SUMMARY

Medicinal plants, since times immemorial, have been used in virtually all cultures
as a source of medicine. About eighty percent of the world population depends on
herbal based alternative systems of medicine. Herbal medicine is now expanding its
base at a faster rate due to the great inputs from ethno medicinal practices being pooled
from all over the world. Medicinal herbs are moving from fringe to mainstream use as a
greater number of people endeavor to opt for herbal formulations over the allopathic
compounds, since these are devoid of side effects and cost effective.
Adhatoda vasica, also known as Malabar nut tree is part of the Acanthaceae
plant family. It is a common small evergreen, sub-herbaceous bush. In Ayurveda, the
ancient system of Indian medicine it is commonly known as vasa (Chopra, 1982) which
is a well known plant in indigenous system of medicine. Adhatoda leaves have been
used extensively in Ayurvedic Medicine for over 2000 years primarily for respiratory
disorders. It is propagated by tender stem cuttings. Stem cuttings of 15-20cm long & 3-4
nodes are ideal for planting. Adhatoda is obtained from commercial sources or collected
from open fields. Charaka Samhita has classified the drug under mucolytic &
expecatorant drug. The roots, leaves & flowers are active principles of the plant possess
a number of pharmacological properties & are used in cough, chronic bronchitis,
rheumatism, asthma & bronchial asthma.
Medicinal plants are the most important source of life saving drugs for the
majority of the worlds population. The biotechnological tools are important to select,
multiply and conserve the critical genotypes. In-vitro regeneration holds tremendous
potential for the production of high-quality plant-based medicine. The continuous use of
plant derived products for health care problems has resulted in the establishment of
various pharmacological laboratories
Plant tissue culture is the culture and maintenance of plant cells or organs in
sterile, nutritionally and environmentally supportive conditions (in vitro). Plant cell and
tissue culture include the cultural techniques for regeneration of functional plants from
embryonic tissues, tissue fragments, calli, isolated cells, or protoplasts. It has

applications in research and commerce. In commercial settings, tissue culture is often

referred to as micro-propagation, which is in fact one of the techniques in tissue culture.
Micro-propagation refers to the production of whole plants from cell cultures derived
from explants (the initial piece of tissue put into culture) or meristem cells.
Further research and conservation of all plant species including medicinal plants is
needed to preserve natures natural drugs. Advances in plant tissue culture will enable
rapid multiplication and sustainable use of medicinal plants for future generations.
Tissue culture techniques have been shown to have definite and indispensable
advantages over the conventional vegetative propagation, as it ensures an extremely
rapid rate of multiplication. Tissue culture technique is not season dependent and
requires only a limited quantity of plant tissue as a source of initial explants. It can also
aid in the production of disease free plants.
Plant material of A. vasica was collected from healthy and disease free plants growing
wildly nearby the college premises. After thorough sterilization by the standard method
mentioned earlier, nodal segments (1 to 3 cm long) were excised from the juvenile twigs
and branches. Each nodal segment essentially contained one or more nodes.
Five explants were cultured on MS medium supplemented with various Auxins (2, 4-D,
IAA and NAA) in different concentrations. Plain MS (without any hormone) was set as
control. It has been found that good response of direct shoot induction was obtained on
2, 4-D (2 mg/l); IAA (4 mg/l) and NAA (1 mg/l). Maximum response was obtained on
NAA (1 mg/l) supplemented medium upon which mean no. of shoots obtained was 5.2.
Shoots induced were morphologically normal and green.

Experiments were conducted by culturing nodal segments on MS medium

supplemented with various concentrations of different Cytokinins like, 6-benzylamino
purine (BAP) and Kinetin (Kn). Both BAP and Kn proved to be very effective in inducing
the shoot buds. Effect of Kn was more promising when compared with BAP, wherein, a
mean no. of shoot induced was 5.2. However, on BAP supplemented medium,
maximum 4-8 shoot5 buds were obtained. The hormonal concentrations at which
maximum response was obtained were BAP (1 mg/l) and Kn (1.5 mg/l). The shoot buds
proliferated was normal, green and healthy.
In another set of experiments, leaves were used as explants. The objective of culturing
leaf pieces was to explore the callus induction potential of the explants. Leaf pieces of
A. vasica were collected from wild plants growing within college premises. For culturing,
juvenile, soft and young leaves were collected and sterilized by standard method of
sterilization. On an average five leaf pieces (size- 5 mm to 10 mm) were cultured on
nutrient medium supplemented with Auxins and Cytokinins alone and in various
combinations.
When leaf pieces were cultured on MS medium supplemented with Auxins- 2, 4-D, NAA
and IAA at various concentrations, maximum response of callus induction was obtained
on 2, 4-D (2.5 mg/l) with highest mass of callus. On IAA supplemented medium, best
response was obtained at 2 mg/l while higher concentrations of NAA (4 mg/) callusing
was optimum. However, the callus induced on Auxin supplemented medium was found
to be non-morphogenic type when sub-cultured.
Shoot buds induced from apical meristems were sub-cultured for elongation on MS
medium supplemented with Auxins and Cytokinins in various combinations. Auxins used

were NAA and IAA whereas BAP was used to satisfy the requirements of Cytokinins. Of
the various concentrations used, higher level of NAA (3 mg/l) with moderate level of
BAP (1 mg/l) was found to be optimum for elongation of shoots. The percentage
response of shoot elongation and mean no. of shoot length were maximum at these
levels- 84% and 11.2 cm, respectively.
Plants with roots were transferred during two weeks, after washing of the agar with
distilled water and to pot with a mixture of sand, soil and organic manure in equal ratio
(1:1:1). Potted plantlets were covered with transparent polythene membrane to ensure
high humidity and watered every three days for two weeks in order to acclimatize plants
to field conditions. After two weeks the acclimatized plants were transferred to pots
containing normal garden soil and maintained in greenhouse under natural day length
conditions.