Int.J.Curr.Microbiol.App.

Sci (2016) Special Issue-3: 44-49

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Special Issue-3 (February-2016) pp. 44-49
Journal homepage: http://www.ijcmas.com

Original Research Article

Invitro Micropropagation of Piper betle L.

M.Elamathi*

Dept. of. Botany and Biotechnology, Bon Secours College for Women, Thanjavur
*Corresponding author

ABSTRACT

Keywords Plant tissue culture has provided new insights into plant biology and has become an
important tool for the improvement of crop species. Recent progress made in tissue
Piper betle L, culture technique has indicated, the potential application of biotechnology in the
Micro improvement of many crop in herbs. From this study concluded that shoot tip and
propagation, leaf base explants of Piper betle are ideal for establishing in vitro culture. MS basal
Callus, medium supplemented with 1mg/l of IAA and 0.5mg/l BAP is suitable induction of
Growth multiple shoots in shoot tip and leaf base explants in Piper betle. Further works on
regulators induction of embryoids and development of hardening method need to be carried
out for successful in vitro culture in this important Indian medicinal plant.

Introduction
frontiers of biotechnology of scientific and
Recently medicinal plants occupy an economic importance. Development In the
important place in health care, cosmetics a technology of plant tissue culture science the
food industries throughout the world. Herbal bioengineering experiments by White (1934
drugs are more preferred than allopathic & 1937 ) Skoog and Murashige and Skoog
drugs because of higher efficiency (1962) have contributed in establishing a
affordability, easy availability and causing strong foundation for the application of this
less or side effect. Even western world begin versatile technology. Important medicines
to use herbal drugs and herbal formulation are economical plants are becoming extinct
described in traditional medicines like, and endangered due to heavy unscientific
chines medicines and traditional medicines and unsustainable exploitation and low
like Aurveda and siddha literature for curing propagation response. Tissue culture
various diseases. Apart from traditional methods valuable tools to propagate rapidly
system of medicines, various Indian and to generative new varieties of medicinal
communities use many medicinal plants for plants.
therapeutic purpose and this unmodified
system is called “Folk medicine” or Plant tissue culture has provide new insights
Ethnomedicne. into plant biology and has become an
important tools for the improvement of crop
In recent years plant biotechnology has species . Recent progress made in tissue
made an impressive progress as one of the culture technique has indicated, the potential

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application of biotechnology in the mastitis, leucorrhoea, otorrhoea, ring worm,

improvement of many crop in herbs. Plant swelling of gum, rheumatism, abrasion, cuts
tissue culture technique has been and injuries etc as folk medicine while the
successfully utilized o generating genetic root is known for its female contraceptive
variability for selecting better genotypes in effects (Chopra et al., 1956: Khanra, 1997).
many crops in many crops in plants. Herbs The betle leaves really do not have any
are staging a come.ack and an herbal match as a cheap, natural and easily
renaissance is blooming across the world. available appetizer, digestive , mild
They have been successfully utilized on stimulant, aphrodisiac and refreshing
generating genetic variability for selecting mastication. Chewing of betel leaves
better genotype in improvement of many produce a sense of well- being , increased
crop in plants. Herbs are staging a come alertness, sweating , salivation, hot sensation
back and an herbal renaissance is blooming and energetic feeling with exhilaration. It
across the world. they have been prized for also increases the capacity to exercise
their medicinal, flavoring and aromatic physical and mental functions more
qualities for countries and yet for a while efficiently for a longer duration but it may
they were over’s had owed by the synthetic produce a kind of psychoactive effect
products of the modern cultivation but once causing a conduction of mild addiction to
having realized their serious side effects habituation and withdrawal symptoms
people are going back to nature with hopes (chu,2001: and Jain,1996).
of safety and security.
Materials and Methods
Secondary metabolites from plants namely
alkaloids, flavonoids, saponins and terpenes Collection of Plant
have played a vital role in pharmaceutical,
cosmetic, perfumery, drying ad flavor The plant source piper betel (L.)
industries. These drug ,flavor , essential, oils (piperaceae),commonly known as Indian
, and colors derived from plants have no betle leaves was collected from
apparent in plant primary metabolism but Thiruvaiyaru at Thanjavur Dt, India.
often play an adaptive role . Secondary
metabolites are commercially feasible and as Methodology
such for enhancing the in vitro production of
natural products. Selection of Explants

The present work deals an in vitro method Leaf explants and apices were used for the
for hairy root induction of Piper betel Linn. present study. The first fully expanded
Herbal improvement through biotechnology leaves in the shoot apex were collected from
means provides new hopes as success in the garden grown plant. The explants were
improving them conventional breeding is excised with the help of sterile forceps and
limited due to their narrow genetic base and blade. The nodes were cut in to 0.5-1.0 cm
sexual incompatibility for the wild relatives. sized segments and care was taken that each
explants include the midrib portion. Apical
Betel leaf is traditionally known to be useful shoot buds measuring 10-15mm in length
for the treatment of various diseases like bad with 2-3 lea primordial attached were also
breath, boils and abscesses, conjunctivitis, used. The selected explants were sterilized
constipation, headache, hysteria, itches, by 0.1% of Mercury Choloride.

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Preparation of Medium growth (898 mg fr.wt.0 1mg/1 IBA conc.

Followed by leaf base explants showed more
Murashige and skoog’s (1962) (MS) than twenty four, fold growth increment
medium was prepared with different growth after, 4 weeks culture period, no callus or
regulators such as IAA, BAP, 2,4 D and morphogenesis observed in all the
NAA at various concentrations. Regular concentration of IBA.
observation at an interval of two days was
made for the formation of callus, change of 2,4-D at different concentrations induced the
colour and initiation of the root and shoot . highest growth increment and formation of
callus in almost all the explants (table- III).
Results and Discussion Callus was initiated in all the explants after
2 weeks in culture. Shoot tip explants were
Different explants like leaf base, leaf lamina, more proliferate and registered the highest
node and shoot tip of piper betle were growth 919519 mg fr.wt.) at 1.5mg/l 2,4-d
cultured on different concentration of concentration. At this concentration all other
hormones (IAA,IBA,2,4-D, BAP kinetin). It explants also showed a maximum
was observed that all explants showed growth.MS medium supplemented with
growth response like enlargement, ignition 1mg/l 2,4 –D also induced good growth on
of callus and formation of shoot and root. all the explants. Nodal explants showed the
therefore all the above explants of piper least growth in all the 2,4-D concentrations.
betle were used throughout the present
study. The different concentration BAP induced
more growth, compared to other auxins and
Effect of different auxins and cytokinins on cytokinins such as IAA,IBA,2,4-D and
growth of different explants of piper betle kinetin. Profuse growth (1009mg fr.wt.) was
were observed and given in I-V. induced by the 0.5 mg/l of BAP in shoot tip
explants. At the above concentrations of
Out of different concentrations (0.5-2.0 BAP, nodal explants at different
mg/l) of IAA used after 4 weeks of culture , concentration of BAP was in the following
maximum growth of all the the explants was order 0.5>1.0>1.5>2.0 (table-iv). In this
observed on 1mg/l followed by 0.5mg/l . Of medium also, shoot tip and leaf base
all the explants registered the highest growth explants showed the best growth response.
(925.80 mg fr.wt) followed by leaf base , at I
mgh/l concentration of IAA. Nodal explants Next to BAP, kinetin induced more growth
showed the least growth increments in all in all the explants, maximum amount of
IAA concentrations. Shoot tip and leaf base growth (984 and 890 mg fr.wt) produced at
explants registered more than twenty four the conc.0.5mg/l and 1.0 mg /l at shoot tip
fold growth increment in fresh weight and explants (Table V). The least growth was
dry weight basis on MS medium obtained by the nodal explants. The growth
supplemented with1mg/IAA callus or gradually decreased as the concentration of
morphogenesis were not observed. kinetin increased.

When IBA was supplemented in different The organogenetic pattern of different

concentrations to MS medium, all the explants (lamina, leaf base, node and shoot
explants showed maximum growth tip) on MS medium supplemented with
increment at 1mg/l conc. Among explants, different concentrations and combinations of
shoot tip explants registered the highest auxins and cytokinins was observed. In
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lamina explants cultured on MS medium Induction of multiples shoots are absent in

with BAP 0.5mg/l induced callus (fig.2) and nodal explants, cultured containing medium
BAP 2mg/l + 2,4-D 1.5 mg/l produced BAP 1.5mg/L+2,4-D 2mg (Fig:8)
callus, shoot and root and the frequency was
90,71 and 90% respectively (Table – VI: In leaf base explants inoculated on medium
Fig: 3-5). with BAP 0.5mg/l+IAA 1mg/l and BAP
1.5mg/l+2,4-D 2mg /l produced multiple
The nodal explants produced the least shoots only. Which frequency was 95,90 and
amount of growth. The medium with BAP 89%of the leaf base and 92,95 and 61%
1mg/l +2,4-D 1.5mg/l induced white and respectively(Fig. 7&9).
friable callus in nodal explants (Fig:6).

Fig.1 Piper Betle L.

Callus, Shoot and Root Induction of Sample

Fig-2 Medium: MS + BAP 0.5 mg /l +IAA 1.5mg /l.

Fig-4 Medium : MS + BAP 2.0 mg/l + 2,4-D 1.5mg/l ,
Fig-5 Medium : MS +BAP 2.0 mg/l+ 2, 4-D 1.5mg/l.
Fig -7 Medium: MS + Bap 0.5 mg/l +IAA -1.0MG/L
Fig-8 Medium : MS + BAP- 1.5 mg/l + IAA-2.0mg/l.
Fig-9 Medium: MS+ BAP-1.5 mg/L +2,4-D-2.0 mg/L

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Fig- 10 Medium: Ms +bap-0.5 mg/l +2,4-D-0.5 mg/l

Fig-11 Medium MS +BAP 0.5 mg / 1=2-4 D1.5mg/l
Fig-12 Medium: MS+Bap 0.5 mg/l+IAA 0.5mg /l
Fig -13 Medium: MS+BAP0.5 mg /l+IAa 0.5mg/l.
Fig -14 Medium:MS + BAP 0.5 mg/L +IAA 1.0 mg/l
Fig-15 Medium; MS+Bap 0.5 mg/l =IAA1.0mg/l
Fig-16 Medium: MS + BAP 0.5 mg/l +IAA 1.0mg/l

Fig-17 C.S. of shoot forming callus showing vasculature to new shoots

Fig-18 C.S of multiple shoot Forming callus showing braching vasculature to new
shoots
Fig-19 C.S of nodal region from field grown plant showing oil giobules
Fig- 20 C.S of nodal region from in vitro plantlet showing absence of oil globules

Shoot tip and leaf base of piper betle are on different concentrations of BAP,IAA and
suitable for induction of morphogenesis in 2,4-D induced profuse growth (Fig: 10-16).
culture. Tue shoot tip explants were cultured Callus, multiple shoots and roots are formed

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on medium containing BAP 0.5mg/l +IAA

1mg/l (Fig 15). Frequency was 90,92 and
94% respectively. Among the two explants
(leaf base and shoot tip) shoot tip showed
best response.

References

1.Chopra,R.N., Nayar ., S.L : Glossary of

Indian Medicinal plants, pp.194.
(1956)
2.Chu, N.S. Effect of betel chewing on the
central and autonomic nervous
systems. J. Biomedical sci., 8 (3):
229-236 (2001).
3. Khanra,S “Betel leaf based industry “.
Nabanna Bharati,30(@):169 ( 1997)
4.Murashige,T and skoog,F., 1962.A revised
medium for rapids growth and
bioassays with tobacco tissue
cultures. Plant physiology. 15: 473-
497.
5.Skoog, F., 1994.growth and organ
formation I tobacco tissue culture .
Am. J.Bot.,31:19-24
6.White,P.R.,1934.Potentially unlimited
growth in excised tomato root tips in
a liquid medium, Plant physiology.,
9: 585 -600.
7.White ,P.R.,1937. Vitamins B in the
nutrition of excised tomato roots. Plant
physiology 12: 803-811.

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