a rapid micropropagation protocol of atropa acuminata

Indian Journal of Biotechnology

Vol 15, October 2016, pp 576-580

A rapid micropropagation protocol of Atropa acuminata Royle ex Lindl.―A

threatened medicinal plant species of Kashmir Himalaya

Farhana Maqbool*, Seema Singh, Zahoor A Kaloo and Mahroofa Jan

Plant Tissue Culture Laboratory, Department of Botany, University of Kashmir, Hazratbal, Srinagar 190006, India

Received 12 June 2015; revised 10 November 2015; accepted 16 November 2015

In the present study, different explants, viz., petiole and nodal explants, of Atropa acuminata Royle ex Lindl. were used

to develop an efficient micropropagation protocol for the conservation of this medicinally important plant. The petiole

explants produced the maximum amount of callus on MS medium supplemented with BAP (3 mg/L) within 18 d in 80%

cultures. Further, shoot regeneration was obtained when these calli were subcultured onto MS medium supplemented with

BAP (5 mg/L), with a mean shoot length of 2.2±0.19 cm in 40% cultures within 48 d. Similarly, from nodal explants, the

maximum amount of callus was achieved on MS medium supplemented with BAP (2 mg/L) in 80% cultures within 17 d.

When these calli were transferred onto MS medium supplemented with BAP (2 mg/L), shoot regeneration was obtained with

a mean shoot length of 2.0±0.20 cm in 80% cultures within 14 d. Root differentiation with 100% response was obtained

within 18 d in in vitro grown shoots on MS medium augmented with IBA (0.5 mg/L) with a mean number of 21.6±6.9 roots.

The in vitro raised plantlets were then successfully acclimatized and hardened in compost under greenhouse conditions

within 3 wk with 80% response. The hardened plants were successfully transferred to the field conditions.

Keywords: Atropa acuminata, callus, explants, micropropagation, root regeneration, shoot regeneration

Introduction Atropa acuminata Royle ex Lindl. (Family:

Solanaceae), commonly known as Indian Belladonna, is

a perennial plant that grows about 1.6 m tall. It has

simple leaves, which are ovate with entire margins. The

flowers are solitary, bell-shaped and yellowish white in

colour. They are hermaphrodites and are pollinated by

insects1. Flowering period is from June to July and the

seeds ripen from August to October. The black fruits are

berries. It is found in the Western Himalayan ranges,

extending from Kashmir at the altitude of 1800-3600 m

asl to the adjoining hills of the Himachal Pradesh up to

2500 m asl. In North West Himalaya, it is distributed in

Kashmir, Muzzafarabad and Chakrata2. The rhizome of

this plant has been traditionally used as a sedative

antidote in cases of mushroom or toadstool poisoning,

analgesic, antispasmodic, hallucinogenic, mydriatic,

narcotic, diuretic and anodyne; arthritis related

inflammatory disorders, muscle and joint pain, muscle

spasms, sore throat and ulcerative colitis3-7

. Aerial parts

of the plant have been used to treat innumerable

ailments, such as, acute infections, anxiety and chicken

pox8. A. acuminata plants serve as one of the most

important source of medicinally important tropane

alkaloids, including atropine, scopolamine and

hyoscyamine9. The drugs atropine and hyoscyamine

extracted from the plant act as stimulants to the

sympathetic nervous system and are employed as

antidotes to opium6. The plant also contains highly

oxygenated oleanane triterpenes, such as, 2α,3α,24

trihydroxyolean-12-ene-28,30-dioic acid and

2α,3α,24,28-tetrahydroxyolean-12-ene10

. Monoterpene,

sesquiterpene, phenylpropanoid, flavonoid and quinine

are present as main constituents11

.

Ranking at the top of medicinal plant inventory from

North West Himalaya, A. acuminata figures among 59

critically endangered taxa and negative list of exports in

India, and has been prioritized for immediate

conservation and large scale multiplication12

. Unabated

as the plant extraction continues to be, far are not days

when this precious legacy will be lost forever. It is

indeed a crisis situation for the species, which calls for

the salvage of whatever is left. It is, therefore, that the

present study for its in vitro propagation and

conservation has been taken up.

Materials and Methods A. acuminata plants was collected from Gulmarg

and Daksum of Kashmir Himalaya and were

―――――

*Author for correspondence:

[email protected]

MAQBOOL et al

transplanted at the Kashmir University Botanical

Garden (KUBG), Srinagar. The speci

collected and processed for herbarium preparation and

latter deposited at Kashmir University Herbarium

(KASH) under Voucher Specimen No. 1913

[Ref.No.F1 (Specimen vouchers. CBT) KU/2013].

Different explants were collected from plants grown

at KUBG (Fig. 1). Petiole and nodal segments

(explants) were first thoroughly washed under

running tap water in order to remove dirt and dust

followed by washing with detergent labolene (1% v/v)

and surfactant Tween-20 (1% v/v). The detergent was

removed by washing the explants with double

distilled water. Then they were treated under laminar

air flow hood with chemical sterilant 2 or 4% sodium

hypochlorite for 8-10 min. This was followed by

washing with autoclaved double distilled water and

finally the explants were inoculated on sterilized

nutrient medium.

Medium and Culture Conditions Murashige and Skoog’s (MS) medium, gelled with

8% agar was supplemented with different

concentrations of auxins and cytokinins

individually and in combination. Auxins like 2, 4

IAA, NAA and IBA, and cytokinins like BAP and

Kn, were used in concentration range of 0.1

The pH of the media was adjusted to 5.8 before

autoclaving at 121°C and 15 lb. The cultures were

incubated at 22±4°C and exposed to a

12 h dark.

Results and Discussion

Callus Production and Shoot Regeneration

Petiole Explants

Callus production was achieved from petiole explants

by using different growth hormones (BAP, Kn, IAA,

NAA, IBA & 2, 4-D) individually as well as in different

combinations. However, optimal callus regeneration was

induced when MS medium was supplemented with BAP

(3 mg/L) (Fig. 2a) and BAP (3 mg/L) in combination

with IAA (2 mg/L) (Fig. 2b). The callus was compact

and light green in colour in both the combinations (MS+

AP & MS+BAP+IAA), and was obtained in 80 and 60%

cultures within 18 and 38 d, respectively (Table 1).

Petiole explant produced maximum amount of callus on

MS medium supplemented with BAP (3 mg/L) as

compared to BAP (3 mg/L) in combination with IAA

(2 mg/L) and also in terms of per cent culture response

and number of days taken for callus production. Similar

results were also obtained from the petiole explant of

Salvia canariensis L. but they used NAA ins

et al: MICROPROPAGATION OF A. ACUMINATA

transplanted at the Kashmir University Botanical

. The specimen was

collected and processed for herbarium preparation and

latter deposited at Kashmir University Herbarium

Specimen No. 1913

[Ref.No.F1 (Specimen vouchers. CBT) KU/2013].

Different explants were collected from plants grown

Petiole and nodal segments

were first thoroughly washed under

running tap water in order to remove dirt and dust,

followed by washing with detergent labolene (1% v/v)

v/v). The detergent was

washing the explants with double

distilled water. Then they were treated under laminar

air flow hood with chemical sterilant 2 or 4% sodium

10 min. This was followed by

washing with autoclaved double distilled water and

nts were inoculated on sterilized

Murashige and Skoog’s (MS) medium, gelled with

8% agar was supplemented with different

concentrations of auxins and cytokinins, both

ombination. Auxins like 2, 4-D,

and cytokinins like BAP and

were used in concentration range of 0.1-5 mg/L.

The pH of the media was adjusted to 5.8 before

autoclaving at 121°C and 15 lb. The cultures were

incubated at 22±4°C and exposed to a 12 h light and

Callus production was achieved from petiole explants

by using different growth hormones (BAP, Kn, IAA,

D) individually as well as in different

combinations. However, optimal callus regeneration was

induced when MS medium was supplemented with BAP

(3 mg/L) (Fig. 2a) and BAP (3 mg/L) in combination

with IAA (2 mg/L) (Fig. 2b). The callus was compact

light green in colour in both the combinations (MS+

AP & MS+BAP+IAA), and was obtained in 80 and 60%

cultures within 18 and 38 d, respectively (Table 1).

Petiole explant produced maximum amount of callus on

MS medium supplemented with BAP (3 mg/L) as

ared to BAP (3 mg/L) in combination with IAA

(2 mg/L) and also in terms of per cent culture response

and number of days taken for callus production. Similar

results were also obtained from the petiole explant of

L. but they used NAA instead of

IAA13

. Petiole derived callus when subcultured on MS

medium supplemented with BAP (5 mg/L) (Fig. 3a) and

BAP (3 mg/L) in combination with IAA (2 mg/L) (Fig.

3b), it regenerated shoots with a mean shoot length of

2.2±0.19 and 1.99±0.19 cm in 40 and

within 48 and 53 d, respectively (Table 2). Shoots

obtained from MS medium supplemented with BAP (5

mg/L) was healthier and with greater length as

compared to shoots achieved from BAP (3 mg/L) in

combination with IAA (2 mg/L) and also in terms

cent culture response.

Nodal Explants

Nodal segments were inoculated on MS medium

containing different growth hormones (BAP, Kn,

IAA, NAA, IBA & 2, 4-D) individually or in different

Fig. 1―A. acuminata mother plant.

Fig. 2―Callus production from petiole explant on MS medium

containing: (a) BAP (3 mg/L); & (b) BAP (3 mg/L)+IAA

(2 mg/L).

Table 1―Effect of plant growth regulators on callus production

from petiole explant

Treatments No. of days taken for

callus production

MS basal -

MS+BAP (3 mg/L) 18

MS+BAP (3 mg/L)

+IAA (2 mgL)

38

(10 replicates per treatment)

577

Petiole derived callus when subcultured on MS

medium supplemented with BAP (5 mg/L) (Fig. 3a) and

BAP (3 mg/L) in combination with IAA (2 mg/L) (Fig.

3b), it regenerated shoots with a mean shoot length of

2.2±0.19 and 1.99±0.19 cm in 40 and 20% cultures

within 48 and 53 d, respectively (Table 2). Shoots

obtained from MS medium supplemented with BAP (5

mg/L) was healthier and with greater length as

compared to shoots achieved from BAP (3 mg/L) in

combination with IAA (2 mg/L) and also in terms of per

were inoculated on MS medium

containing different growth hormones (BAP, Kn,

D) individually or in different

mother plant.

Callus production from petiole explant on MS medium


Effect of plant growth regulators on callus production

from petiole explant

of days taken for

callus production

% culture response

-

80

60

578 INDIAN J BIOTECHNOL, OCTOBER 2016

combinations. MS medium fortified with BAP (2

mg/L) (Fig. 4a) and BAP (3 mg/L)+IAA (2 mg/L)

(Fig. 4b) proved effective in differentiating compact

and cream coloured callus in 80 and 60% cultures

within 17 and 21 d, respectively (Table 3), and

maximum amount of callus was obtained on MS

medium supplemented with BAP (2 mg/L). Callus

obtained from nodal explants when subcultured on

MS medium supplemented with BAP (3 mg/L)

(Fig. 5a) and BAP (2 mg/L) (Fig. 5b) regenerated

shoots with a mean shoot length of 2.0±0.20 and

1.9±0.23 cm in 80 and 70% cultures within 14 and 15

d, respectively (Table 4). Shoots regenerated on MS

medium augmented with BAP (3 mg/L) was healthier

and with greater length as compared to BAP (2 mg/L)

and also in terms of number of days taken for shoot

regeneration. Similar results were obtained from the

shoot regeneration of Solanum nigrum L. on MS

medium supplemented with BAP (3 mg/L)14

.

Rooting of Regenerated Shoots For induction of roots, in vitro cultured shoots were

placed onto both full and half strength MS medium

supplemented with auxins like IAA and IBA

individually at various concentrations and in

combinations with cytokinins like BAP. About

21.6±6.9 and 6.1±0.87 mean number of roots were

obtained in 100 and 60% cultures within 18 d on full

strength MS medium supplemented with IBA

(0.5 mg/L) and IBA (1 mg/L), respectively (Figs 6a).

Roots were also regenerated on half strength MS

medium supplemented with IBA (0.5 mg/L). Roots

regenerated from full strength MS medium enriched

with IBA (0.5 mg/L) was greater in number as

compared to IBA (1 mg/L) and ½ MS+IBA (0.5

mg/L), and also in terms of per cent culture response.

However the per cent cultures showing rooting as

Table 2―Effect of plant growth regulators on shoot regeneration

from petiole derived callus

Treatments Mean length of

shoots (cm)±SE

No. of days taken

for shoot

regeneration

% culture

response

MS+BAP (5 mg/L) 2.0±0.19 48 40

MS+BAP (3mg/L)

+IAA (2 mg/L)

1.9±0.19 53 20


Table 3―Effect of plant growth regulators on callus production

from nodal explant

Treatments No. of days taken

for callus production

% culture

response

MS basal - -

MS+BAP (2 mg/L) 17 80

MS+BAP (3 mgL)

+IAA (2 mg/L)

21 60

(10 replicates per treatment

Table 4―Effect of plant growth regulators on shoot regeneration

from nodal derived callus

Treatments Mean length of

shoots (cm)±SE

No. of days

taken for shoot

regeneration

% culture

response

MS+BAP (2 mg/L 2.0±0.20 14 80

MS+BAP (3 mgL) 1.9±0.23 15 70


Fig. 3―Shoot regeneration from petiole explant on MS medium


(2 mg/L).

Fig. 4―Callus production from nodal explant on MS medium

containing: (a) BAP (2 mg/L); & (b) BAP (3 mg/L)+IAA (2

mg/L).

Fig. 5―Shoot regeneration from nodal explant on MS medium

containing: (a) BAP (2 mg/L); & (b) BAP (3 mg/L).

MAQBOOL et al: MICROPROPAGATION OF A. ACUMINATA 579

well as average number of roots/cultures declined

(Table 5). The present results are in agreement with

the study of Ahuja et al15

who obtained root

regeneration in A. acuminata by using full strength

revised tobacco medium supplemented with IBA.

Acclimatization/Hardening

For acclimatization, 18-d-old well developed

plantlets were taken out of the culture vials. The

medium adhering to the basal portion of plantlets was

washed off with double distilled water. After washing

they were transferred to jiffy pots and earthen pots

containing autoclaved soil and sand in 1:1 ratio

(Fig. 7a) as well as compost (Fig. 7b). The transferred

plantlets were maintained under controlled conditions

of temperature (22±4oC) and relative humidity (60%)

in greenhouse. These plantlets were watered at regular

intervals. The hardening of the plants was achieved

within 3 wk with 80% survival rate in compost, and

60% survival rate in soil-sand mixture within 4 wk.

The in vitro raised plantlets were successfully

acclimatized/hardened under greenhouse conditions

and hardened plants were successfully transferred to

the field conditions. The in vitro culture strategies

developed could be used as tools to increase the

alkaloid content and enhance secondary metabolites

in A. acuminata.

Conclusion

An efficient and rapid micropropagation protocol

of A. acuminata plants was developed using petiole

and nodal segments as explants. Among all the plant

growth regulators, BAP was proved to be the most

effective for both callus induction as well as shoot

regeneration. However, best rooting was achieved on

MS medium augmented with IBA. The plantlets

grown in compost were hardened and acclimatized in

a greenhouse with 80% survival rate.

References

1 Nasir Y J, Atropa acuminata Royle ex Miers, in Hook.

Flora of Pakistan, 1 (1972) 138.

2 Dhar U & Kachroo P, Alpine flora of Kashmir Himalaya

(Scientific Publishers, Jodhpur), 102 (1983) 353-764.

3 Rhodes J B, Abrams J H & Manning R T, Controlled

clinical trial of sedative―Anticholinergic drugs in patients

with the irritable bowel syndrome, J Clin Pharmacol, 18

(1978) 340-345.

4 Grieve M, A modern herbal (Pengium, Middleburg) 1984.

5 Chiej R, The Macdonald encyclopedia of medicinal plants

(Macdonald and Co. Ltd., London) 1984.

6 Kaul M K, Medicinal plants of Kashmir and Ladakh (Indus

Publications, New Delhi, India) 1997, p 173.

7 Shanafelt T D, Barton D L, Adjei A A & Loprinzi C L,

Pathophysiology and treatment of hot flashes, Mayo Clin

Proc, 77 (2002) 1207-1218.

8 Bettermann H, Cysarz D, Portsteffen A & Kümmell H C,

Bimodal dose-dependent effect on autonomic, cardiac

control after oral administration of Atropa belladonna,

Auton Neurosci, 90 (2001) 132-137.

9 Phillipson J D & Handa S S, N-oxides of hyoscyamine and

hyoscine in the Solanaceae, Phytochemistry, 14 (1975)

999-1003.

10 Mehmood M A, Anis I, Khan P M, Riaz M, Makhmoor T

et al, Highly oxygenated triterpenes from the roots of

Atropa acuminata, Nat Prod Let, 16 (2002) 371-376.

11 Jayakanthi J, Dhanarajan M S, Sarumathy K & Vijay T,

The protective potential effects of Atropa acuminata on

acetaminophen induced hepatotoxity and oxidative stress in

albino rats, Int J Pharm Pharmaceut Sci, 3 Suppl 5 (2011)

197-203.

Table 5―Effect of plant growth regulators on rooting from

regenerated shoots

Treatments Mean no. of

roots

Mean no. of

days taken

for rooting

% culture

response

MS+IBA (0.5 mg/L) 21.6±6.9cm 18 100

MS+IBA (1 mg/L) 6.1±0.8cm 18 60

1/2 MS+IBA(0.5 mg/L) 3.4±0.4cm 20 40


Fig. 6―Rooting of regenerated shoots on MS medium containing:

(a) IBA (0.5 mg/L); (b) IBA (1 mg/L); & (c) 1/2 MS+IBA

(0.5 mg/L).

Fig. 7―Regenerated plants potted in earthen pots containing:

(a) soil and sand; & (b) compost.

580 INDIAN J BIOTECHNOL, OCTOBER 2016

12 Wani P A, Nawchoo I A & Wafia B A, Improvement of

sexual destination in Atropa acuminata Royle

(Solanaceae)―A critically endangered medicinal plant of

Northwestern Himalaya, Pak J Biol Sci, 10 (2007)

778-782.

13 Mederos-Molina S, In vitro callus induction and plants

from stem and petiole explants of Salvia canariensis L.

Plant Tissue Cult, 14 (2004) 167-172.

14 Kavitha M S, Wesely E G & Mahalingam P, Direct

multiple shoot regeneration from shoot tip and nodal

explants of Solanum nigrum L. a medicinal herb, J Ornam

Hortic Plants, 2 (2012) 65-72.

15 Ahuja A, Sambyal M & Koul S, In vitro propagation and

conservation of Atropa acuminata Royle ex Lindl.―An

indigenous threatened medicinal plant, J Plant Biochem

Biotechnol, 11 (2002) 121-124.

a rapid micropropagation protocol of atropa acuminata

Documents