plant growth regulators and sucrose requirements for in ... · and treated with 0.1% solution (w/v)...
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WSN 49(2) (2016) 283-294 EISSN 2392-2192
Plant growth regulators and sucrose requirements
for in vitro induction of shoots from different explants of Atalantia monophylla (L.) Corr. Serr.
M. Manokari*, Mahipal S. Shekhawat
Biotechnology Laboratory, Department of Plant Science, M.G.G.A.C. Mahe, Pondicherry, India
*E-mail address: [email protected]
ABSTRACT
An efficient in vitro regeneration and conservation system depend on the healthy culture
induction from the suitable explants. Culture induction is a significant stage when very small plant
material exists from the rare species. It is necessary to develop culture induction protocol from various
explants to conserve the valuable plant species. Atalantia monophylla is a rare species with life giving
properties. Shoots were induced from the shoot tip, node and internode explants. Among these the
nodal shoot segments were reported most appropriate explant for the induction of shoots from the
nodal meristems. MS medium (Murashige and Skoog) proved better than the Woody Plants (WP)
medium in bud breaking. Sucrose at 3% level was optimum concentration for the establishment of cultures
than the other concentrations evaluated. Shoot tips responded on MS medium supplemented with 0.5 mg
L-1
each of BAP and Kin, nodal shoot segments responded better on MS medium augmented with 1.0 mg
L-1
BAP . Half strength MS medium supplemented with 2,4-D (1.0 mg L
-1) induced maximum responsive
callus (87%) from the internode explants.
Keywords: Atalantia monophylla; in vitro; rare; explants; PGRs
1. INTRODUCTION
Atalantia monophylla (L.) Corr. Serr. (syn: Limonia monophylla L.) is one of the
important species of the family Rutaceae. It is commonly known as Wild lemon, Jungli
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Nimbu, Aranyanimbuka, Banjamir nimbu, Bannimbu, Kattunarakam, Kattuelumichai etc.
(Kandappa et al., 2015). This species is reported as rare and endemic to southern peninsular
India (Sukumaran and Raj, 2007). It is a large thorny shrub grows up to 2.5 meters in height.
Leaves are simple, alternate, oblong and entire with crenulate margin. Flowers are white,
small and arranged in axillary racemes. Fruits are small berries with minute seeds
(Sankaranarayanan et al., 2010).
The leaves and bark of this plant are traditionally used in the treatment of vitiated
kapha, vata, flatulance, hemiplegia, arthritis, skin diseases, bacterial infections and
malignancy (Panda, 2004; Kumar and Narayana, 2010). The essential oil obtained from the
berries is reported to cure chronic rheumatism, paralysis and inflammations (Sukumaran and
Raj, 2010). The herbal extract made from the leaves is used in hemiplegia due to the presence
of an active ingredient compound liniment. The boiled leaves are used to cure glandular
swelling (Sankaranarayanan et al., 2010). The essential oil contains higher terpene esters
(azulene group). The plant roots are antispasmodic and exploited due to the presence of
alkaloids, atalaphylline and atalaphyllidine. The root bark yields limonoid and atalantin
(Kirtikar and Basu, 1999).
A. monophylla is used to control Spodoptera litura, Helicoverpa armigera, Earias
vittella (Baskar et al., 2009; Muthu et al. 2010), Culex quinquefasciatus, Anopheles stephensi,
and Aedes aegypti (Sivagnaname and Kalyanasundaram, 2004). Besides, the plant is reported
to posses larvicidal and pupicidal (WHO, 1975), insecticidal (Grainge and Ahamed 1988,
Sukumar et al. 1991), mosquitocidal (Sivagnaname and Kalyanasundaram, 2004), ovicidal
(Baskar and Ignacimuthu, 2012), antifungal, antioxidant and cytotoxic (Kandappa et al.,
2015) activities.
The conventional propagation through seeds and stem cuttings of this species is not
efficient as the rate of seeds germination and rooting ability is very poor. Vegetative
propagation requires some special climatic conditions than the existing harsh environmental
conditions. Therefore, the distribution of this species is limited and restricted in certain
geographic zones. The biotechnological interventions have been used to conserve endangered,
rare, ornamental and medicinal species. In vitro conservation methods gained significance for
vegetatively propagated and non-orthodox seed yielding plant species for conservation and
production of pathogen-free plantlets (Engelmann, 2011).
Bud breaking or induction of shoots is an initial but very significant stage in the case of
certain plant species when very small plant material exists in nature. The present
communication describes the chemical factors affecting shoots induction in rare and endemic
plant A. monophylla using different types of explants.
2. MATERIALS AND METHODS
2. 1. Plant material selection
The field surveys were conducted and the plants were procured from the southern
districts of the Coromandel Coast of India, lies on the geographical coordinates of 11° 55' 48"
N, 79° 49' 48" E. The plants were identified using standard floras (Gamble, 1921; Matthew,
1982). Healthy and actively growing plants were collected and maintained in the greenhouse
to get disease free planting materials.
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2. 2. Explants and sterilization
Different types of explanting materials namely leaves, apical shoot tips, nodal shoot
segments and internodes were used to establish the cultures. The explants were washed
thoroughly with 2% solution (v/v) of Tween-20®
followed by running tap water for 10 min
and treated with 0.1% solution (w/v) of Bavistin (systemic fungicide; BASF India Ltd., India)
for 6-8 min, then washed thrice with distilled water. The explants were then dipped in 70%
ethanol for 1 min, followed by surface sterilized with 0.1% solution (w/v) of HgCl2 for 4-5
min under laminar air flow cabinet. The sterilized explants were washed with autoclaved
double distilled water for 5-6 times to remove the adhered traces of HgCl2. The explants were
inoculated and cultured on different basal media supplemented with different concentrations
and combinations of plant growth regulators (PGRs).
2. 3. Nutrient media and culture conditions
Two types of nutrient media were used for the present study. These include MS basal
medium (Murashige and Skoog, 1962) and Woody Plants (WP) medium (Llyod and McCown,
1980). Sucrose (Hi-Media, Mumbai) was added as a source of carbohydrate. Different
concentrations of sucrose (1%, 2% and 3%) were tested to find out optimum concentration of
carbon requirement for the establishment of cultures. Additives (50 mg L-1
of ascorbic acid
and 25 mg L-1
each of arginine, adenine sulphate and citric acid) were incorporated in the
culture medium. Culture medium was solidified by 0.8% agar to support the proper position
of the plant material in the medium. The pH of the medium was adjusted to 5.8 ±0.02 using
0.1 N NaOH or 0.1 N HCl prior to autoclaving for 15 min at 121 ºC and 1.1 kg cm-2
. The
cultures were kept in growth room under controlled conditions at 23 ±2 °C to 28 ±2 °C
temperature with illumination of 20-50 µmol m-2
s-1
Spectral Photon Flux Density (SPFD) and
60-70% relative humidity (RH). The light was provided by fluorescent tubes and incandescent
bulbs (Philips, India). Positive air pressure and temperature was maintained by air
conditioning system.
2. 4. Effect of plant growth regulators on culture induction
To investigate the effect of plant growth regulators on bud break and the establishment
of cultures in vitro, the explants were inoculated vertically and horizontally on MS medium
containing different concentrations of plant growth regulators (BAP; 6-benzylaminopurine,
Kin; Kinetin, IAA; indle-3 acetic acid and NAA; α-naphthalene acetic acid), ranging from 1.0
to 3.0 mg L-1
. Leaf explants (1 cm long) were inoculated directly into the medium (maximum
one per culture tube) after trimming both the ends to induce callus. The medium employed for
callus induction was augmented with different concentrations of 2,4-Dichlorophenoxy acetic
acid (2,4-D). The explants inoculated on medium devoid of growth regulators were served as
control. The culture vessels were properly capped and sealed after inoculation. Explants were
harvested throughout the year to study the seasonal response on establishment of the cultures.
2. 5. Observations and data analysis
All the experiments were set up in randomized block design with a minimum of 20
replicates per treatment, and experiments were performed three times. The observations were
taken after four weeks of inoculation. The culture induction response represents the efficiency
of explants on a specific medium after number of days of inoculation as mentioned in the
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results. The data were statistically analyzed using analysis of variance (ANOVA), and
differences among the mean values were compared with Duncan’s multiple range test (P<
0.05) using SPSS ver. 16. The results were expressed as mean ±Standard Error (SE).
3. RESULTS AND DISCUSSION
3. 1. Seasonal collection of explants for culture induction
The in vitro response of explants was greatly affected by the season/month of collection
of the explants in the establishment of cultures of A. monophylla. Maximum percentage of
response (95%) was observed during the months of October-December from all the explants
evaluated (Fig. 1). Less percentage of culture responsiveness was observed during summer
months (April-June) of the year. The physiological state of explants under special in vitro
conditions was determined by the season of explants collection and the influence of the plant
growth regulators. The seasonal response of explants in cultures were reported in Azadirachta
indica (Arora et al., 2010), Celastrus paniculatus (Phulwaria et al., 2013), Schleichera oleosa
(Saha, 2013), Morinda citrifolia (Shekhawat et al., 2015b), Hemidesmus indicus (Shekhawat
and Manokari, 2016b) and Blyttia spiralis (Patel et al., 2016).
Fig. 1. Seasonal effect of explants collection on induction of shoots.
3. 2. Effect of different types of explants in culture establishment
Selection of appropriate explants is a significant step to avoid exploitation of somatic
tissues from the rare and conservation prioritized species. For the establishment of cultures,
different explants like leaves, shoot tips, nodes and internodes were cultured on different
media (MS and WP medium) supplemented with different combinations and concentrations
of growth regulators. The in vitro growth and morphogenesis is largely governed by plant tissue
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culture medium, and it generally comprises of inorganic salts, organic compounds, vitamins,
additives such as ascorbic acid, adenine sulphate, arginine and citric acid, and gelling agent (agar)
etc. The problem of phenolic exudation was tried to control by incorporation of additives,
activated charcoal and by periodic subculture. Among the different methods tried, subsequent
subculture with 8 days interval on fresh medium amended with additives (50 mg L-1
of
ascorbic acid and 25 mg L-1
each of arginine, adenine sulphate and citric acid) resulted with
better response. Addition of activated charcoal in the medium delayed the culture induction
repose from the explants.
3.3. Culture establishment from shoot tip explants
Two different basal media (MS, WP) were tested to optimize the appropriate culture
medium for A. monophylla. Shoot tips showed elongation up to 3.0 cm within 10 days of
incubation on MS medium (Fig. 2A and B). However, shoot tips cultured on WP medium
responded after 20 days for elongation up to 2.4 cm and only 72% of explants responded on
WP medium. Shoot tip elongation was observed on MS medium supplemented with 0.5 mg L-
1 each of BAP and Kin. Higher percentage of response (92%) with three shoots was observed
from the shoot tip explants when cultured on MS medium augmented with 0.5 mg L-1
each
BAP and Kin (Table 1). The combined effect of cytokinins for shoots induction was reported
in Artemisia absinthium (Shekhawat and Manokari, 2015) and Glinus lotoides (Teshome and
Feyissa, 2015). At higher concentration of cytokinins, the induction response was suppressed.
Shoot tips were reported better explants in micropropagation of Citrus hystrix (Eng et al.,
2014) and Vitex trifolia (Ahmed and Anis, 2014).
Fig. 2A. Culture induction using shoot tip explants.
Fig. 2B. Shoot bud elongation -Microscopical view (scale bar 50µm).
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Table 1. Effect of different concentrations of cytokinins (BAP and Kin) on shoot induction
response of shoot tip explants.
Conc. of cytokinins
(mg L-1
)
Response
(%)
Number of shoots
(Mean ±SE)
Shoot length (cm)
(Mean ±SE)
Control 0.00 0a 0.0±0.00
a 0.00±0.00
a
BAP
0.1 56d 2.3±0.00
d 1.12±0.12
b
0.5 83g 2.8±0.12
e 2.60±0.07
c
1.0 80g 2.4±0.20
d 2.03±0.20
c
1.5 69f 2.0±0.21
c 1.19±0.24
b
2.0 51c 1.7±0.19
c 1.04±0.11
b
Kin
0.1 50c 1.6±0.12
c 1.00±0.14
b
0.5 59e 2.1±0.13
b 1.93±0.20
c
1.0 55d 2.0±0.20
c 1.22±0.19
b
1.5 52c 1.8±0.00
c 0.93±0.11
b
2.0 46b 1.2±0.13
b 0.85±0.00
b
BAP + Kin
0.1 60e 2.0±0.10
c 2.13±0.15
c
0.5 92i 3.0±0.17
e 3.04±0.11
d
1.0 89h 2.5±0.21
b 2.59±0.33
c
1.5 73f 2.2±0.11
b 2.10±0.10
c
Note: Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance of
variation between the concentrations was studied using DMRT at 5% level (P < 0.05). Mean values represented
in corresponding column followed by same alphabets are not significantly different.
3.4. Culture induction from nodal shoot segments
Freshly sprouted nodal shoot segments were found most suitable explants for the
induction of multiple shoots in A. monopylla. Similar findings regarding suitability of nodal
shoot segments as explants were reported in Citrus limon (Rathore et al., 2007), Terminalia
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catappa (Phulwaria et al., 2012), Salvadora oleoides (Shekhawat et al., 2012) and Morinda
coreia (Shekhawat et al., 2015a). Among different concentrations and combinations of
cytokinins tested, 1.0 mg L-1
BAP was observed more effective with respect to bud break
from the nodal explants. Cent percentage bud break was achieved within ten days of
inoculation on full strength MS medium supplemented with 1.0 mg L-1
BAP. The highest
(12.4±0.20) number of shoot buds was observed with 3% sucrose and additives when
incorporated with 1.0 mg L-1
BAP (Fig. 3A to C). The influence of additives in the culture
medium in establishment of cultures and better multiple shoots induction was reported in
number of plant species such as Stevia rebaudiana (Sridhar and Aswath, 2014), Blyttia
spiralis (Patel et al., 2016) and Hemidesmus indicus (Shekhawat and Manokari, 2016b).
Comparatively less percentage of response was observed on Kin alone and BAP+Kin in
shoots induction from the nodal explants. The superiority of BAP over Kin for bud breaking
response from nodal shoot segments was reported in Citrus limon (Rathore et al., 2007). The
percentage of response and number of shoots differentiated were less on WP medium at
higher concentrations of cytokinins with additives (Table 2 and Fig. 4).
Fig. 3A. Bud breaking in nodal shoot explant.
Fig. 3B. Microscopical view of buds (scale bar 50µm).
Fig. 3C. Multiple shoots induction from nodal explants.
Table 2. Effect of different concentrations of cytokinins (BAP and Kin) and IAA (0.1 mg L-1
)
on shoot induction response of nodal explants.
Conc. of cytokinins
(mg L-1
)
Response
(%)
Number of shoot
buds (Mean±SE)
Shoot length (cm)
(Mean±SE)
Control 0.00 0a 0.0±0.00
a 0.00±0.00
a
BAP
0.1 89d 10.5±0.00
c 2.19±0.13
c
0.5 93e 11.2±0.12
f 2.66±0.27
d
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1.0 100f 12.4±0.20
g 4.36±0.31
f
1.5 91e 12.0±0.21
g 3.00±0.20
e
2.0 86d 10.0±0.19
e 2.12±0.14
c
Kin
0.1 72c 7.16±0.07
c 1.47±0.10
b
0.5 79e 8.31±0.11
d 1.98±0.29
c
1.0 87d 8.50±0.10
d 2.10±0.11
c
1.5 74c 7.94±0.22
c 1.90±0.15
c
2.0 66b 6.29±0.15
b 1.74±0.21
b
BAP + Kin
0.1 63b 7.00±0.27
c 2.13±0.15
c
0.5 79d 7.93±0.10
d 3.04±0.11
e
1.0 85d 6.18±0.10
b 2.59±0.33
d
1.5 75c 5.22±0.00
a 2.10±0.10
c
Note: Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance of
variation between the concentrations was studied using DMRT at 5% level (P < 0.05). Mean values represented
in corresponding column followed by same alphabets are not significantly different.
Fig. 4. Effect of different media on response of different explants.
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3. 5. Callus induction from leaf and internode explants
Callus formation was observed in leaf and internodal explants on full strength MS
medium supplemented with different concentrations of 2,4-D within week. The initial dark
incubation of explants on 2,4-D for 2 days was found as limiting factor in callus induction.
The callus was induced from the leaf explants within 3 weeks and after 10 weeks from the
internode explants. Among the different strengths of MS medium and concentrations of
growth regulators tested, half strength MS medium augmented with 1.0 mg L−1
2,4-D with
additives was reported superior. All the concentrations of 2,4-D induced callus, from leaf and
internode explants. Maximum percentage (87%) of callus was regenerated from internode
explants than leaf explants (73%). Callus produced on this medium was fast growing, green,
friable and had the potential to regenerate shoots on shoot differentiation medium (Table 3
and Fig. 5A and B).
Fig. 5A. Callus induction from internode explants.
Fig. 5B. Callus tissues under photomicroscope (scale bar 50µm).
Table 3. Effect of 2,4-D on induction of callus from the leaf and internode explants.
Conc. of 2,4-D (mg L-1
)
Callus induction response
(%)
Leaf Internode
Callus characteristics
0.0 0a 0
a No callus induction
0.25 47c 74
b Creamy white, slow growing
0.5 52d 79
c Pale white, slow growing
0.75 69e 84
d Pale green, slow growing
1.0 73f 87
e Green, friable, fast growing
1.5 63e 80
d Brown, poor growth
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2.0 58d 78
c Slow growing
3.0 43b 71
b Brown, no growth
Note: Mean separation was analyzed by ANOVA using SPSS software (version 16.0) and significance of
variation between the concentrations was studied using DMRT at 5% level (P < 0.05). Mean values represented
in corresponding column followed by same alphabets are not significantly different.
Callus initiated with leaf explants on 2,4-D medium was creamy white, unorganized and
less proliferative which turned brown and died within 4 weeks. Further increase in
concentration of 2,4-D in the medium did not show any progressive callus proliferation.
Successful regeneration of plantlets from callus using cotyledons explants on MS medium
supplemented with 2,4-D was reported in Citrus jambhiri (Savita et al., 2011), leaf explants in
Citrus limon (Kasprzyk-Pawelec et al., 2015) and seeds, internode and apical shoot tip
explants in Citrus sinensis (Azim et al., 2011). Reduced strength of MS salts for the induction
of callus was reported in Ulex europaeus (Ramirez et al., 2012).
4. CONCLUSION
Culture establishment being the first step of any micropropagation protocol requires
clear understanding of plant responses on various physiochemicals, in vitro culture
environments including nutrient media, growth regulators, types of culture vessel etc. The
culture establishment from the appropriate explants is challenging in development of a
regeneration system for the rare species. The present study explains the suitable explant type,
seasonal response of explants and optimum medium for culture initiation in A. monophylla.
The developed method could be used as a tool for the conservation of this species which is
endemic in nature with limited distribution.
References
[1] H.R. Kandappa, K. Pillay, V.S.R. Obulam, V.G.K. Sharma, P. Govender, Tropical
Journal of Pharmaceutical Research 14 (2015) 487-493.
[2] S. Sukumaran, A.D.S. Raj, Indian Forester 133 (2007) 1254-1266.
[3] S. Sankaranarayanan, P. Bama, J. Ramachandran, P.T. Kalaichelvan, M. Deccaraman,
Journal of Medicinal Plants Research 4 (2010) 1089-1101.
[4] H. Panda, Asia Pacific Business Press, Inc., Delhi, 2004, pp. 166-167.
[5] R.B. Kumar, B.S. Narayana, Ethnobotanical Leaflets 14 (2010) 95-107.
[6] S. Sukumaran, A.D.S. Raj, Indian Journal of Traditional Knowledge 9 (2010) 294-299.
[7] K.R. Kirtikar, B.D. Basu, Bishen Singh Mahendra Pal Singh Publication, Dehradun,
1999, pp. 1655-1656.
World Scientific News 49(2) (2016) 283-294
-293-
[8] K. Baskar, S. Kingsley, S.E. Vendan, M.G. Paulraj, V. Duraipandiyan, Chemosphere 75
(2009) 355-359.
[9] C. Muthu, K. Baskar, S. Kingsley, S. Ignacimuthu, Journal of Central European
Agriculture 11 (2010) 23-26.
[10] M. Sivagnaname, M. Kalyanasundaram. Mem Inst Oswaldo Cruz, Rio de Janeiro 99
(2004) 115-118.
[11] WHO-World Health Organization 1975, WHO/VBC/75.583. Mimeographed document.
[12] M. Grainge, S. Ahmed, John Wiley & Sons, New York, 1988, p. 41.
[13] K. Sukumar, M.J. Perich, L.R. Boobar, Journal of American Mosquito Control
Association 7 (1991) 210-217.
[14] K. Baskar, S. Ignacimuthu, Journal of Agricultural Technology 8 (2012) 861-868.
[15] F. Engelmann, In Vitro Cell and Developmental Biology of Plants 47 (2011) 5-16.
[16] T. Murashige, F. Skoog, Physiologia Plantarum 15 (1962): 473-497.
[17] G. Llyod, B.G. McCown, International Plant Propagator’s Society Combined
Proceedings 30 (1980) 421-427.
[18] K. Arora, M. Sharma, J. Srivatsava, S.A. Ranade, A.K. Sharma, Agro-forestry
ecosystems 78 (2010) 53-63.
[19] M. Phulwaria, M.K. Rai, A.K. Patel, V. Kataria, N.S. Shekhawat, AoB PLANTS, 2013,
doi: 10.1093/aobpl/pls054.
[20] D. Saha, Indian Journal of Natural Products and Resources, 4 (2013) 102-109.
[21] M.S. Shekhawat, N. Kannan, M. Manokari, C.P. Ravindran, Journal of Applied
Research in Medicinal and Aromatic Plants 2 (2015b) 174-181.
[22] M.S. Shekhawat, M. Manokari, Indian Journal of Plant Physiology, 2016b, DOI
10.1007/s40502-016-5.
[23] A.K. Patel, D. Lodha, K. Ram, S. Shekhawat, N.S. Shekhawat, In Vitro Cell and
Developmental Biology of Plants, 2016, DOI 10.1007/s11627-015-9738-1.
[24] M.S. Shekhawat, M. Manokari, Chinese Journal of Biology, 2015,
http://dx.doi.org/10.1155/2015/273405.
[25] S. Teshome, T. Feyissa, American Journal of Plant Sciences 6 (2015) 1329-1340.
[26] W. H. Eng, M.A. Aziz, U.R. Sinniah, Pakistan Journal of Botany 46 (2014) 1453-1458.
[27] R. Ahmed, M. Anis, Physiology and Molecular Biology of Plants 20 (2014) 385-392.
[28] J.S. Rathore, M.S. Rathore, M. Singh, R.P. Singh, N.S. Shekhawat, Indian Journal of
Biotechnology 6 (2007) 239-244.
[29] M. Phulwaria, K. Rm, Harish, A.K. Gupta, N.S. Shekhawat, Journal of Forest Research
17 (2012) 202-207.
[30] N. S. Shekhawat, S. Mohnot, M. Phulwaria, Harish, S. Shekhawat, Journal of
Sustainable Forestry 31 (2012) 620-632.
World Scientific News 49(2) (2016) 283-294
-294-
[31] Shekhawat, M.S., Kannan, N. and M. Manokari, South African Journal of Botany 100
(2015b) 43-50.
[32] T. M. Sridhar, C.R. Aswath, American Journal of Plant Sciences, 5 (2014) 192-199.
[33] B. Savita, G.S. Singh, A.K. Virk, Nagpal, Physiology and Molecular Biology of Plants
17 (2011) 161-169.
[34] A. Kasprzyk-Pawelec, J. Pietrusiewicz, E. Szczuka, Acta Scientiarum Polonrum Cultus
14 (2015) 143-153.
[35] F. Azim, M.M. Rahman, S.H. Prodhan, S.U. Sikdar, N. Zobayer, M. Ashrafuzzaman,
International Journal of Agricultural Research, Innovation and Technology 1 (2011)
64-68.
[36] I. Ramirez, F. Dorta, A. Cuadros-Inostroza, H. Pena-Cortes, Electronic Journal of
Biotechnology 15 (2012), DOI: 10.2224/vol15-issue4-fulltext-4.
( Received 15 May 2016; accepted 05 June 2016 )