In vitro micropropagation of Colecus
arometicus- An important medicinal plant
Iffat Khan1*, Kirti
Jain2
1Sarojini Naidu Govt. College, Shivaji Nagar, 462016, Bhopal (M.P.), India.
2Govt. Science and Commerce College, Benazir Jahangirabad, 462016, Bhopal (M.P.), India.
ABSTRACT:
In vitro micropropagation of Coleus aromaticus
using shoot meristems was developed. Coleus arometicus
is an important medicinal plant, contains an active ingredient colecus. Multiple shoots were induced in vitro from Apical
and Auxiliary meristems. Murashige
and Skoog’s medium supplemented with BAP 0.5 to 3.0
mg/l, 2.0 mg/l BAP + 0.5 mg/l Kn, 1.0 mg/l BAP + 1.0
mg/l Kn, 1.0 mg/l BAP + 2.0 mg/l Kn. MS + 2.0 mg/l
BAP was found to be most suitable for shoot tip multiplication (75%). For
rooting the shoots were excised and transferred to MS medium combination with
ether 10 g/l sucrose or 1 mg/l NAA + 20g sucrose or 200 mg activated charcoal.
Rooted plantlets were then transferred to pot covered with polyethylene bags
for one week and then grown in the green house. These hardened plants have been
successfully established in soil.
INTRODUCTION:
Coleus arometicus is a very
important medicinal plant, comes under Lamiaceae
family. This "Power" herb has an active ingredient in it called Colecus. C. arometicus a
perennial plant with strong camphor-like odor that was first discovered in the
lower elevations of India. It grows well in warm temperate or subtropical
areas.
It has been used for over 3,000 years in Ayurvedic Medicine. In
ancient Sanskrit texts it is known as Makandi and was
said to have been used for supporting healthy functioning heart and lungs,
Allergies, Asthma, Bladder infection, Eczema, Glaucoma, Hypertension, Irritable
Bowel Syndrome (Spastic Colon) menstrual cramps, weight loss and a number of other functions in the body.
Coleus has long been used in traditional Indian (Ayurvedic) medicine, but
gained popularity when chemical extract of the root called Colecus
demonchitis, helps in glaucoma, congestive heart
failure, and other conditions. The root is used for a myriad of medicinal
purposes. In recent years, Coleus arometicus
has gained recognition as the only known plant source of the diterpene. Other uses include treatment of ulcers, boils,
swellings, urogenital and wound healing properties.
It is used by almost all local people for its therapeutically efficacy against
common cold, cough, fever, headache and indigestion1.
The demand of this powerful herb is increasing day by day and thus a
large multiplication of this plant is necessary. Micropropagation
using meristem, shoot tip, and nodal explants is the most
favored method for producing clones of elite varieties of crops. Sometimes
slight difference in the genetic is from undiffentiated
tissue like callus or cell suspensions. Somatic embryos derived from such
tissue may also show variations that are basically tissue culture induced. Such
variations are termed somaclonal variation. Normally
these variations are screened for beneficial attributes and are utilized in
crop improvement programs.
Much work has not been done in the tissue culture protocol development
of Coleus arometicus, so an advance protocol
needs to be developed for the proper and rapid mass micropropagation
of Coleus arometicus. Thus, in the present
work an appropriate and efficient protocol is trying to being developed.
Plant tissue culture is an emerging tool for plant biotechnology and a
potential tool for micro propagation of some economically important crops of
agriculture, horticulture, forestry rare and threatened plants. This micropropagation procedure should be useful for conservation
as well as production of this important medicinal plant.
MATERIALS
AND METHODS:
This technique involves sterilization, inoculation and regeneration of
plant cells, tissues of organ under aseptic conditions in culture vials
containing synthetics nutrient medium. Both the chemical composition of the
medium and the controlled conditions (light, temperature, humidity aeration,
etc.) effectively control the expression of any genotypic and phenotypic
potential in explants.
Explants preparation and inoculation
Apical and Auxiliary meristems were used as
explants excised from the plant growing in green house of institute. Explants
were washed with soap solution followed by tap water for 25min.It was surface
sterilized with 0.1% (w/v) mercuric chloride solution for 10 min and followed
by rising with sterilized distilled water for 4-5min.Now inoculate the explants
in the medium for the initiation of shoots.
The pH of the medium was adjusted to 5.75 with 0.1 N NaOH
or 0.1 N HCl solution prior to adding 0.8% (w/v)
agar. The cultures were incubated at a temperature of 25±2ºC.
Multiplication of shoots
After 10-15 days the bud break were initiated in the medium. Initiated
healthy shoots were excised and used for multiplication.
Different media used for
multiplication -
Medium 1 MS + 1.0
mg/l BAP
Medium 2 MS + 2.0 mg/l BAP
Medium 3 MS + 2.0 mg/l BAP + 0.5 mg/l KN
Medium 4 MS +3.0 mg/l BAP
Medium 5 MS + 2.0 mg/l BAP + 1.0 KN
Medium 6 MS + 1.0 mg/l BAP + 2.0 KN
Medium 7 MS + 2.0 KN mg/l
The measurement was taken on the basis of % age of shoot response,
number of multiple shoot developed, shoot length and callus formed from each
ten replicates.
Root induction
Regeneration multiple shoots were separated in different rooting
medium. Shoots of 2-3cm were transferred to MS medium containing either 10g/l
sucrose or 1 mg/l NAA + 20g sucrose or 200 mg activated charcoal. Rooted
plantlets were then transferred to pot covered with polyethylene bags for one
week and than grown in the green house.
RESULT AND DISCUSSION:
Micropropagation using shoot
tip is preferred in recent times over conventional vegetative propagation
because of rapidity of multiplication within a short period of time. The
advantage of shoot tip culture is that the shoot has already been
differentiated in vivo. Therefore to establish a complete plant, only
elongation of the existing shoot and root differentiation is required. The most
significant advantage of shoot tip culture is that, in a relatively short time
a large number of true-to-true disease free plants could be produced.
In Coleus arometicus for shoot tip
multiplication was obtained maximum in BAP alone. The optimum concentration was
found to be 2.0 mg/l BAP. The dose of cytokinin is
critical in shoot organogenisis2. Geetha et
al. reported high frequency shoot multiplication from cotyledonary
nodes of pigeonpea in the presence of BAP3.
The shoot regeneration frequency increase with increase in concentration of cytokinins concentration above 2mg/l drastically decreased
shoots bud regenration. However in Coleus arometicus higher concentratins
of BAP in the medium decrease multiple shoot induction and BAP alone was
satisfactory in including shoot multiplication. BAP in combination with Kin
decrease shoot multiplication. BAP alone in medium induced bud break and
multiple shoot formation in Mentha4 (table 1, table 2 and Fig. 1).
On the contrary, synergistic effect of two cytokinins
on shoot multiplication was shown in Citrus aurantifolia5 and
in Kaempferia galanga6. Kartha reported high frequency shoot multiplication and
elongation from shoot apical meristem of Lycopersicon esculentum7. He
suggested meristem proper culture as an efficient
plant propagation system producing fewer polyploids
or chromosomally aberrant plants. Genetic stability is conserved in the case of
plants derived from shoot tip culture8.
The active meristem located on the nodes
could attribute to the formation of multiple shoot in the present
investigation, Continuous presence of cytokinins in
the medium resulted in shoot length supperssion that
has also been reported in guava9.
Rooting of the micro propagated plants was usually achieved in auxin containing medium10. Root formation is an
energy demanding process and thus exogenous supply of carbohydrates is
required. However this being the last stage of vitro culture it is important to
transform the plant from heterotrophic to autotropic
mode of nutrition. Thus the supply of exogenous sugars should be reduced at
this time. In Colecus arometicus
the reduction of sugar to 2% level decreased rooting. In most species, reducing
sugar from 3%-2% level does not make any difference. In Coleus arometicus the use of NAA has been used for root
initiation. The multiple shoot production protocol developed in this study may
be utilized for the cultivation practices of this commercially important
medicinal plant.
FINDING
AND CONCLUSION:
The explants show better response when collected and inoculated from
the month of August to March. But when collected in other month the response of
the explants is poor. The maximum explants were sterilized when treated for 4-5
minutes with 0.1% HgCl2 solution, shows 80-95% sterile culture. When
the treatment of the explants is below 4 minutes and above 5 minutes the
percentage of sterilization is low and dehydrated respectively. But when the explants were
treated with 70% alcohol for any duration the explants got dehydrated. Thus
treatment of alcohol is avoided in the explants of Coleus arometics.
Figure
1: Colecus arometicus; a and b-habit ; c-initiation of root ;
d-multiplication of root, e and f-root
induction in shoot.
Table 1 Effect of growth regulations on multiplication of shoots in Colecus arometicus
|
Medium +
Growth hormones mg/I |
% age of
shoot induction |
No. of
shoots per culture |
Average
shoot length in c.m. |
Callussing |
|
MS + 0.5
BAP |
75% |
1-3 |
1-2 |
- |
|
MS + 1.0
BAP |
70% |
1-2 |
2 cm |
- |
|
MS + 1.5
BAP |
68% |
1-1.5 |
2 cm |
- |
|
MS + 2.0
BAP |
60% |
1-2 |
1-2 |
- |
|
MS + 0.5
KN |
40% |
1 |
1-2 |
+ |
|
MS + 1.0
KN |
42% |
1 |
1-2 |
++ |
|
MS +
0.5 BAP + 0.5 KN |
45% |
1 |
1 |
++ |
|
MS +
1.0 BAP + 0.5 KN |
45% |
1-2 |
1 |
+ |
Table 2
Effect of growth regulations on multiplication of shoots in Colecus arometicus
|
Medium + Growth hormones mg/l |
% age of response |
Average number of shoots |
Average shoot length in cm. |
Callussing |
|
MS + 1.0 BAP |
70% |
15 |
2-3 |
- |
|
MS + 2.0 BAP |
75% |
15-20 |
2-3 |
- |
|
MS + 2.0 BAP + 0.5
KN |
60% |
8-10 |
2-5 |
+ |
|
MS + 3.0 BAP |
30% |
4-5 |
3-5 |
- |
|
MS + 1.0 BAP + 1.0 KN |
20% |
5-8 |
1-2 |
+ |
|
MS + 1.0 BAP + 2.0
KN |
20% |
4-9 |
1-2 |
++ |
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Received on 17.02.2012
Modified on 28.02.2012
Accepted on 10.03.2012
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Research J.
Science and Tech. 4(1): Jan.-Feb. 2012: 18-21