After sterilization, the outer leaves were
removed and 3 cm long shoot buds were excised and
prepared for inoculation.
Culture Media Preparation and Culture Establishment:
Murashige and Skoog medium were used for all the
experiments consisted of the basic macro and micro
nutrients and organic supplements. In all cases MS stock
solutions [9] supplemented with 30 gm/l sucrose as a
carbon source, 8 gm/l agar was used. BAP (0.0, 0.5, 1.0,
1.5 and 2.0 mg/l) in combination with consistent
concentrations of NAA (0.5 mg/l) for initiation and
proliferation of shoots were prepared. The pH was
adjusted to 5.8 by addition of 0.1% N HCl and 0.1% NaOH
as required. The basal medium was then poured into the
Magenta culture vessels and medium was autoclaved at
15 pounds per square inch (psi) and 121C for 20 minutes.
Finally, all the autoclaved culture media were allowed to
cool at room temperature and stored in culture rooms
until further use.
85
The surface sterilized and trimmed explants were
cultured on a shoot initiation medium consisting of MS
base (Murashige and Skoog, 1962) and BAP at (0.0, 0.5, 1,
1.5 and 2.0 mg/l) in combination with NAA at 0.5 mg/l
concentration. The culture vessel was sealed with a
parafilm and kept in a growth room under 16:8 h (light:
dark) photoperiod regime with light intensity of 2000-2500
lux at 25 2C and 70% relative humidity (RH). Sub
culturing was done by transferring the new micro plantlets
in to fresh media within 2 weeks of interval. Growth
performance of the shoots(number of days for shoot
initiation, number of leafs, number of shoots and shoot
length were recorded per a week.
Rooting of Shoots: The shoots with more than 1.0 to 1.8
cm in height were taken out from the jar and then placed
in half strength MS media supplemented with (0, 0.75,
1.5, 2.25 and 3 mg/l) concentration of IBA. The pH was
adjusted to 5.8 with 0.1% N NaOH and 0.1% N HCl prior
to autoclaving at 121C and 15 pounds per square inch
(psi) for 20 min. The culture room was maintained at
25 2C temperature uniform light was provided by
using florescent tubes 2000-2500 lux at 25 2C and 70 %
relative humidity (RH) over a light/dark cycles of 16/8 hrs.
Number of roots and root length were measured after a
month of culturing.
Acclimatization: Young rooted papaya plantlets were
taken out from the glass Jars and washed with running
tap water gently to remove traces of agar. Plantlets with
actively growing roots were planted in to a pot having
coco peat soil. After four days in the coco peat soil the
plantlets were transferred to plastic pots containing a
mixture of garden soil, cow dung and sand with 2:1:1 ratio
and covered with polyethylene to retain high humidity.
Plantlets were covered with plastic sheets and maintained
in shade netted greenhouse for one month. Finally,
survival capacity of the plantlets was recorded.
RESULTS AND DISCUSSION
Establishment and Proliferation of Shoots from Bud [H1]:
As indicated in T(1), there is a significant difference
(p<0.01) among the combined effects of cytokinin ( BAP)
and auxin (NAA) concentrations on the number of days
to shoot emergence, number of leaves shoot number,
as well as shoot length. However; BAP at 0.5, 1.0 and
1.5 mg/l were not statistically significantly different from
 World J. Agric. Sci., 11 (2): 84-88, 2015
each other. The fastest shoot initiation was recorded on
MS medium supplemented with 0.5 mg/l BAP + 0.5 mg/l
NAA; where shoots were proliferated within 9.5 days
of Inoculation. Whereas, the longest days for shoot
initiation (13.5 days) were recorded on MS media
supplemented with 2.0 mg/l BAP + 0.5 NAA which was
significantly different from the other treatments.
The number of days for shoot initiation decrease at lower
concentration of the hormone combination but increased
when the concentration of the hormone is increased.
This is might be due to the combined effect of the lower
endogenous and lower exogenous hormone to initiate
shoots within a short period of time as compared to
the combined effects of the higher concentration of
exogenous hormone with lower concentration of
endogenous hormone. The result is in agreement with
those obtained by Hidaka et al. and Kabir et al., [10, 11].
Multiple leaves formation in all treatments revealed
that, the maximum number of leaves (21) was counted from
MS media supplemented with 1.0 mg/l BAP + 0.5 mg/l
NAA. While the minimum number of leaves (17) was
counted on MS media supplemented with 2.0 mg/l BAP +
0.5 NAA mg/l which were significantly different from the
other treatments. The leaves produced on the MS medium
containing 2.0 mg/l BAP and 0.5 mg/l concentration were
pale with prominent midribs and some misshaped
leaves were observed. This might be due to symptoms of
BAP toxicity on leaf formation at higher concentration.
In contrast, plants on the other mediums had dark-green
leaves with good quality shoots. Similar report was
reported by Drew [12] on leaf morphology of in vitro
shoots of papaya.
Different concentrations of BAP with combined
effects of the same concentration of NAA showed a
significant effect on the number of shoots. The present
finding is in line with Reuvani et al., Davis et al. and Drew
et al., [13-15]. The highest numbers of shoots (16) were
counted on the medium supplemented with MS medium
with 1.0 mg/l BAP + 0.5 mg/l NAA. However, the lowest
numbers of shoots (9.5) were from the MS media
supplemented with 0.5 mg/l BAP+ 2 mg/l NAA which
was significantly different from the other treatments
(Table 1). Similar works were carried out by Hidka et al.,
Hossain et al., Islam et al. and Panjaitan et al., [11; 16-19].
The number of shoots produced decreased when the BAP
concentration was increased from 1.0 mg/l to 2.0 mg/l and
decreased at 0.5 mg/l. This difference on number of
shoots might be related with the physiological and
inhibitory effects of synthetic cytokinins of BAP at high
concentration [20-21].
86
The longest shoot (1.7cm) was measured on MS
medium supplemented with BAP 1.0 mg/l + 0.5 mg/l NAA
and the shortest (1.12cm) was recorded on MS medium
supplemented with 0.5 mg/l BAP + 2.0 mg/l NAA. As BAP
concentration increased from lower to higher, the mean
shoot length decreased significantly. Berrie [20] also
described that the supra-optimal amount of the cytokinine
concentration are inhibitory to shoot growth. On the other
hand, at lower concentration of BAP the mean length of
the shoots was decreased; this might be due to the lower
effect of cytokinine to elongate shoots at lower
concentration. This is in agreement with Islam et al., [17].
Rooting of in Vitro Grown Shoots: Among the Five
treatments the maximum numbers of roots (16.25) and best
rooting was obtained from 1.5 mg/l of IBA and with many
branches (Table 2). The least mean number of roots
(9.00 and 7.75) were shown on the medium containing
2.25 mg/l and 3.0 mg/l concentration of IBA. This was due
to the formation of the callus at the base of the roots and
also might be the toxic effects of IBA at higher
concentration. Similar finding was reported by Mousumi
[22]. The results affirmed that, using relatively low
(1.5 mg/l) concentration of IBA allowed shootlet explants
to form roots at greatest number; thus, these conditions
would be recommended for optimal root formation.
These results were in line with Anandan et al.,
Rogayah et al. and Rohman et al., [21: 23-24].
Best treatment for root length (3.92 cm) was found on
MS medium supplemented with 1.5 mg/l concentrations of
IBA (Table 2). The Shortest root (2.25 cm) was recorded
on the medium having 3.0 mg/l IBA. Increase in
concentration of IBA promoted root length up to certain
level and beyond that, inhibiting effect on root length was
recorded [25]; [7]. No variation were observed on MS
medium containing 0.75 mg/l and 1.5 mg/l concentrations
of IBA having mean root length of 3.72 and 3.92Cm,
respectively.
Acclimatization: Lowest survival rate were recorded from
the seedlings transferred to the greenhouse and planted
in to plastic pots containing garden soil, sand and cow
dung with 2:1:1 ratio (Table 3). Hence, only 40% of the
acclimatized seedlings were survived in the greenhouse.
This was due to, the effect of the green house that had no
well regulated temperature, relative humidity and light
intensity level. Likewise, some of the survived seedlings
also showed symptoms of leaf tip necrosis. On the other
hand, root damage during transfer, transplantation and
 World J. Agric. Sci., 11 (2): 84-88, 2015

Table 1: Effects of different concentrations of BAP on the different morphogenetic responses of Carica papaya L.
BAP NAA Duration forshootemergence Numberofleaves (n) Number of shoots(n) Shootlength(cm)
0.0 0.0 0.0c 0.0d 0.0d 0.0d
0.5 0.5 9.5b 18.25bc 14.5ab 1.37b
1.0 0.5 10.5b 21.0a 16a 1.7a
1.5 0.5 10.75b 19.5ab 12.75b 1.45b
2.0 0.5 13.25a 17.0c 9.5c 1.12c
LSD 1.75 2.34 2.21 0.21
CV 10.33 8.03 10.89 9.69
Mean 11.00 18.93 13.18 1.41
Means with same letter (s) in the same column are not significantly different at 1%, CV= Coefficient of Variation (%), n = number, cm= centimeter, LSD=
Least Significant Difference

Table 2: Effects of various concentration of IBA for the formation of roots on 2. Van Droogenbroeck, B., P. Breyne, P. Goetghebeur,
the in vitro grown microshoots of Carica papaya L.
E. Romeijn-Peeters, T. Kydnt and G. Gheysen, 2002.
IBA Number of roots (n) Root length per Shoot (cm)
AFLP analysis of the genetic relationships among
0.0 0.0d 0.0d
0.75 12.50ab 3.72ab
papaya and its wild relatives (Caricaceae) from
1.5 16.25a 3.92a Ecuador. Theoretical and Applied Genetics,
2.25 9.00bc 3.05bc 105: 289-297.
3.0 7.75c 2.25c 3. Bruce, S. and C.A. Peter, 2008. Handbook of
LSD 3.78 0.79 Environmental Physiology of Fruit Crops. 1st Ed.
CV 21.60 15.47
pp: 217.
Mean 11.37 3.31
Treatment means within a column followed by the same letter are not
4. Jennylynd, B. and J.T. Ngarmsak, 2010. Processing of
significantly different at 1%. Where, n= number, CV= Coefficient of fresh-cut tropical fruits and vegetables: A technical
Variation (%),expl= explants, cm= centimeter, LSD =Least Significant guide.Food and Agriculture Organization of the
difference. United Nations Regional Office for Asia and the
Pacific Bangkok.
Table 3: The survival percentage of acclimatized papaya
5. Samson, J.A., 1986. Tropical Fruits. (2nd edition).
No of plants transplanted No of survived plants Survival percentage
20 8 40%
Longman Scientific and Technical, pp: 256-269.
6. USDA, (United States Department of Agriculture),
uprooting from the media were also another factor for the 2009. National nutrient data base for standard
lowest survival rate of the seedlings. Similar results were refence, release 22, nutrient data laboratory
obtained by Kabir et al., Islam et al. and Anandan et al., homepage, http://www.ars.usda.gov/ba/bhnrc/ndl.
[11: 17: 21]. Accessed on December10, 2012.
7. Saker, M.M., S.A. Bekheet, H.S. Taha and
CONCLUSION A.A. Reda, 1999. In vitro propagation of
papaya (CaricapapayaL.) www. acgssr.org/
In general, it is concluded that, the in vitro protocol Biotechnology/V2N2December1999/fullpaper/p22.
described in this study for propagation of hermaphrodite Accessed on February19, 2013.
Carica papaya cv. L. Maradol, using shoot bud, is simple, 8. Lai, C.C., S.D. Yeh and J.S. Yang, 2000.
reproducible and micro propagating and rooting of in Enhancement of papaya axillary shoots
vitro proliferated shoots were successfully achieved; proliferation in vitro by controlling the available
even though, half of the acclimatized plantlets were not ethylene. Botanical Bulletin of Academia Sinica,
survived. Thus, long term goals and efforts towards 41: 203-212
enhancing Carica papaya L. response to in vitro 9. Murashige, T. and F. Skoog, 1962. A revised
manipulation should be continued. medium for rapid growth and bioassays with
tobacco tissue cultures. Physiolology Plantarum,
REFERENCES 15: 473-497.
10. Hidaka, T., S. Komori, M. Yamada and H. Fukamachi,
1. Ming, R., Q. Yu and P.H. Moore, 2007. 2008. Mass production of papaya (Carica papaya L.)
Sex determination in papaya. Seminars in Cell and saplings using shoot tip culture for commercial use.
Developmental Biology, 18: 401-408. South Pacific Studies, 28: 2.

87
 World J. Agric. Sci., 11 (2): 84-88, 2015
11. Kabir, A.H., M.A. Bari, A.K.M.N. Hunda,
M.A. Rezvy and I. Mahfuz, 2007. Effects of growth
regulators and carbon source on axillary shoot
proliferation from shoot tip explant and successful
transplantation of papaya (Carica papaya L.).
Biotechnology, 6(2): 268-272.
12. Drew, R.A. 1992. Factors affecting growth re-sponses
to fructose and other sugars, p:. 65-82. In: Tissue
culture and field evaluation of papaya
(Carica papaya L.). PhD Diss., Murdoch Univ.,
Murdoch, Western Australia.
13. Reuveni, O., D.R. Shlesinger and U. Lavi, 1990.
In vitro clonal propagation of dioecious Carica
papaya. Plant Cell, Tissue and Organ Culture,
20:41-46. Netherlands: Kluwer Academic Publisher.
14. Davies, P.J., 1987. Plant hormones and their role. In:
Davies, P. and J. Dordrecht (eds.). Plant Growth and
Development, pp: 1-11. The Netherlands,
Martinus Nijhoff.
15. Drew, R.A., 1988. Rapid clonal propagation of papaya
in vitro from mature field grown trees. Hort Science,
23: 609-611
16. Hossain, M., S.M. Rahman, R. Islam and O.I. Joarder,
1991. High efficiency plant regeneration from
petiole explants of Carica papaya L. through
organogenesis. Plant Cell Report, 13: 99-102.
17. Islam, R., S.M., Rahman, M. Hossain and
O.L. Joarder, 1993. In vitro propagation of papaya
through culture of lateral buds from mature field
grown plants. Plant Tissue Culture, 3: 47-50.
18. Islam, M., 2001. In vitro plant regeneration and
improvement of Carica papaya L. Through
somaclonal variation. PhD Thesis. Plant Breeding
and Biotechnology Laboratory, Department of
Botany, University of Rajshahi, Bangladish.
88
19. Panjaitan, S.B., M.A. Aziz, A.A. Rashid and
N.M. Saleh, 2007. In vitro Plantlet Regeneration from
shoot tip of field-grown hermaphrodite papaya
(Carica papaya L.). International Journal of
Agriculture and Biology, 6: 827-832.
20. Berrie, A.M., 1984. Germination and growth
regulators. In: Advanced plant physiology.
Malcom, W.B. (ed.), English Language Book Society.
Long man, pp: 987
21. Anandan, R., S. Thirugnanakumar D. Sudhakar and
P. Balasubramanian, 2011. In vitro organogenesis
and plantlet regeneration of (Carica papaya L.).
Journal of Agricultural Technology, 7(5): 1339-1348
22. Mousumi, M., G. Sukumar and M.B. Barid, 1994.
Callus culture and plantlet production in Carica
papaya (Var. Honey Dew). Plant Cell Reports,
13: 390-393.
23. Rogayah, S., J.O. Abdullah, P. Namasivayam,
P. Muda and U.K.A. Bakar, 2012. Better rooting
procedures to enhance survival rate of field
grown Malaysian Eksotika papaya transformed
with 1-Aminocyclopropane-1carboxylic acid Oxidase
Gene. Improvement Science Research Network
Biotechnology, pp: 1-10.
24. Rohman, M.M., Md.N. Islam, Md.S. Alam,
M.R. Ahmad and K. Paul, 2007. Lateral bud culture of
papaya (Carica papaya L.) for clonal propagation.
Biotechnology, 6(3): 339-343, Japan.
25. Protul, K.R., K.R. Shyamal and M.D.H. Lokman, 2012.
Propagation of papaya (Carica papaya L.) CV.
SHAHI through in vitro culture. Bangladesh Journal
of Botany, 41(2): 191-195.