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International Journal of Agricultural

Science and Research (IJASR)


ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 5, Oct 2016, 285-292
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REGENERATION OF SECHIUM EDULE (JACQ) SW. FROM STERILE IN


VITRO NODAL EXPLANTS AND ASSESSMENT OF CLONAL
FIDELITY USING ISSR AND RAPD MARKERS
DHARMALINGAM THILAGAM, BELUR SATYAN KUMUDINI &
SHIRAGAMBI HANMATAGOUDA MANOHAR
Department of Biotechnology, C.P.G.S, Jain University, 3rd block, Jayanagar Bangalore, India
ABSTRACT
An efficient regeneration protocol was developed for Sechium edule (Jacq.) Swartz. an important vegetable crop
in the Cucurbitaceae family. Effect of different concentrations of auxins and cytokinins were evaluated on shoot
proliferation and root induction using sterile nodal explants. Maximum number of nodes and length of the shoots was
observed on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) 2 M. The shoot length and
number of nodes were increased by subculturing in the fresh culture medium containing BA 2 M. At the end of 4 weeks
of incubation, grown shoots were sub-cultured into half-strength MS medium supplemented with indole-3-butyric acid
IBA 10 M. In vitro grown plantlets were transferred to plastic cups containing autoclaved soil: cocopeat: compost (1:1:1)
and successfully transferred to field conditions with 75 % survival. The genetic fidelity of in vitro plantlets was assessed by
Random Amplified Polymorphic DNA (RAPD) and Inter Specific Sequence Repeat (ISSR) markers. Out of 20 ISSR and
20 RAPD markers, 7 RAPD and 6 ISSR primers produced clear, distinct and scorable bands yielding average of 5.14 and
5.16 bands per primer respectively. Banding patterns of in vitro plantlets were monomorphic similar to the mother plant
confirmed true-to-type nature of the in vitro plantlets.

Original Article

(IBA) 10 M for root induction. Longest root length 4.86 cm was recorded in full-strength MS medium supplemented by

KEYWORDS: Sechium Edule, Nodal Explants, In vitro Regeneration, Genetic Fidelity, Cucurbitaceae

Received: Sep 04, 2016; Accepted: Sep 29, 2016; Published: Oct 04, 2016; Paper Id.: IJASROCT201633

INTRODUCTION
Sechium edule (Jacq.) Swartz. or Chayote, is one of the important vegetable crops in the family
Cucurbitaceae (Saade 1996), native to Mexico and Central America (Newstrom 1991). It has the highest diversity
profiles for fruit and plant characteristics (e.g. size, texture, colour, flavour, skin type, presence of spines, different
leaf forms, leaf vein structure, vines and flowers) as reported by Cadena-Iniguez et al. (2008).
Due to its diverse potential biological activities in medicine, it gained importance in the recent years.
The seeds are classified recalcitrant and cannot be stored by drying unlike orthodox seeds (Abdelnour-Esquivel and
Engelmann 2001). Due to this reason, germplasm has to be stored in field conditions and the possibilities of losing
them are on the higher side as witnessed in Mexico (Saade 1996). So, an alternative method of preserving the
germplasm has to be standardized. Conservation of germplasm has become necessary in the Plant Genetic
Resources field in order to avoid certain medicinal and nutritious plants becoming endemic species. As chayote is
loaded with several medicinal values, fibre content and essential/non-essential amino acids, conservation has

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Dharmalingam Thilagam, Belur Satyan Kumudini &


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become important to maintain its genetic diversity.


Due to the low percentage of success in conventional propagation where the pest attack is high as the plant
havent developed resistance against the pest infection during plantation and after harvest, in turn leading to the
propagation of diseased plants and existence of heterogeneity in the fruits.
Micropropagation helps to obtain numerous clones in shorter time and helps in conserving the plants. In fact, its
an effective substitute to establish innumerable plants having recalcitrant seeds. Chayote micropropagation was done using
nodal explants (Esquivel et al. 2002), hypocotyls, shoot tip explants (Guangdong 1997) and embryo
(Cruz-Martineza et al. 2012). But, there are no clear reports for the in vitro regeneration of Sechium edule using in vitro
nodal explants. In order to avoid the heterogeneity due to genetic diversity and for synthetic seed technology
(seeds are recalcitrant; cannot be stored), micropropagation becomes the basic tool to produce innumerable identical
clones, thus retrieving the homogeneity.
In the present study, an efficient micropropagation protocol for the in vitro regeneration of Sechium edule using
its sterile in vitro nodal explants were evaluated and genetic fidelity of the in vitro raised plantlets were assessed by
molecular markers.

MATERIALS AND METHODS


Matured S. edule vegetables were collected from the market, Bangalore. The sprouted vegetables (15-20 days)
were planted in the CPGS campus garden, Jain University, Bangalore.
The shoots having 5-6 nodes were taken from the matured sprouted vegetables and surface sterilized by using the
protocol Esquivel et al. (2002). Single node with auxiliary bud (1 cm) was cut from the shoot segments and inoculated in
test tubes (Borosil, India) containing MS (Murashige and Skoog 1962) medium supplemented with 3 % sucrose, 0.8 %
agar (Hi-Media, Mumbai) and BA (concentration range- 0.5 1, 2, 5, 10 M). After 4 weeks, nodes from in vitro shoots
were taken and cultured in MS medium fortified with auxins indole-3-butyric acid (IBA), indole-3-acetic-acid IAA,
-naphthaleneacetic acid NAA, 2, 4- dichlorophenoxyacetic acid (2, 4-D) and cytokinins (6-benzyladenine (BA),
thidiazuron (TDZ), 2-isopentenyl adenine (2-iP) and Kinetin (Kn) in the concentrations of 0.5, 1.0, 2.0, 5.0, 10.0 M.
The pH of the MS medium was adjusted to 5.8 followed by autoclave at 1.2 Kg/cm2 pressure and 121 oC for 15 min. All
cultures were maintained at 16 h photoperiod with 3000 lux light intensity at 252 oC.
After 4 weeks, regenerated shoots were transferred to medium containing IBA and NAA in the full and
half-strength MS medium. The nature of callus, regeneration frequency, shoot length (cm), number of nodes, number and
length (cm) of root produced per shoot were recorded for each treatment. Plants with healthy roots were transferred in poly
bags containing autoclaved soil: sand: vermiculite in the ratio of 1:1:1 (Thiruvengadam et al. 2006)
DNA was extracted from the leaves of 4 selected hardened plantlets and mother plant following Cetyl
trimethylammonium bromide (CTAB) method (Doyle 1991). Amplification of DNA sample was carried out in 25 l
volume by Manohar et al. (2013). The optimized PCR condition for ISSR and RAPD analysis was carried out as per
Jain et al. (2015). The amplified products were electrophoresed in 1.2 % agarose gels in 0.5X TBE buffer and were
compared to 100 bp DNA ladder.

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All the experiments were conducted with 9 replicates per treatment and repeated thrice. The data were analyzed
statistically using Graphpad Prism 6.01 (GraphPad Software. Inc., California, USA). The significance of differences
among means was carried out using Two-way Analysis of variance (ANOVA) tests at P 0.05. The results were
represented as a means SE of three treatments.

RESULTS AND DISCUSSIONS


Disease free sprouts of S. edule were used for the in vitro establishment of plants. Ex vitro nodes were cut, surface
sterilized and inoculated into MS medium supplemented with various concentrations of BA.
Effect of auxins and cytokinins were evaluated on the shoot proliferation of S. edule using in vitro sterile nodal
explants. Single shoot emerged from the nodal explants for which the data on the length of shoots, number of nodes in a
shoot, callus and root induction frequency was represented in Table 1. Among the different hormones with varied
concentrations, the best shoot induction without callus formation was found in MS media containing BAP 2 M
(Figure 1a). Influence of BAP in shooting has been observed in Trichosanthes dioica (Modgil et al. 2004) and Cucumis
sativus (Ahmad and Anis 2005). KN and 2iP were also showing shoot regeneration associated with the callus formation at
the base of nodal explants (Figure 1c & 1d). The length of the shoot was higher in BA compared to KN and 2iP in 4 weeks
of culture. A synergetic effect of BA and IAA were tested where the shoot induction was similar to the BA with negligible
significance (Figure 1b). The combination of cytokinins and auxins produced shoots along with more mass of intervening
callus (data not shown). TDZ at lower concentrations induced multiple adventitious buds but failed to regenerate shoots
when inoculated in different concentrations of BA.
For the root induction, full strength and half strength MS + IBA, MS + NAA were used. The mean number of root
per shoot was 16.5 and mean length of the root per shoot 4.86 cm after 4 weeks was observed in IBA 10 M
(Figure 1e, Table 2). The effectiveness of IBA in rooting has been reported in S. edule (Esquivel et al. 2002) and
Momordica dioica (Rai et al. 2012). GA3 of different concentrations were used to examine the elongation of internodes.
But GA3 inhibited elongation and leaf enlargement (data not shown) and this is contradictory to reports in Cucumis melo
(Kathal et al. 1988), Citullus vulgaris (Srivastava et al. 1989), where elongation of shoots occurred in presence BA but not
GA3.
Plantlets with healthy roots were transferred to poly bags containing autoclaved soil: sand: vermiculite (1:1:1).
After 2 weeks of growth in lab conditions (Figure 1f), plants were transplanted ex vitro and kept under greenhouse
conditions (Figure 1g) for 4 weeks followed by transferring to the field conditions (Figure 1h) under shade for few days
followed by exposure to sunlight gradually. Thus the rooted in vitro plants were successfully established in field conditions
with 75 % survival rate which was similar to the survival rate of the transferred plantlets of chayote was 75 % reported by
Guangdong et al. (1997).
RAPD and ISSR markers were used to prove the genetic uniformity in the in vitro clones (Verma et al. 2013).
To confirm the genetic stability, DNA of 4 randomly selected hardened plants was compared with the DNA of the mother
plant. 20 RAPD primers used in this analysis gave rise to average 5.14 monomorphic bands. Band profiles of the in vitro
plantlets and the mother plant were generated using a total of 20 ISSR primers with an average of 5.16 monomorphic
bands (Table 3). Monomorphic band pattern obtained in RAPD OPD 11 and ISSR UBC 807 was shown in Figure 2 & 3
respectively.
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Micropropagation through nodal segments has been reported earlier in Sechium edule by Esquivel et al. (2002) to
bring the mass multiplication and homogeneity in the morphology of fruits for the selected desired phenotypes.
Therefore, this study clearly showed the influence of auxins and cytokinins in vitro regeneration of S. edule by in vitro
nodal explants in the Indian cultivars due to its intraspecific variation (Cadena-Iniguez et al. 2007) and to prove the genetic
uniformity between the mother plant and tissue cultured plants, genetic fidelity assessment were carried out.

CONCLUSIONS
Conservation of chayote genetic resources in fields, germplasm exchange through seeds is difficult to maintain as
the risks of disease and pest attacks are high. Due to the entire fruits/seed propagation, there is no homogeneity in the fruit
morphology and yield. Thus, this report on micropropagation evaluated the influence of auxins and cytokinins on the in
vitro regeneration of S. edule through nodal explants by the standardization of plant growth hormones for the direct
organogenesis to eliminate the somaclonal variation and to overcome the heterogeneity in the fruits. To show the
true-to-type nature of micropropagated plants, genetic fidelity analysis was performed which was the first report on clonal
fidelity assessment on S.edule. These reports can be used for crop improvement in terms of genetically identical clones of
the desired phenotypes/traits of this plant.

ACKNOWLEDGMENTS
Authors thank the management of the Jain University, Centre for Post Graduate Studies, Bangalore, India for
providing the necessary facilities and University Grants Commission - Rajiv Gandhi National Fellowship scheme
(F1-17.1/2014-15/RGNF-2014-15-SC-TAM-56956 /SA-III), Government of India for providing the financial assistance.
REFERENCES
1.

Abdelnour-Esquivel A, Engelmann F (2002) Cryopreservation of chayote (Sechium edule JACQ. SW.) zygotic embryos and
shoot-tips from in vitro plantlets. CryoLetters 23 (5):299-308

2.

Ahmad N, Anis M (2005) In vitro mass propagation of Cucumis sativus L. from nodal segments. Turk J Bot 29 (3):237-240

3.

Cadena-Iiguez J, Arvalo-Galarza L, Avendao-Arrazate CH, Soto-Hernndez M, Ruiz-Posadas LdM, Santiago-Osorio E,


Acosta-Ramos M, Cisneros-Solano VM, Aguirre-Medina JF, Ochoa-Martnez D (2007) Production, genetics, postharvest
management and pharmacological characteristics of Sechium edule (Jacq.) Sw. FPJ 1 (1):41-53

4.

Cruz-Martneza VO, Castellanos-Hernndez O, Acevedo-Hernndez G, Rodrguez-Sahagn (2012) A Evaluation of different


methods for in Vitro plant regeneration of Sechium edule.

5.

Doyle J (1991) DNA protocols for plants. In: Molecular techniques in taxonomy. Springer, pp 283-293

6.

Esquivel AA, Ramrez C, Engelmann F (2002) Micropropagacin de chayote (Sechium edule Jacq. SW) a partir de brotes
vegetativos. Agronoma mesoamericana 13 (2):147-151

7.

Guangdong WXLBW (1997) In Vitro Plantlet Regeneration from Hypocotyl of Sechium edule Swortz [J]. Acta Universitatis
Agriculturae Boreali - Occidentalis 1

8.

Jain JR, Kumudini BS, Manohar SH (2015) Standardization of DNA isolation and RAPD-PCR protocol from Sechium edule.
Int J Adv Lif Sci 8 (3):359-363

9.

Kathal R, Bhatnagar S, Bhojwani SS (1988) Regeneration of plants from leaf explants of Cucumis melo cv. Pusa Sharbati.
Plant Cell Rep 7 (6):449-451

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10. Modgil M, Modgil R, Kumar R (2004) Carbohydrate and mineral content of chayote (Sechium edule) and bottle gourd
(Lagenaria Siceraria). J Hum Ecol 15 (2):157-159
11. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15
(3):473-497
12. Newstrom LE (1991) Evidence for the origin of chayote, Sechium edule (Cucurbitaceae). Econ Bot 45 (3):410-428
13. Rai GK, Singh M, Rai NP, Bhardwaj D, Kumar S (2012) In vitro propagation of spine gourd (Momordica dioica Roxb.) and
assessment of genetic fidelity of micropropagated plants using RAPD analysis. Physiol Mol Biol Plants 18 (3):273-280
14. Saade RL (1996) Chayote. Sechium edule (Jacq.) Sw. Promoting the conservation and use of underutilized and neglected
crops. 8. Institute of Plant Genetics and Crop Plant Research, Gatersleben/International Plant Genetic Resources Institute,
Rome, Italy.
15. Srivastava D, Andrianov V, Piruzian E (1989) Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad.
cv. Melitopolski). Plant Cell Rep 8 (5):300-302
16. Thiruvengadam M, Rekha K, Jayabalan N (2006) An efficient in vitro propagation of Momordica dioica Roxb. ex Willd
Philippine Agricultural Scientist (Philippines)
17. Verma KS, Kachhwaha S, Kothari S (2013) In vitro plant regeneration of Citrullus colocynthis (L.) Schard. and assessment of
genetic fidelity using ISSR and RAPD markers. Ind J Biotech 12:409-414

APPENDICES
Table 1: Influence of Auxins and Cytokinins on Shoot Proliferation using
Nodal Segments of Sechium Edule after 4 Weeks of Inoculation

IAA

IBA

NAA

24D

0.5
1.0
2.0
5.0
10.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
BAP
0.5
1.0

0
0
0
0
0
0.5
1.0
2.0
5.0
10.0
0
0
0
0
0
0
0
0
0
0
KN
0
0

0
0
0
0
0
0
0
0
0
0
0.5
1.0
2.0
5.0
10.0
0
0
0
0
0
TDZ
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.5
1.0
2.0
5.0
10.0
2iP
0
0

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100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

Shoot
regeneratio
n
frequency
(%)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
88.88
77.77
0
0

Root
regeneratio
n
frequency
(%)
++
+
+
+
+++
+
++
++
++
+++
+
+
++
++
+++
-

33.8

100

Callus
induction
frequenc
y (%)

Length of
shoots/node
(cm)
(MeanSE)

Number of
nodes/shoot
(MeanSE)

1.030.22a
0.670.12ba
1.380.14ac
0.760.12ac
1.040.09a
0.610.09a
0.620.09a
0.630.09a
0.540.10a
0.540.10a
0.380.05a
0.470.10a
0.470.10a
0.410.15a
0.410.12a
0.380.09a
0.310.06a
0.200.05a
-

1.830.31a
1.290.90a
4.160.65bd
3.830.78c
4.350.78d
2.360.40a
1.710.30a
2.330.41a
1.660.37a
1.850.40a
1.300.29a
1.710.21a
1.360.28a
1.720.38a
0.850.12a
1.690.32a
1.330.29a
0.550.15ba
-

1.300.29a

3.330.60a
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2.0
5.0
10.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0.5
1.0
2.0
5.0
10.0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0.5
1.0
2.0
5.0
10.0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0.5
1.0
2.0
5.0
10.0

33.8
22.8
100
100
88.8
88.8
100
88.8
88.8
100
100
100
100
100
100
100
100
100
100

1.180.39a
1.670.28a
0.310.04b
0.390.14cb
1.160.02a
1.220.16a
1.070.14a
1.160.14a
0.800.17a
0.140.05a
0.160.02a
0.830.15a
0.440.20a
0.320.09a
0.470.09a
0.910.26a

100
100
100
100
100
88.8
88.8
100
66.6
66.6
88.8
0
0
0
100
100
100
88.8
100

3.780.57ab
4.890.73b
2.110.11ac
1.670.37c
2.560.18a
3.330.44a
2.110.35a
2.220.36a
1.330.33ab
1.560.18a
1.110.11ab
1.330.24ac
1.110.20ad
3.000.78a

Data recorded at the end of 4 weeks. Values represent the MeanSE of three treatments, each with 9 replicates.
Means within a column followed by the same letter are not significantly different at P 0.05 according to Two-way
ANOVA tests.
Table 2: In Vitro Rooting of Shoots on Full-Strength and Half
Strength MS Medium Fortified with Different Auxins
Auxins
Full Strength
MS

IBA
NAA

Half strength
MS

IBA
NAA

Conc.
(M)

Root induction
frequency (%)

Mean number of
roots/shoot

5.0
10.0
5.0
10.0
5.0
10.0
5.0
10.0

100
100
100
100
100
100
100
100

13
16
15
14
12
16
14
16

Mean length of roots


(cm)
(MeanSE)
4.480.25a
4.860.39a
4.670.28a
3.710.16a
4.290.23a
4.270.21a
3.620.31a
3.680.34a

Data recorded at the end of 4 weeks. Values represent the MeanSE of three treatments, each with 12 replicates.
Means within a column followed by the same letter are not significantly different at P 0.05 according to Two-way
ANOVA tests.
Table 3: List of Screened Primers with Their Sequence, Ta, Monomorphic
Bands Generated in RAPD and ISSR Markers
Serial No.
1
2
3
4
5
6
7

RAPD Primers
OPD-03
OPD-11
OPD-13
OPD-20
OPE-02
OPE-14
OPE-15

Sequence (5-3)
GTCGCCGTCA
AGCGCCATTG
GGGGTGACGA
ACCCGGTCAC
GGTGCGGGAA
TGCGGCTGAG
ACGCACAACC

Ta (oC)
35
35
35
35
35
35
35

Number of Monomorphic Bands


4
6
6
5
6
4
5

Serial No.

ISSR primers

Sequence (5-3)

Ta (oC)

Number of Monomorphic bands

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2
3
4
5
6

UBC-807
UBC-841
UBC-847
UBC-861
UBC-885
UBC-887

Table 3: Contd.,
(AG)8C
52
(GA)8YC
54
(CA)8RC
54.8
(ACC)6
60.5
BHB(GA)7
51.9
DVD(TC)7
51.1

291

7
6
4
5
4
5

Figure 1: (a-f) In vitro Regeneration of S. Edule from Sterile Nodal Explants (a) Shoot Regeneration in BA 2M
(b) Shoot Regeneration in BA 2M + IAA 1M (c) Shoot Regeneration in 2iP 0.5 M (d) Shoot Regeneration in
KN 2M (e) Shoot Elongation and Rooting in IBA 10M (f) Plantlets for Hardening in Poly Bags for 2
weeks (g) Plants in Greenhouse after 30 days (h) Acclimatized plant after 45 Days with Big Leaves

Figure 2: RAPD Band Profile of S.edule Plantlets Produced from the Primer OPD 11.
M- Marker (100 bp). Lane 1: Mother Plant; Lanes 2 to 5: Micropropagated Plantlets

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Dharmalingam Thilagam, Belur Satyan Kumudini &


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Figure 3: ISSR band Profile of S.edule Plantlets Produced From the Primer UBC 801.
M- Marker (100 bp). Lane 1: Mother Plant; Lanes 2 to 5: Micropropagated Plantlets

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