Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
It bring me a great felicity and proud to gratify every individual who helped me in making the
project a success and giving their invaluable time to me.
I express my deep indebtedness to Dr. Z. A. Haider, Associate Dean, College of Biotechnology,
Birsa Agricultral University, Ranchi-6 for his continous encouragement and providing necessary facilities
to complete this work.
I convey my sincere gratitude and thanks to Dr. Joydev Gangopadhyay, Principle & Director,
DIST, Dugapur, for providing the permission to complete my dissertation.
I wish to avail this to express my heartfelt devotion and profound respect to our teachers,
Mr, Abijit Sarkar, HOD of Biotechnology, D.I.S.T (Durgapur) and Dr.Anita Paul for generating great
enthusiasm inside me. I render my thanks to all the faculty of D.I.S.T.
I am thankful to my seniors and friends, who have co-operated me from time to time throughout
my study.
Last but not least, I wish to avail this opportunity to express my heartfelt devotion profound respect
to my worthy novel Parents and family member, whose inspirational value and sacrifice helped me a lot
in achieving the goal.
Above all I offer my heartfelt devotion and much more thanks to the Almighty for his sacred blessing
bestowed on my life.
CONTENT PAGE NO
ACKNOWLEDGMENT
1. INTRODUCTION 1-3
2. OBJECTIVE OF PROJECT 4
LABORATORY EQUIPMENT
SURFACE STERILIZATON OF EXPLANT
TRASFER OF EXPLANT
MULTIPLICATION OF SHOOTS
ROOTING AND HARDENING
6. OBSERVATION 16-17
8. CONCLUSION 21
9. BIBLIOGRAPHY 22
INTRODUCTION
The plant cell, tissue and organ are cultured in a defined medium under strict
aseptic conditions and incubated under artificial environment (in vitro) to induce
growth and development. The developmental fate of the plant tissues to be
manipulated towards desired ends is by inclusion of growth regulators (phyto
hormones) and other chemicals in the medium.
Tissue Culture is considered a cost-effective technology but with a capable impact
which is clearly visible in the form of improved yields, supply of virus-free clones
of true-to-type cultivars with provision of improved germplasm. It also considered
as a„Lab to Land‟ technology.
High multiplication rate e.g. 106 of plants per year from a single explant. And
very small explants can be used.
Micropropagation using meristem and shoot culture to produce large numbers
of identical individuals.
Large scale growth of plant cells in liquid culture as a source of secondary
products.
Crossing distantly related species by protoplast fusion and regeneration of the
novel hybrid.
Production of diploid plants from haploid cultures to achieve homozygous
lines more rapidly in breeding programs.
DISADVANTAGES
Micropropagation is not always the perfect means of multiplying plants, conditions
that limit its use include:
It is very expensive, and can have a labor cost of more than 70% .
As monoculture is produced after micropropagation, in case of infection whole
crop can get damaged.
An infected plant sample can produce infected progeny. This is uncommon if
the stock plants are carefully screened and vetted to prevent culturing plants
infected with virus or fungus.
Not all plants can be successfully tissue cultured, often because the proper
medium for growth is not known or the plants produce secondary metabolic
chemicals that stunt or kill the explant.
Sometimes plants or cultivars do not come true to type after being tissue
cultured; this is often dependent on the type of explant material utilized during
the initiation phase or the result of the age of the cell or propagule line.
Some plants are very difficult to disinfest of fungal organisms.
The major limitation in the use of micropropagation for many plants is the cost of
production. For this reason, many plant breeders do not utilize micropropagation
because the cost is prohibitive other breeders use it to produce stock plants that are
then used for seed multiplication.
Mechanisation of the process could reduce labour costs, but has proven difficult to
achieve, despite active attempts to develop technological solutions.
OBJECTIVE OF PROJECT
Due to the prevailing reasons there is a huge need for in vitro propagation of
Rauwolfia serpentina to satisfy the growing commercial demand of the plant for
the production of life supporting alkaloids and conservation of this valuable
endangered plant itself. Hence improvements in plant tissue culture techniques for
the mass propagation of R. serpentina are highly desirable.
The present study was undertaken to develop a more efficient protocol for rapid
in vitro propagation & multiplication of Rauwolfia serpentina i.e., the
standardization of low cost media and proportion of phytohormones for induction
of efficient multiplication of apical as well as nodal and axillary tissue of field
grown plant. The present work deals with species Rauwolfia serpentina. The total
work has been carried out at Plant Tissue Culture Laboratory, College of
Biotechnology, BAU campus, Ranchi.
DESCRIPTION OF SARPGANDHA
Status: The natural reserves of this plant are declining, especially after reports of
its medicinal properties appeared in literatures. International Union for the
Conservation of Nature and Natural Resources (IUCN) has kept this plant under
endangered status.
Distribution: The snake-weed genus includes about 50 species; this has fairly
wide area of distribution, including the tropical part of the Himalayas, the Indian
peninsula, Sri Lanka, Burma, and Indonesia.
Botany: An erect perennial shrub with a long, irregularly, nodular, yellowish, root
stock.
Leaves: In whorls of 3, thin, lanceolate, acute, bright green above and pale
beneath.
Fruit: Drupe, single or didynamous, shining black, the inflorescenece with red
pedicels and calyx and white corolla.
Natural Components: The root contains ophioxylin (an alkaloid having orange
colored crystalline principle), resin, starch and wax. The total alkaloid yield is
0.8%. Five crystalline alkaloids isolated are ajmaline, ajmalicine, serpentine,
serpentinine, and yohimbine.
Cultivation: This plant is under cultivation in India, Sri Lanka, and Java.
Experiments on cultivation are in progress in the United States.
Climate: It grows luxuriantly well where the rainfall is 2500 mm or more. The
areas having more equable climatic variations seem to be more suited than the
areas having higher climatic variations.
Soil: It prefers soil with plenty of humus and rich in nitrogenous and organic
matter with good drainage. Alkaline soils are not suitable for commercial
cultivation.
Maturity Period: 3 Years. At this time the sub aerial parts dry and main root
reach a depth of 0.9 meters.
Medicinal uses
Reserpine is an alkaloid first isolated from R. serpentina which was widely used as
an antihypertensive drug. Other plants of this genus are also used medicinally, both
in conventional western medicine and in Ayurveda, Unani medicine. Alkaloids in
the plants reduce blood pressure, depress activity of central nervous system and act
as hypnotics.
The root of Rauwolfia sp. was popular from Asian times, both in India and on the
Malay Peninsula, as an antidote to the stings of insects and bites of poisonous
reptiles. It has been also used as anti pyretic, an oxytoxic, a sedative and a
palliative for insanity.
In 1891, Dymock detected the presence of an alkaloid and a yellow resin in the
root of R.sepentina.
In 1940, Vakil made the first recorded reference to the therapeutic application
of Rauwolfia sp. in case of human hypertension.
Around the same time, Gupta, Deb and Kahali, reported the application of
Rauwolfia sp. in mental disorder.
In 1944, Bhatia and Kapur reported after the administration in animals of the 2
alkaloids isoajamaline and neoajamaline,stimulation followed by depression
of central nervous system and lowering of blood pressure.
In 1952, Muller, schlittler and Bein isolated reserpine, which accounted for
approximately 50% of the activities of Rauwolfia sp. root.
In 1953, Bein, Muller and associates found resepine to possess marked and
long lasting hypotensive vasodepression and sedative, hypnotic properties.
In 1953, Ford and Moyer after treating 25 cases of essential hypertension with
combined Rauwolfia sp. and hexamethonium therapy were able to report
adequate reduction of pressure levels of large number of cases.
In 1996, Sudha and Seeni has been achieved micropropagation from explant of
Rauwolfia sp. micrantha Hook F cultures.
In2008, Bhatt R, Arif M,Gour A.K, Rao P.B,devised protocol optimization for
in vitro propagation.
►LABORATORY REQUIREMENTS
All the constituent were added to double distilled water and the volume was
adjusted to1 liter. Later growth regulators are added to form various RS
(Rauwolfia serpentina) media formulation for growth.
The pH of the media was adjusted between 5.6 to 5.8, N/10 HCl and N/10
NaOH was used if necessary.
The media was boiled and agar was then added. It was homogenized by
boiling and continuous stirring. Then fill the culture bottles with media.
METHODS
This part of the procedure was carried out in a sterile working area, or with
meticulous aseptic technique.
The explant material is then surface sterilized, usually in multiple courses of
bleach and alcohol washes and finally rinsed in sterilized water.
Now this small portion of surface sterilized plant tissue is placed on a growth
medium, typically a medium containing sucrose as an energy source and one or
more plant growth regulators in specific combination. Usually the medium is
thickened with agar to create a gel which supports the explants during growth.
At first sterilized cotton was divided into three different parts, the first part was
used to sterilize the platform of the laminar flow, the glassware were sterilized
with the second part dipped into ethanol.
The hands were also sterilized with third part, all prior to the inoculation of the
explants.
For cutting the explants inside the laminar airflow chamber, sterilized brown
paper was used.
This shoot tips as well as buds were cut to about 1× 1.5 cm small pieces with
the help of sterilized scalpels and surgical blade on brown paper to make them
exposed for up taking the nutrients from the appropriate media contained in the
bottles.
The bottles were opened at an angle of 450 and 1-2 pieces of explants were
placed on the medium with the upper epidermis pressed gently against the
surface of the agar to make a good contact.
The bottles were capped tightly and transfer to the culture room at 3000 lux of
light for 16 hrs. and at 25 C.
Observations were recorded weekly.
After some days the excised explants which may be shoot tips axillary bud
and meristem is cultured aseptically on nutrient medium and incubated in
culture room for providing a control environmental condition such as
temperature, light and humidity. Under the appropriate condition the
explants will start regeneration by means of multiplication within the
meristematic cells which forms small leaf primordia, shoots and sprouting
apical bud.
Explant tissue should now being to divides its meristematic cells and shows
multiplication which ultimately gives rise to multiple shoot formation.
The shoot primordia grow out into multiple shoots which can be propagated
further by nodal cutting, the axillary bud of each segment will grow out in
culture to form yet another shoots.
Various hormonal regimes (RS media) shows different response kept in
controlled environmental condition.
Following the successful growth of plant tissue, the establishment stage may
be repeated, by taking tissue samples from the plantlets produced in the first
stage.
Root growth does not always occur in the earlier stages in the plant cell
culture, and is of course a requirement for successful plant growth in vitro
by transferring the plantlets to a growth medium containing auxin in
subsequent stages.
Since duration of project work was only 30 days, it was not possible to carry
the complete micro propagation stages of my work. But it can be carrying
out further.
“HARDENING” refers to the preparation of the plants for natural growth
environment, in which culture plant is subjected to hardening in sterilized
soil under control humidity condition.
Until this stage, the plantlets have been grown in “ideal conditions”,
designed to encourage rapid growth.
Thus in this way a plantlet is obtained which possesses both shoots and
young leaves having a root system for its autotrophic growth in subsequent
environment.
After hardening plants are ready to grow in the field.
OBSERVATION
These pictures show typical results, after about 4 weeks on each medium. To
summaries, multiple adventitious buds formed on the medium, leading to many
small shoots on the upper surface where the leaf is not in contact with the medium.
Fig: Nodal cut on RS2 media, Fig: Shoot tips on RS5 media, after
after 14 days of incubation. 21 days of incubation.
Fig: Shoot tips on RS10 media, after 28 days of incubation.
MS media + of shoots/explants.
Amongst the different sets of media, the best shoot multiplication was
observed from the RS2 media containing BAP (2mg/L).
►DISCUSSION
The smaller size of explants were chosen due to fact that smaller size of explants
provide less chance of contamination, as well as it contain large amount of
meristmatic tissue. During initiation the explants did not show any leaching or
browning of tissues.
The explants cultured on MS basal medium supplemented with different
Combinations of BAP and AdSO4 showed varied response for regeneration. Plant
hormones affect gene expression and transcription levels, cellular division and
growth. Plant hormones are in small amounts can promote and influence the
growth, development, and differentiation of cells and tissues. Explants culture on
MS basal medium without any PGR supplementation has no growth. This was
possibly due to significant role of PGR over multiplication.
The plant regeneration via adventitious organ arising directly from explants
requires mainly cytokinin or high cytokinin to low auxin ratio and the suitable
combination of growth regulators. Cytokinins are derived from adenine and
produce two immediate effects on undifferentiated cells; the stimulation of DNA
synthesis and increased cell division (Ting, 1982). Cytokinins also produce a
delayed response in undifferentiated tissue which is the formation of shoot
primordia. In the media supplemented with BAP and small amount of AdSO4
showed significant shoot multiplication. Further it was observed that the
supplement of citric acid (1 mg/L) extensively promote the multiplication and
regeneration. It was also noticed that the high concentration of cytokinin and other
regulator result in moderate or no response.
►RESPONSE ON BUD BREAKING
For this the MS basal media was supplement with four different concentration of
benzyl amino purine (BAP) and adenine sulphate are taken and observation was
recorded in following interval of time period.
►DISCUSSION
Exogenously supplied cytokinin in the nutrient medium placed a major role for the
breaking of apical buds from shoot. Addition of Adenine sulphate in the nutrient
medium induces a bud breaking in most of the cases when supplement with BAP.
The exogenous cytokinin in combination with an AdSO4 does markedly increase in
response with gradual increase in time.
It was observed that minimal amount of adenine sulphate with both 1mg/ L and
2 mg/L of BAP showed positive result. But the higher amount of both growth
regulator leads to subsequent decrease in expression. Further the more productive
result was observed when 2 mg/l of BAP and 50 mg/L of adenine sulphate was
used for bud breaking.
CONCLUSION
Since duration of my project work was only 30 days, it was not possible to observe
the complete results of my work. However, initiation and multiplication of shoot
tips and bud breaking was observed. The present study describes a well
documented and reliable protocol for R. serpentina from shoot explants with much
higher rate of multiplication. This protocol can be used as a basic tool for
cultivation and study of Sarpagandha plant. The shoot tips (explants) of Rauwolfia
sp. which were cultured on MS media supplemented with BAP,AdSO4 and Citric
acid to standardized the optimum concentration as well as combination for
multiplication of the explants. Four concentration of BAP was used alone and two
conc. with association of two types of PGR viz, adenine sulphate and citric acid.
The duration of incubation was 7, 14,21and 28 days.
Under critical observation, we observe that different concentration and
combination effected the growth and multiplication of explants, at optimum
concentration the percentage of shoot multiplication in Rauwolfia sp. is maximum.
Our conclusion is that micropropagation of plant is affected by combination
of different hormones and their concentration in culture media. Of which the BAP
(1 mg/L), AdSO4(50 mg/L) and citric acid (1 mg/L) was found to be most
favorable for shoot proliferation and multiplication in Rauwolfia serpentina. And
plants need phyto hormones at very specific time during plant growth and at
specific concentration.
BIBILIOGRAPHY
http://en.wikipedia.org/wiki/Rauwolfia sp.
http://www.biotechnologyonline.gov.au/pdf/biotech/plant_tissue_culture_in_class.