Beruflich Dokumente
Kultur Dokumente
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
www.tjprc.org
editor@tjprc.org
286
287
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.
editor@tjprc.org
288
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.
4.
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
289
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
www.tjprc.org
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
editor@tjprc.org
290
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
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
Serial No.
ISSR primers
Sequence (5-3)
Ta (oC)
1
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
www.tjprc.org
editor@tjprc.org
292
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