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




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




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.

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