COMPREHENSIVE REPORT

OF
READY-482
Micropropagation of R. Serpentina by using growth
hormone Kinetin (KIN)

SUBMITTED BY
Mr Abhishek Annaso Pawar
(REG. NO-BSRU/19/0249)

SUBMITTED TO
COLLEGE OF AGRICULTURE BIOTECHNOLOGY,
(B.TECH BIOTECHNOLOGY), SARALGAON, TAL-
MURBAD, DIST,THANE, MH, PINCODEs-421401

AFFILIATED TO
DR.BALASAHEB SAWANT KOKAN KRUSHI VIDYAPEETH,
DAPOLI, DIST-RATNAGIRI, MH, INDIA 2023-2024

1
Micropropagation of R. Serpentine by using growth hormone
Kinetin
Submitted by
Mr Abhishek Annaso Pawar
REG. NO. BSRU/19/249

Research Guide
Assoc.R.J.Janephalkar
(Department of plant
Biotechnology)

Submitted To
COLLEGE OF AGRICULTURE BIOTECHNOLOGY,
(B. TECH BIOTECHNOLOGY) SARALGAON, TAL-
MURBAD, DIST- THANE, MH,

Affiliated to
Dr.B.S.K.K.V, DAPOLI.
2023-24

2
 CERTIFICATE
This is to certify that the thesis entitled “Micropropagation of R.Serpentine by
using growth hormone Kinetin (KIN). Submitted by Mr. Abhishek Annaso Pawar
(BSRU/19/0249) has successfully completed student READY-482 project formulation,
execution and presentation of College of Agriculture Biotechnology Saralgaon.

Assoc.R.J.Janephalkar
(Research Guide)
College Of Agril. Biotechnology, Saralgaon

EVALUATION COMMITTEE

Member Member
Miss khatke S.D. Miss. Sonawane A.H
Department of Plant Tissue Culture Department of Plant Tissue Cultur
Agri Biotechnology, Saralgaon. Agri Biotechnology, Saralgaon.

Mr. Janephalkar R.J

(Training Co-Ordinator)
College of Agri Biotechnology,
Saralgaon.

Member Chairman
Mr. Janephalkar R.J Dr. Sawardekar S.V

College of Agri Biotechnology, Saralgaon. Plant Biotechnology Centre

(Principal) Dr. B.S.K.K.V.Dapoli

3
 CERTIFICATE
This is to certify that the thesis entitled “Micropropagation of R.Serpentine by
using K i n e t i n ( K I N ) . Submitted by Mr Abhishek Annaso Pawar
(BSRU/19/0249) has successfully completed student READY-482 project formulation,
execution and presentation of College of Agriculture Biotechnology Saralgaon.

Assoc.R.J.Janephalkar
(Research Guide)
College Of Agril. Biotechnology,
Saralgaon

4
 ACKNOWLEDGEMENT

I would like to acknowledge and give my warmest thanks to my supervisor

Assoc. R.J.Janephlkar made this work possible. His guidance and advice carried me through
all the stages of writing my project. I would also like to thank my committee members for
lettings my defence be an enjoyable moment, and for your brilliant comments, thank you.

Also, I am grateful to Asst.Prof. A.H.Sonwane and Asst.Prof. S.D.Khatke Department

of Plant Biotechnology for his ongoing mentorship and never-ending supply of fascinating
tasks. This humble approach to research and science is an inspiration. This approach is
evident in his simple but obvious writing style, which I aspire to emulate throughout my
career.

I would also like to give special thanks to my friends and my family as a whole for their
continuous support and understanding when undertaking my research and writing my project.
Your prayer for me was what sustained me this far.

I am forever thankful for the unconditional love and support throughout the entire thesis
process and every day.

Place: Saralgaon Mr.Abhishek Annaso Pawar

Date:

5
 CONTENTS

●
CHAPTER ●
TITLE ●
PAGE.NO

12
1
●
●
●
INTRODUCTION

22
2
●
●
●
REVIEW OF LITRETURE

31
3
●
●
●
MATERIAL AND METHOD

42
4
●
●
●
RESULT AND DISCUSSION

44
5
●
●
●
SUMMARY AND CONCLUSSION

49
6
●
●
●
REFERENCE

50
7
●
●
●
GLOSSARY

6
 LIST OF TABLES

●
Sr. No. ●
Ta ●
Name of table ●
P
ble. a
No. g
e
N
o
.
●
1 ●
1 ●
Taxonomic Classification ●
17
●
2 ●
2 Materials
● ●
33
●
3 ●
3 ●
Chemicals ●
34
●
4 ●
4 ●
Chemical composition of stock ●
35
●
solution
●
5 ●
5 ●
Component of Murashige and ●
36
Skoog’s
●
medium (MS) (1962) preparation of
stock General composition of basal
MS
●
(Murashige and Skoog) medium
●
6 ●
6 ●
Preparation of stock solution of ●
37
growth
●
hormones
●
7 ●
7 ●
Media combination and no. of ●
48
explant
●
inoculated for shoot induction
●
8 ●
8 ●
Effect Of Different Concentrations ●
49
Of
●
NAA For Root Induction.
●
9 ●
9 ●
Effect of different hardening ●
50
mixture
●
on R.Serpentina

7
 LIST OF PICTURES/FIGUERS

Sr. No. Name of Picture Page

No.
18
1 Serpentina

40
2 Stock preparation

40
3 Media Preparation

40
4 PH adjustment

5 41
Explant cutting

6 42
Pouring of MS Media

7 42
Inoculation of explant

8
 ABRRIVATION

●
µg ●
Microgram
●
µM ●
Micromolar
●
BAP ●
Benzyl amino
●
purine
●
Cm ●
Centimetre
●
D ●
Day
●
EDTA ●
Ethylene diamine
●
tetra acetic acid
●
Fig ●
Figure
●
G ●
Gram
●
Hrs or H ●
Hours
●
HCL ●
Hydrochloride
●
L ●
Liter
●
M ●
Molar
●
MW ●
Molecular weight
●
mg ●
Milligram
●
mg/l or gm/L ●
Milligram per liter
●
Min ●
Minute
●
MI ●
Milliliter
●
MS ●
Murashige and
●
skoogs medium
●
N ●
Normal
●
NAA ●
α-Napthalene
●
acetic acid
●
pH ●
Hydrogen ion
●
concentration
●
T ●
Treatment
●
v/v ●
volume/volume
●
w/v ●
weight/volume

9
INTRODUCTION

10
CHAPTER.NO 1

INTRODUCTION

Rauvolfia serpentina is an important medicinal plant to treat insomnia,

hypochondriasis, ailments of the central nervous system and high blood pressure. R. serpentina
is economically important because of the presence of reserpine and rescinnamine group of
alkaloids used in the allopathic systems for the treatment of hypertension, cardiovascular
diseases and as a sedative tranquilizing agent. Interestingly, the powdered root of Rauvolfia
serpentina has been in use in India for at least 2,000 years to treat mental illness (Srivastava,
1995). Reserpine is considered a sympathomimetic agent, one that targets the sympathetic
nervous system. Reserpine has been found to lower blood pressure in remarkably low oral
doses. In Ayurvedic system of medicine, Rauvolfia serpentina roots is part of complex
formulation for treatment of insomnia, epilepsy, asthma, acute stomach ache and painful
delivery of child besides controlling high blood pressure and insanity (Trivedi, 1995). The
plant is widely distributed in India but nowhere gregarious. Natural populations are
encountered in widely separated localities having different habitat conditions. It grows as an
under shrub in the sub-Himalayan tract, Karnataka and Goa and eastern and western Ghats
up to an attitude of 1200 m. Over 95 per cent of the medicinal plants used by the Indian
pharmaceutical industry are today collected from the wild (FRLHT, 1997). Over 70 per cent
of the plant collections involve the use of roots, bark, wood, stem and in some areas the
whole plant, leading to destructive harvesting. Rauvolfia serpentina is also no exception. If
not carefully monitored, this practice could lead to the depletion of genetic stocks and
ultimately to the diversity of medicinal plants. Simultaneously it is also important to look
at the ex-situ conservation of medicinal plants through In vitro studies are required to be
taken up in this species due to the following reasons. Plants from the tropics are worth
between $ billion to $ 47 billion, annually, to the global pharmaceutical industry sarpagandha
alone, (Rauvolfia serpentina), is the base for
$ 260 million worth trade in hypertension and schizophrenia drugs. Large quantities of roots
of this plant were collected from forest areas during sixties, leading to a great depletion of
11
natural stock in the country (Sarin,1986). At present Rauvolfia serpentina reserves are
becoming increasingly exhausted while the requirements of pharmaceutical industry of
importing countries in Rau anatine, reserpine, ajmalicine etc cannot be met (Kunakh and
Akhimova, 1989) Due to the nonavailability of R.serpentine commercial material is often
adulterated with the stems of the drug, roots of other Rauvolfia species mainly R.canescens
Linn. and Clerodendrum species.

However the roots of R.serpentine can be easily distinguished from the others based on
the surface character of the root. Despite their wide geographical distribution and edaphic
tolerance, Rauvolfia serpentina species have not lent themselves to easy cultivation due to
various factors, which influence their propagation, growth and development and also their
alkaloid content (Anonymous, 1969). Cloning of elite plants would be advantageous for
increased alkaloid production (Raja and Hebel, 1996) Poor seed viability and very low
germination percentage that may be ascribed largely to the presence of cinnamic acid
derivatives in the seeds (Mitra, 1976) which could be an inhibiting factor. For rare species that
are in decline, in situ conservation may not be adequate protection (Rajora and Mossier, 2000).
Tissue and cell culture and cryopreservation provide excellent avenues and opportunities for
ex situ conservation of these genetic resources. Low temperature storage of in vitro stock
material commonly used for conservation of plant germplasm (Pruski et al, 2000). This
method, if properly adjusted to specific genotypes, can substantially reduce labour and media
costs The present paper deals with in vitro regeneration and conservation of R.serpentina , as
a possible approach to conserve genetic diversity of this species.

Rauvolfia serpentina was utilized in India as a folk medication for quite a long
time to treat a wide number of ailments, eg. snake and insect bites, febrile conditions, malaria,
stomach pain, and diarrhea. It is additionally utilized as a uterine energizer, febrifuge, and
remedy for nervous system disorders. The conventional use of this plant are loose bowels,
fever, cut, injuries, stomach-ache, periods related problems. The plant is utilized in
gastrointestinal issues by the "kavirajes" of Puthiaupazilla of Rajshahi area of Bangladesh. The
paste is made up of sarpgandha roots, buds with milk and applied on the snake bit affected
area in Karnataka. Its roots are administered in snake bite in Orissa. In Tamil Nadu this
plant is utilized in regular intervals of three days for this purpose (50 grams /day).

12
1.2 HEALTH BENEFITS

 Anti-bacterial:
The findings of one of the in-vitro studies demonstrated that reserpine content is
effective against many of the human pathogens like Salmonella typhimurium,
Escherichia coli, Citrobacterfreundii, Proteus vulgaris, Enterococcus faecalisand
Staphylococcus aureus.
 Hypoglycemic:
The methanolic extract of Rauwolfia serpentina was checked for its hypoglycemic
activity in alloxan-induced diabeticrats. Result showed that methanolic extract
exhibited dhypoglycemic, hypolipidemic and hepatoprotective activities in alloxan-
induced diabeticrats. The methanol root extract of Rauwolfia serpentina was evaluated
for the same in another in-vivo study which showed that methanolic extract showed
glycemic, antiatherogenic, coronary risk, and cardio protective indices in alloxan-
induced diabetic mice.
 Anti-diarrheal:
An in-vivo study conducted on mice models in which diarrhea was artificially induced
by administering castor oil to check the anti-diarrheal activity showed that methanolic
extract of Rauwolfia serpentina is associated with anti-diarrhoeal activity an helped in
reducing intestinal weight and fluid volume.
 Antiarrhythmic:
in-vivo study on rats and cats demonstrated the tajmaline derived from biomass grown
in tissue culture, exhibit antiarrhythmic properties exactly in the same way as ajmaline
derived from the natural root of Rauwolfia serpentine. Ajmaline as class I
antiarrhythmic agent derived from the roots of the plant is very helpful in diagnosing
Brugada Syndrome (hereditary cardiac disorder.
 Anxiolytic:
The findings of the reported clinical study suggested that reserpine, alseroxylon and the
whole crude root exhibited anxiolytic action in ambulatory patients showed Rauwolfia
serpentina use as an anxiolytic agent.

13
 Fig :- R.Serpentina

Table . No 1: Taxonomic Classification :

●
Kingdom Plantae ●
Phylum Tracheophytes
Subphylum
●
Angiospermae
●
Class ●
Magnoliopsida
●
Order ●
Gentianales
●
Family ●
Apocynaceae
●
Genus ●
Rauvolfia
●
Species ●
serpentina
●
Common names ●
Sarpagandha

14
Leaves: In whorls of 3, thin, lanceolate, acute, bright green above and pale beneath.

Flowers: in irregular corymbose cymes, white, often tinged with violet.

Fruit: Drupe, single or did ymous, shining black, the inflorescence with red pedicels and
calyx and white corolla.

Flowering Time: March to May in Indian conditions.

Natural Components: The root contains ophioxylin (an alkaloid having orange coloured
crystalline principle), resin, starch and wax. The total alkaloid yield is 0.8%. Five crystalline
alkaloids isolated are ajmaline, ajmalicine, serpentine, serpentinine, and yohimbine.

Ayurvedic Preparations: Sarpagandha ghanavati, sarpagandha yoga, Sarpagandha churna,

Maheswari vati etc.

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.

Propagation: Can be propagated both through seeds and vegetatively, but propagation by seed
is preferred.

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.

Rauwolfia serpentina is an important medicinal plant (shrub) belonging to the Apocynaceae

family. This plant is occurring naturally in India and Bangladesh and is found to grow wild in
the Asian continent. It has been reported to contain 50 indole alkaloids that are mainly
localized in the root bark. Among these alkaloids, reserpine, yohimbine, serpentine,
deserpidine, ajmalicine and ajmaline are used to treat hypertension and breast cancers.
Reserpine, used as a natural tranquilizer was found to have several times greater hypertensive
activity than the crude
15
plant extract . Rauwolfia serpentina holds an important position in the pharmaceutical world
because of its immense anti-hypertensive properties resulting from the presence of reserpine in
the oleoresin fraction of the roots. Poor seed viability, low seed germination rate, and
enormous genetic variability are the major constraints for the commercial cultivation of R.
serpentine through conventional mode. Many higher plants are major sources of natural
products used as pharmaceuticals, agrochemicals, flavour and fragrance ingredients, food
additives, and pesticides. The search for new plant derived chemicals should thus be a
priority in current and future efforts toward sustainable conservation and rational utilization
of biodiversity. In the search for alternatives to production of desirable medicine
compounds from plants, biotechnological approaches, specifically, plant tissue cultures, are
found to have potential as a supplement to traditional agriculture in the industrial
production of bioactive plant metabolites. Cell suspension culture systems could be used
for large scale culturing of plant cells from which secondary metabolites could be extracted.
The advantage of this method is that it can ultimately provide a continuous, reliable source of
natural products. The roots, the leaves and the juice of R .serpentina have been considered of
medicinal importance from the very early times and have attracted the attention of the
practitioners of the indigenous system of medicine. It has been used as an anthelmintic, as an
antidote against snake bite and bites of other poisonous insects, in diarrhoea, dysentery,
cholera and also as an ecbolic. Rauwolfia root is reported to contain 0.7 – 3.0 % of total
alkaloids in the dry mass and the amount vary with time and source of collection. The rate
of plant propagation is important for commercial cultivation to meet the pharmaceutical
demand for reserpine. Chemical synthesis of reserpine has not been adopted due to its high
cost compared to extraction from the natural source. While roots of R .serpentina is the main
source of the alkaloids mentioned above, indiscriminate harvesting of the roots has
threatened the survival of the plant. However, high demand for the alkaloids necessitates
rapid production of roots within a short time frame. Therefore, root cultures are a
potentially useful in vitro system for commercial production of secondary metabolites.
Rauwolfia serpentine roots are generally obtained through shoot organogenesis callus
morphogenesis, or by Agrobacterium rhizogenes-mediated transformation. The
induction of callus growth and subsequent differentiation and organogenesis is accomplished
by the differential application of growth regulators and the control of conditions in the culture
medium. With the stimulus of endogenous growth substances or by addition of exogenous
growth regulators to the nutrient medium, cell division, cell growth and tissue differentiation
are induced. There are many reports on the regeneration of various medicinal plants via callus

16
culture. Satheesh and Bhavanandan have reported the regeneration of shoots from callus of
Plumbagorosea using appropriate concentrations of auxins and cytokinin’s. Mantell and Hugo
have also reported a high frequency of shoot, root, and micro tuber production from
Dioscoreaalata depending on the culture medium used, the type of explant from which the
calli originated, and the photoperiod.

1.3 Plant Tissue Culture:

Plant tissue culture techniques are a part of large group of strategies and
technologies, ranging through molecular genetics, recombinant DNA studies, genomic
characterization, gene transfer techniques, aseptic growth of cells, tissues, organs and in vitro
regeneration of plants etc. In vitro techniques are used mainly for the regeneration of organs or
somatic embryos for propagation, disease elimination and pathogen free plant multiplication,
transformation and regeneration of transgenic plants for improvement in their traits. It is
possible to achieve rapid propagation of selected and elite genotypes and important in a short
time. Micropropagation is the first and major commercial application of tissue culture
techniques. It is currently used in a large number of medicinally important plants ranging from
a herb to a big tree, both in angiosperms and gymnosperms. through enhanced axillary bud
formation, organogenesis or somatic embryogenesis. Micropropagation of elite, selected or
recalcitrant genotypes generally involve four distinct stages: explants establishment,
regeneration and proliferation, rooting and acclimatization and finally transplanting ex vitro.
The micropropagation technology has a vast potential to produce plants of superior quality,
isolation of useful variants in well adapted high yielding genotypes with better disease and
stress tolerance capacities. In the present investigation, two important medicinal plants, Rand
R. serpentina has been selected for their large-scale propagation and conservation purposes
through tissue culture techniques.

Conventional cultivation is affected by various environmental and climatic factors,

but micropropagation insures a good regular supply of medicinal plants, using minimum space
and time. Micropropagation is the process of vegetative growth and multiplication from plants
tissues or seeds in aseptic and favourable conditions on artificial growth media. Tissue culture
is based on concept of totipotency; the ability of plant cells and tissues to develop into whole
new plant. Gottlieb Haberlandt, a German botanist is considered as the father of plant tissue
culture, was the first to get the experimental evidence on totipotency by culturing plant cells
on Knop’s The highest frequency (73%) of shoot organogenesis was observed when rhizome

17
was taken as the explant in case of Acorus calamus along with the treatment of KIN
(Bhagat, 2011) . First node (shoot tip) of Commiphora wightii performed better on KIN and
GA3 supplemented medium (Tejovathi et al., 2011) . The juvenile leaf explants of
Rauvolfia serpentine responded efficiently in invitro regeneration (Singh et al. 2009) . In
Thymus hyemalis Lange maximum proliferation was observed for nodal explants (Nordine et
al., 2013)

Tissue culture success mainly depends on the genotype, age, types and position of
explants in the mother plant. The advantages of In vitro micro propagation of medicinal plant
are listed below:

4. ADVANTAGES

 Rapid clonal propagation and multiplication.

 Controlled and altered environmental conditions
aids to meet specific needs of the plant.
 Availability of planting material throughout the year
irrespective of seasonal and regional variation
 Identification and production of clones with
desired characteristics (somoclonal
variation).
 Conservation of threatened plant species.
 Preservation of genetic material by cryopreservation.

Micropropagation of medicinal plants remained neglected till a miracle drug plant of India,
Rauvolfia serpentina (L.) Benth plants were produced from its somatic callus tissue. Presently
there are reports on organogenic differentiation in somatic callus tissue of medicinal plants, but
there are only few reports on endangered medical plants, where complete plants have been
regenerated in vitro.

5. Role Of Growth Regulator:

KINETIN(KIN):-is considered as the most widely used cytokinin for shoot induction,
proliferation and multiplication in wide variety of plant species. The edge of KIN over other
cytokinin’s is well documented in many plants including Fragaria Xananassu. (Kang el al..
1994), Eucalyptus impensa (Bunn 2005), Psoralea corylifolia (Jeyakumar and Jayabalan
2002), Bupleurum kaoi (Chin et a/., 2006) and Rauvolfia serpentina (Faisal el al.. 2006).
Also in Cassia siamea (Sreelatha et al, 2007), highest shoot multiplication was observed
18
on MS medium containing K I N . Further in Rauvolfia serpentina (Harisararaj et al,
2009) reported good shoot proliferation on KIN than BA. The successful node cultures were
developed in Paraserianthes falcutaria (Sasmitamihardja et al, 2005) on the medium
supplemented with BAP. Similaru nodal segment of various leguminous tree species showed
good axillary bud proliferation on MS medium supplemented with KIN including Albizia
chinensis ( Sinha et ai. 2000). Pterocarpous marsupium (Chand and Singh 2004) and Albizia
lehbek (Pereen et ai. 2012). Maximum shoot multiplication in Terminalia bellerica
(Metha et ai. 2012). was observed when nodal segments were cultured on MS medium
supplemented with KIN.

1.6 Secondary Metabolites :

During metabolism plants produce enormous number of compounds as part of

defense mechanism (Bennett and Wallsgrrove, 1994) . These compounds do not play essential
role like primary metabolites, hence they are called secondary metabolites. With the rapid
increase in world population, extreme pressure on the available cultivable land, there is fast
disappearance of natural habitats for medicinal plants together with environmental and
geopolitical instabilities; it has become increasingly difficult to acquire plant-derived
compounds (Mulabagal and Tsay, 2004) . In vitro tissue culture offers an effective and
potential alternative of production of bioactive compound because the amount of secondary
metabolites produced in this technique can be even higher than in parent plants (Rao et al.,
2002) . Amount of secondary metabolites was found significantly higher, in in vitro
plantlets and callus compared to in vivo plantlets of Swertia chirayita. Higher heavy metal
accumulation in in vitro as compared to in vivo plantlets correlated higher secondary
metabolite production supporting that they play regulatory role in influencing the plant
secondary metabolism ( Kumar et al. 2014 ) . An efficient and reproducible method for the
induction of callus and hairy roots from explants of Rauwolfia serpentina is standardized to
produce secondary metabolites in vitro. Hairy roots were induced from leaf explants and
these leaf explants were infected with Agrobacterium rhizogenes to induce hairy roots for
the production of secondary metabolites in large scale (Shetty et al. 2014) . Plant cell cultures
are an attractive alternative source to whole plant for the production of secondary
metabolites. Plant cell cultures have the following advantages over conventional
agricultural production viz., independent of geographical and environmental variations,
defined production system with continuous supply of products, uniform quality, yield,
rapidity of production and production of novel compounds. In recent years, various plant cell
culture systems have been exploited for the enhancement
19 of high value
metabolites (Rao et al., 2002) . Homogenous cell suspension culture of Celastrus paniculatus,
was established and multifold production of secondary metabolites like alkaloids and total
phenols was obtained under the influence of monochromatic lights under in vitro condition
(Billore et al., 2016) . The unorganized callus and cell suspension cultures contained fewer
amounts of all phenolic compounds than re-differentiated shoots. The capacity of cell
suspension culture to accumulate phenolics was enhanced after the application of salicylic acid
and yeast extract thus provide a chance to improve yield (Owis et al., 2016) . Jasmonic acid
has been used to modulate the production of various secondary metabolites in plant tissue
culture techniques. Jasmonic acid (JA) is most effective in eliciting total phenolic production
of callus cell suspension cultures of Celastrus paniculatus. The total phenolics production was
maximum when 250 μM, 250 μM and 50 μM of jasmonic acid was treated for 24, 48 and 72
hours respectively. Salicylic acid (SA) has also been used to enhance in vitro regeneration in
several plant species. The total phenolics production was maximum when 100 μM, 250 μM
and 50 μM of Salicylic acid was treated for 24, 48 and 72 hours respectively (Anusha et al.
2016) . Among copper salts, copper sulphate and copper chloride were successfully used as
abiotic elicitors in number of plant cell culture. The total phenolics production was maximum
when 250 μM, 100 μM and 250 μM of CuSO4 was treated for 24, 48 and 72 hours
respectively (Kasparova M. et al. 2008).

20
7. OBJECTIVES:
8.
The present research work has been carried out with following objective:

1. To optimize culture conditions for regeneration, multiplication, and

regenerant differentiation in somatic tissue through direct organogenesis
of R . Serpentina.

2. To standardized the technique for in vitro rooting in regenerated microshoots

and acclimatization of plantlets in natural conditions.

3. To assess the physiological and biochemical changes during acclimatization

period in micro propagated plantlets.

4. To study the comparative metabolic profile of secondary metabolites in leaves

of in vitro raised and sonar plants through HPLC analysis.

21
REVIEW OF LITREATURE

22
CHAPTER.NO 2

REVIEW OF LITREATURE

2.1 Historical background :

In the history of tissue culture, the pioneer step was made by Henri-Lous Duhumel du
Monceau in 1756, who during his pioneering studies on wound-healing in plants, observed
callus formation (Gautheret, 1985). The science of the tissue culture cemented its roots from
the path breaking research in botany like the discovery of the cell theory. In 1839, Schleiden
and Schwann proposed that cell is the basic, structural and fundamental unit of an organism.
They visualized that plant cell is capable of autonomy and therefore capable to generate the
whole plant from the single plant cell (totipotency). This idea was tested from time to time by
various researchers, but the work of Vochting (1878) and on the limits of divisibility of plant
segments was perhaps the most important. He showed that the upper part of the stem segment
produced buds and the lower end produced callus or roots. Thus he suggested the polar
development is the key feature that guides the development of the plant fragments.

In 1902. a German Botanist, Gottlieb Haberlandt was the first to try to obtain
experimental evidence of totipotency by culturing single cells of Lamium purpureiim and
Eichhornia crassipes on Knop's salt solution with sucrose and observed obious growth in
palisade cells. Although, Haberlandt remained unsuccessful in getting the desired results but
he clearly established the concept of totipotency by predicting that one could successfully
cultivate the artificial embryos from vegetative cells and the technique of cultivating isolated
cells in nutrient solution permits the investigation of important problems from a new
experimental approach. Due to this endeavour. Gottlieb Haberlandt is regarded as the father of
tissue culture.

From 1902 to 1930 several attempts were made for organ culture. In 1904, Hanning
isolated embryos of some crucifers like Raphanus caudatus, R. landra, R. stavis etc. and
successfully cultured on the mineral salt and sugar solution under aseptic conditions, thus laid
the foundation of embryo culture. In 1908, Simon regenerated callus, buds and roots from
Poplar stem segments and established the basis for callus culture. Using a different approach,
Kotte (1922), a student of G. Haberlandt and Robbins (1922) succeeded in culturing of isolated

23
root and shoot tips in vitro. White (1934) had established successfully indefinite culture of
tomato root tips, using excised explants with merismatic cells, on the excellent defined
medium containing ingredients of sucrose, mineral salts and yeast extract. At the same time,
in 1934 Roger Gautheret grew callus from cambial tissues isolated from woody plants
including Willow sycamore. These were the first sustained dividing callus cultures. Soon after
Gautheret, Noubecourt 1939 reported the successful growth of plant callus cultures, proving
the one of the assumptions of G. Haberlandt true. Van Overbeeck and his co-workers
in 1941 demonstrated for the first time, the stimulatory effect of coconut milk on embryo
development and callus formation in Datura innoxia.

In 1950, there was an immense advancement in the field of plant tissue culture, after the
discovery of effect of different PGRs on plant development. Skoog and Tsui in 1957
demonstrated the induction of cell division by the addition of adenine in the tobacco plant.
Steward and Reinert (1958) reported the regeneration in callus culture of carrot. The
tremendous increase in the in vitro cultivation of valuable number of plant species was made
by the discovery of different cytokinin’s.

Skoog and Miller (1957) put forth the concept of hormonal control of organ formation
after the discovery of cytokinin. Since then, tissue culture techniques have revolutionized the
improvement in plant species and conservation. The dream of Haberlandt for the cultivation of
somatic embryos became true when the first report appeared on somatic embryogenesis in
carrot tissues by Reinert (1958, 1959) and steward et al.. (1958). In early 1960s, the most
significant breakthrough in the field of tissue culture was the development of well defined
culture medium which originally devised for the rapid growth and bioassay for the tobacco
callus (Murashige and Skoog 1962). This medium contains all the desired salt composition
which is widely used to induce organogenesis and regeneration of various plant parts in
cultured tissues. Even today this medium is recognized most effective and is commonly used
medium in plant tissue culture.

Maheshwari and his co-workers became actively engaged in in vitro techniques in 1960
and laid the foundation of new mile stone by raising haploids, through anther culture of
Datura innoxia (Guha and Maheshwari, 1964, 1966). In 1971, Takebe et al.. first
demonstrated the totipotency of protoplast and regenerated the tobacco plants from
mesophyll protoplasts. Thrope, in (1980), reported de novo organogenesis by interchanging
auxin and cytokinin ratio in the medium. In addition, explants such as cotyledonary node,
hypocotyls, callus and thin
24
superficial cell layers have been used in traditional morphogenetic studies as well as to
produce de novo organs and plantlets in various plant species (Murashige 1974, 1979).

2.2 Micropropagation :

In vivo clonal propagation of plants is tedious, expensive and frequently unsuccessful.

Micropropagation is an alternative method of vegetative plant propagation and produces plants
in a rapid way not only generating clonal planting stocks, but also effectively captures genetic
variation while by passing long breeding cycles (Giri ei ill.. 2004). Further, micropropagation
is a process that involves exposing plant tissue to a specific regime of nutrients and hormones
under sterile or in artificial conditions outside the mother plant to produce many new plants.

In vitro clonal propagation through tissue culture is referred as micropropagation. Use of tissue
culture technique for micropropagation was first started by Morel (1960) for propagation of
orchids, and is now applied to several plants. The main aim of micropropagation is to provide
a sufficient number of plantlets within a short duration and in a limited space.
Micropropagation offers a viable tool for mass propagation and germplasm preservation of
many medicinal and aromatic plants. As a result. laboratory-scale micropropagation protocols
are available for a wide range of species and at present micropropagation is the widest use of
plant tissue culture technology.

There are three methods by which micropropagation can be achieved. These are
enhancing axillary bud breaking, production of adventitious buds directly or indirectly via
callus and somatic embryogenesis directly or indirectly on explants (Murashige. 1974). Tissue
culture techniques are being used in all types of plants like temperate, tropical, forest and
ornamental, gymnosperms or angiosperms. Micropropagation techniques are preferred over the
conventional propagation methods because a small amount of fissue is required to produce
millions of clonal plants in a short period. Micropropagation generally involves four distinct
stages (Murashige 1974).

i) Initiation of aseptic culture - from explants

ii)
iii)Shoot multiplication of the explants
iv)
v) Rooting of the in vitro regenerated shoots.
vi)
vii)Transplantation - Transfer of the acclimatized plants to green house or field conditions.

25
Debergh and Maene (1981) introduced the stage 0 (preparation of explants) making
micropropagation a five stage process. George Morel (1960) was the pioneer in applying shoot
tip culture as a clonal multiplication tool. After his success in cloning the orchid, Cymbidium,
the clonal multiplication gained momentum in the 1970s. Furthermore, in vitro growth and
development of a plant is determined by a number of complex factors.

1. The genetic makeup of the plant.

2. Nutrients; water, macro and micro elements and sugars.
3. Physical growth factors, light, temperature, pH, O2 and CO2 concentrations.
4. Some organic substances; regulators and vitamins etc.

2.3 Explant type :

The regeneration power of the explants depends upon the size, nature of plant woody
herbaceous, season of collection and healthiness of the plant. Any piece of the plant tissue can
be used as an explant such as shoot apices, hypocotyls, epicotyls, mesocotyl, cotyledon,
cotyledonary node, leaf and stem segments which have been known to possess the potential
for shoot induction in aseptic culture (Bajaj and Gosal, 1981). A popular explant widely
utilized for multiple shoot production is nodal explants with axillary bud of field grown
medicinal plants including Raulvofia Serpentina (Faisal et al, 2005), Psoralen corylifolia
(Faisal and Anis 2006), Capssicum annum (Ahmad et al., 2006), Tylophora indica (Faisal
and Anis, 2007), Cassia angiistifolia (Siddique and Anis 2007), Vitex negundo (Ahmad and
Anis, 2010), Nyctanthes arbor tristis (Jahan et al., 2010a and b), Salix tetrasperma (Khan et al.
2011), Withania somnifera (Fatima and Anis, 2011 Euphorbia continifolia (Perveen et al.
2013), Abrusprecatorius (Perveen et al, 2013), Vitex trifolia (Ahmad et al. 2013) and
Cassia alata (Ahmed et al, 2013).

2.4 Plant Growth Regulators :

Plant growth regulators are the synthetic organic substances which in minute amount
act like the natural hormones, other than the vitamins and micro elements to promote, inhibit,
or otherwise modify the physiological process of plants. These are the main components of
media in determining the growth and developmental pathway of in vitro plant cell totipotency
(Davies, 1995). Auxins, cytokinin’s and their different combinations and interactions are
considered most important PGR sclieme for regulating growth, development and
morphogenesis in vitro regeneration of plants The influence of PGRs and their interaction on

26
in vitro regeneration of different plant species have been reported by Skirvin et al., (1990),
Rout and Das (1997). Komalavalli and Rao (2000), Ahmad and Anis (2007), Siddique and
Anis (2008). Faisal and Anis ( 2006 and 2009). Khan et ai, (2010), Perveen and Anis (2011).
Fatima et al. (2011), Naz and Anis (2012), Seemab et ai, (2012), and Ahmad et ciL. (2013).
Cytokinin’s like 6-benzyl adenine (BA), Kinetin (Kin), and Metatopolin (niT) are adenine
derivatives, promote cell division, shoot proliferation and shoot morphogenesis (Skoog and
Miller, 1953 and Miller, 1961).

Cytokinin’s are the most important additives of the plant tissue culture media which
regulate cell division, stimulate axillary and adventious shoot bud proliferation and
differentiation (Sugiyama, 1999). Tissue culture is all about the modifications of the media
thus the optimization of the culture media is affected by the change in concentrations of the
applied cytokinin’s because their uptake, transport and metabolism differ between varieties
and during the interaction with the endogenous physiology of explants (Staden et al, 2008).
BA is considered as the most widely used cytokinin for shoot induction, proliferation and
multiplication in wide variety of plant species. The edge of BA over other cytokinin’s is well
documented in many plants including Fragaria Xananassu. (Kang el al.. 1994), Eucalyptus
impensa (Bunn 2005), Psoralea corylifolia (Jeyakumar and Jayabalan 2002), Bupleurum kaoi
(Chin et a/., 2006) and Rauvolfia serpentina (Faisal el al.. 2006). Also in Cassia siamea
(Sreelatha et al, 2007), highest shoot multiplication was observed on MS medium containing
BA. Further in Rauvolfia Serpentina (Harisararaj et al, 2009) reported good shoot proliferation
on BA than Kn. The successful node cultures were developed in Paraserianthes falcutaria
(Sasmitamihardja et al, 2005) on the medium supplemented with BAP. Similar nodal segment
of various leguminous tree species showed good axillary bud proliferation on MS medium
supplemented with BAP including Albizia chinensis ( Sinha et ai. 2000). Pterocarpous
marsupium (Chand and Singh 2004) and Albizia lehbek (Pereen et ai. 2012). Maximum shoot
multiplication in Rauvolfia Serpentina (Metha et ai. 2012). Rview of Literature was observed
when nodal segments were cultured on MS medium supplemented with BA. Thidiazuran
(TDZ) (A'- phenyl-A-1, 2, 3-thidiazol-5-yl urea) is a plant bio regulator that induces axillary
shoots bud (Binzel et ai, 1996 and Pardhan et al, 1998). TDZ was reported to be more
effective in lower concentration for shoot bud induction and at higher concentration produces
callus in Hagenia abyssinica (Feyissa et al, 2005). TDZ was also tested best for shoot
induction in Rauvolfia serpentina (Jahan and Anis 2009) on nodal segments. Similar positive
effects were also reported in Cannabis sativa (Lata et al, 2009) when the nodal segments were
cultured on
27
MS medium supplemented with TDZ. A good effect of TDZ was investigated on in vitro
shoot proliferation from nodal segments of Rauvolfia serpentina (Faisal et al, 2005).
Metatopolin (niT), is used in the plant tissue culture either alone or in combination with the
KIN. mT has the direct effect on shoot length enhancement and increase the
secondary metabolite production. In Juglans nigra (Pijut et al, 2007) cultured nodal segments
on MS basal medium supplemented with mT (4.1 |iM), a significant increase in shoot
number, elongation and number of nodes per explants was observed. mT enhanced the
shoot proliferation rate in Uniola paniculata (Carmen valego et al, 2009). In Aloe
arborescent, (Amoo et al. 2012), studied the evalvated roles of different aromatic
cytokinin’s types (mT, mTR, MemT and BAR) on direct organogenesis in vitro and
antioxidant activity of regenerated shoots of secondary metabolite production,
improvement in every aspect was reported and mT (5.0|iM) gave the largest number of
transplantable shoots. In addition, a range of auxins and cytokinin’s in combination played a
significant role in the multiplication of many plant species. Among the different tested
combinations of different cytokinin’s (Kin) with auxins (KIN, lAA and IBA), KIN with
NAA has proven to be optimal for regeneration of shoots from nodal segments as is reported
in Alhizia lebbeck (Perveen and Anis, 2012), Withania somnifera (Fatima and Anis 2012),
Acacia ehrenbergiania (Javed et al, 2013) and Euphorbia cotinifolia (Perveeent'/a/., 2013).

2.5 Subculturing

The rapid rate of shoot regeneration, proliferation and multiplication depends upon the
subculturing of the shoot cultures because to avoid toxic effect of closed vessels 16 four sub-
culturings. In Kaempferia galanga (Shirin et al., 2001 ). observed 13 fold increase in the
plantlet production after every four weeks of subculturing, cultured on MS medium containing
KIN (12.0 \xM) and NAA (3.0 \xM). Mass multiplication of shoots was reported in Rauvolfia
serpentina (Fracaro and Echeverrigaray 2001), without any decline in shoot number up to eight
months and were sub cultured after every four weeks. Significant shoot improvement was
observed in Rauvolfia serpentina (Arya ei al., 2003) through repeated transfer of explants on
the same fresh medium after ever four weeks. Effect of sub culture passage on shoot
multiplication was also observed by Siddique and Anis 2007 in Cassia angustifolia where
shoot number increased up to fourth passage, became stable during the fifth passage, beyond
which a gradual decrease in multiplication rate was observed.

28
2.6 Rooting

Rooting is the main step of the micropropagation and considerable work has been
done to achieve rooting in various plant species like Rauvolfia serpentina (Pereira et al..
2003), Pongamiapinnata (Sugla et al., 2007), Ocimum basilica (Siddique and Anis, 2009) and
Cassia angustifolia (Siddique et al, 2010). In many plant species rooting greatly depends
upon the strength of the MS medium with or without plant growth regulator. For root
induction, optimization of basal medium, strength and hormone concentration is very
effective. MS medium was found satisfactory for root induction in number of plant
species e.g., Vitex negundo (Ahmad and Anis, 2011). Rota graveolens (Ahmad et al, 2012).
Also, good results for root induction were reported on half strength MS medium in Rauvolfia
serpentina (Perveen et al, 2011). Bauhinia tomentosa (Naz et al, 2011), Salix tetrasperma
(Khan et al, 2011), Cassia angustifolia (Parveen et al. 2012), Cuphea procumbens (Fatima
et al, 2012). and Withania somnifera (Fatima and Anis 2012) whereas in Cardiospermum
halicacabum (.lahan and Anis, 2009) one-third concentration of MS salts was effective
for root formation. Review of Literature Addition of different auxin (lAA, NAA and
IBA) have been proven to be more effective for rhizogenesis in number of woody plants
for instance in Liquidambar styraiflua (Pareira et al, 2003), Pterocarpous marsurpium (Anis et
al, 2005), Garcinia indica (Malik et al, 2005) and in Rauvolfia Serpentina (Faisal et al, 2005)
and in Mucuna pruriens (Faisal et al. 2006). 100% rooting was achieved in Withania
sommferu (Fatima and, Anis 2011), in vitro raised shoots when transferred to half strength
MS medium supplemented with 0.5 \iM NAA.

2.7 Hardening and Acclimatization

Acclimatization of the obtained micro propagated plantlets is a critical moment for

establishment of good protocol for mass production. Adaptation of plants in greenhouse, field
or in the nature is another delicate and difficult stage. Easy acclimatization depends primarily
on the formation and establishment of autotrophic growth. Successful results can be achieved
by careful environmental control during acclimatization. Plantlets developed in the in vitro
environment faces different conditions like aseptic environment, low diffused light, high
relative humidity, low temperature and the chemically well defined medium, rich in sugar and
nutrients which is responsible for their heterotrophic growth. These plantlets being very fragile
face a number of problems when they are transferred to ex vitro environment due to high
rates

29
of water loss when taken out from the culture vessels. Even if the water potential of the
substrate is higher than the water potential of the media and due to the poor development of
cuticle and stomata, the plantlet may wilt quickly. In addition, water supply may be limiting
because of low hydraulic conductivity of roots and root stem connections (Fila et al, 1998 and
Pospisilova et al, 2007). The heterotrophic mode of nutrition and poor mechanism of water
control loss make these plantlets vulnerable to transplantation shocks, when directly placed in
the green house or field. Many plants die during this transitional period. Therefore, prior to ex
vitro transplantation, plantlets needs 2-4 weeks of acclimatization with gradual lowering of
humidity and high irradiance to check photo inhibition, caused due to the under developed
physiological system. Acclimatization period of micro propagated plantlets is thus necessary
and very crucial for the better survival in the ex vitro conditions before transferring them in to
the garden, field or green house conditions.

30
MATERIAL AND METHODS

31
CHAPTER.NO 3

MATERIAL AND METHODS

1.Source of plant material and experimental : The plant will be collected

from Priya neursury dapoli , Maharashtra Pincode 415713 used as an experimental material.
The experiment will be conducted in Department of Plant Biotechnology College of
Agriculture Biotechnology, Saralgaon during 2023-2024.

2.Details of KIN at different concentration : The MS medium will be supplemented

with different concentration of KIN ( 0.5,1.5,2.5,3.5,4.5,5.5,6.5,7.5 and 8.5 uM/L ) for the
induction of shoot in marking nut through nodal explants. The Details of statistical design
will be applied for above said objective.

Statistical design : Completely Random Design (CRD)

No.of treatments : 09

No.of replication : 03

Treatments Details : Concentration of KIN

●
Treatment (T) ●
Concentration of KIN uM/L
● T1
●
0.5
● T2
●
1.5
● T3
●
2.5
● T4
●
3.5
● T5
●
4.5
● T6
●
5.5
● T7
●
6.5
● T8
●
7.5
● T9
●
8.5

32
Table : Materials and Chemical

● ● ● ●

●
EQUIPMENT ●
GLASSWARE ●
CHEMICAL ●
OTHER

●
Autoclave ●
Micropipette ●
MS Media ●
Aluminium Foil

●
Laminar air flow ●
Trays ●
70% Ethanol ●
Tissue Paper

●
Weighing balance ●
Conical flask ●
HCL ●
Lamp

●
PH Meter ●
Beaker ●
EDTA ●
Cotton

●
Shaker ●
Test tube ●
Stock Solution ●
Bag

●
Magnetic stirrer ●
Culture tube ●
Bavistin
Solution

●
Hot plate ●
Forcape ●
Liquid detergent

●
Glass Rod ●
Mercuric
chloride

●
Spirit

33
Table Component of Murashige and Skoog’s medium (MS) (1962) preparation of stock
General composition of basal MS (Murashige and Skoog) medium

●
Constitutes ●
Concentration (mg/l)
●
I. Inorganic compound
●
Potassium nitrate (KNO3) ●
38
●
Ammonium nitrate(NH4NO3) ●
33
●
Calcium chloride(CaCl2).2H2O ●
8.8
●
Magnesium sulphate(MgSO4).7H2O ●
7.4
●
Potassium phosphate( KH2PO4) ●
3.4
●
Micro nutrients
●
Manganese sulphate (MnSO4). 4H20 ●
4.46
●
Zinc sulphate (ZnSO4).7H2O ●
1.7
●
Boric acid (H3BO3) ●
1.24
●
Potassium iodide (KI) ●
0.166
●
Molybdic acid (Na2MoO4).2H2O ●
0.05
●
Cobalt chloride (CoCl2).5H2O ●
0.005
●
Copper sulphate (CuSO4).5H2O ●
0.005
●
Iron source
●
Disodium EDTA (Na2-EDTA) ●
3.725
●
Ferrous sulphate (FeSO4).7H2O ●
2.785
●
Organic source
●
Vitamins
●
Nicotinic acid ●
0.05
●
Pyridoxine-HC ●
0.05
●
Thymine-HCl ●
0.01
●
Amino acid
●
Glycine ●
0.2
●
Carbon source
●
Sucrose ●
30
●
Gelling agent
●
Agar ●
8

34
 3. GROWTH HORMONES-

1. Kinetin(KIN)

1.PREPARATION OF STOCK SOLUTION OF

GROWTH HORMONES-

The growth hormones were dissolved in few drops of the solvent and volume was made up to
the required level with double distilled water, filter sterilized. The stock of growth hormones
was prepared in different mg/ml for convenient use and stored at cold condition (4°C).

Table . No 6 : Preparation of stock solution of growth hormones

●
Growth ●
Solution Preparation
●
regulators
●
Solvent ●
Diluents ●
Powder ●
Liquid ●
Sterilizatio
●
Storage ●
storage n
●
KIN ●
1N ●
Water ●
RT ●
2-8°C ●
CA/F
KOH
●
KIN ●
1N ●
Water ●
RT ●
2–8C ●
CA/F
KOH

RT-Room temperature

CA-Co-autoclavable

F-Filter sterilization

To obtain the final working concentration of 1.0 mg/l of plant growth regulator in culture
medium, 1.0 ml of the stock solution was added to 1 lit of medium. It was calculated by using
following formula to meet the requirement as given

Volume of stock solution = Desired hormone conc. × medium volume

Stock solution conc.

35
4. Methodology

1. Collection of Explant: -

R.Serpentina are collected from the from the Priya neursury dapoli , Maharashtra
415713 , in the November 2024 for Micropropagation .

Fig: Ravoulfia Serpentina

2. Stock solution A (Macronutrients)

Stock solution of macronutrients was prepared up to 10 times the concentration of the

final medium in 1000 ml of distilled water (dw). Ten times the weight of the salts required per
litre of the medium were weighed properly and dissolved by using a magnetic stirrer in about
750 ml of distilled water and then made up to 1000 ml by further addition of distilled water
(dw). To make the solution free from all sorts of solid contaminants, it was filtered through
Whatman no. I filter paper. Then it was poured into a plastic container, labelled with marker
and stored in a refrigerator at 40C for later use.

3. Stock solution B (Micronutrients)

The stock solution of micronutrients was made up to 100 times the final strength of
necessary constituents of the medium in 1000 ml of distilled water (dw) as described for the
stock solution of macronutrients. The stock solution was filtered, labelled and stored in a
refrigerator at 40C.

36
3.4.4 Stock solution C (Iron sources)

This was prepared at 100 times the final strength of Fe2SO4 and Na2EDTA in 100 ml
of distilled water and chelated by heating on a heater cum magnetic stirrer. Then the volume
was made up to 1000 ml by further addition of distilled water. Finally, the stock solution was
filtered and stored in a refrigerator at 40C.

3.4.5 Stock solution D (Vitamins)

Each of the desired ingredients except myo-inositol were taken at 10 folds (100x) of their
final strength in a measuring cylinder and dissolved in 750 ml of distilled water. Then the final
volume was made up to 1000 ml by further addition of distilled water. The solution was dispensed into
10 ml aliquots and stored at –200C. Myo-inositol was used directly at the time of media preparation.

Fig: Stock Preparation

37
3.4.6 Media preparation: -

Medium was prepared by dissolving required amount of stock Solutions. Sucrose (30gm) was
dissolved, filtered, mixed in stock solutions measured for the preparation of media and was
made to final volume. The pH of medium was adjusted to 5.8. After that required growth
hormones and agar (8 gm) were added. The flask was wrapped with aluminium foil was
autoclaved at 15 psi pressure for 15-20 min at 121°c. After sterilized the MS media poured in
test tube then observed 48 hrs.

Fig: Glassware for media preparation Fig : pH Calibration

Fig : Media Preparation Fig : Media Pouring

38
3.4.7 Surface sterilization: -

The Nodes wash with running tap water for 15 minutes then the with antifungal agent
Bavistin (0.2-0.5%) for 7-8 minutes after this surface sterilized with 70% ethanol for 1
minute. 0.1% Mercuric chloride (HgCl2) treatment of 2-3 minute and then nodes wash trice
with sterile double distilled water.

Fig : Solution for surface sterilization

3.4.8 Inoculation: -

Before starting the inoculation work, slab of Laminar flow was cleaned with rectified
spirit and culture vessels containing autoclaved media, petri dishes, and spirit, lamps, cotton
and other things required were kept on the slab of transfer chamber. A day before inoculation
of work, transfer chamber was fumigated with fumes obtained by heating formic acid and
potassium permanganate (KMnO4). The forceps, scalpels, needles; scissors were kept in a
glass tube column containing rectified spirits. Nodes were inoculated in the test tube
containing culture media aseptically.

39
 Fig: Inoculated Explant

3.4.9 Incubation: -

Cultured test tube were incubated in culture room . The temperature of room was
maintained at 25± 10°C using air conditioner and light intensity (1200 lux) was provided from
fluorescent tubes (40 watt) and incandescent bulbs (40 watts). A photoperiod of 16h light and
8h dark was provided. Observed and examined the cultures and record the data.

Fig. Incubation

40
3.4.10 Shoot Multiplication:

At these stage it explant has expanded into a cluster of small shoot multiple shoot are
separated and transplanted to new cultured medium. Shoots are subculture 2-8 weeks. Explnat
may be subculture several times to new medium to maximise the quantity shoot produce.

Fig: Initiated Shoots For Multiplication stage

3.4.11 Root Formation :

The rooting stage prepares the regenerated plants for transplanting form in vitro conditions in
controlled condition. This stage may involve not only rooting of shoots , but also conditioning
of the plants to increase potential for acclimatization and survival during transplanting. The
induction of adventitious roots may be achieved in vitro in the presence of auxin .

Fig: Root Formation

41
3.4.12 Hardening:

Once the shoot is well rooted, it makes a complete plantlet. However, the in vitro rooted
plantlets are unable to photosynthesise efficiently and are adjusted to the aseptic conditions
inside the culture vessel. Hence, the plantlet is not ready for the outdoor conditions and has to
be acclimatised to the outer environment, For this the plantlet is first planted onto a sterilised
mix of soil, vermiculite and perlite (2:1:1) in the controlled conditions of the lab’s culture
room. This is called hardening. During hardening the in vitro grown plants develop
their photosynthetic machinery, stomatal functioning, root system and ability to deal with the
micro- organisms .After around a week or two of hardening inside the culture room and the
plantlet is then transferred to the green house for acclimatization, for further enhancing the
plant survival. Once the plant is acclimatised in the green house it can be slowly moved
to the outdoor conditions. Hence, the plants are exposed in stepwise manner to harsher
conditions.

Fig: Hardening

42
RESULT

43
CHAPTER .NO 4

RESULT AND DISCUSSION

4.1 Observation Table :

Table.No 7 : Media combination and no. of explant inoculated for

shoot induction
●
Concentration ●
No.o ●
No.of explant ●
Averag ●
Average ●
Shoot
●
(uM) f ●
showing e ●
shoot ●
Regenra
●
expl shoot
●
no.of length tion (%)
ant Shoo
induction (cm)
●
(3 repeats) t
initia
tion
●
CONTROL ●
10 ●
0 ●
0 ●
0 ●
0 ●
0 ●
0
●
MS+KIN(0.5u ●
10 ●
3 ●
4 ●
5 ●
2.55 ●
1.66 ●
40%
M)
●
MS+KIN(1.5u ●
10 ●
2 ●
5 ●
4 ●
6.66 ●
2.33 ●
36.66%
M)
●
MS+KIN(2.5u ●
10 ●
4 ●
6 ●
5 ●
7.5 ●
3.32 ●
50%
M)
●
MS+KIN(3.5u ●
10 ●
3 ●
4 ●
4 ●
9.5 ●
5.44 ●
36.66%
M)
●
MS+KIN(4.5u ●
10 ●
2 ●
3 ●
3 ●
8.5 ●
4.83 ●
26.66%
M)
●
MS+KIN(5.5u ●
10 ●
6 ●
8 ●
9 ●
11.5 ●
7.16 ●
76,66%
M)
●
MS+KIN(6.5u ●
10 ●
4 ●
6 ●
5 ●
9.8 ●
5.33 ●
50%
M) Chart Title
●
MS+KIN(7.5u ●
10 ●
3 ●
5 ●
6 ●
9 ●
4.55 ●
46.66%
M)
9
●
MS+KIN(8.5u ●
10 ●
2 ●
4 ●
5 ●
84.6
●
2.55 ●
36.66%
M)
6 6 6 6
5 55 5 5 5
4 4 44 4 4
3 3 33 3
000 2 2 2

Rep 1 Rep 2 Rep 3

Graphical Representation on shoot induction

44
Table.No 8:Effect Of Different Concentrations Of KIN For Root Induction
●
Concentration Of ●
No. ●
No.of explant ● Average ●
Average ●
Root
KIN of no.of
●
●
showing ●
●
Root ●
Regenra
●
expl ●
Root
ant Root length tion (%)
initia
inductio (cm)
tion
n
●
(3 Repeats)
●
CONTROL ●
10 ●
0 ●
0 ● 0 ●
0 ●
0 ●
0
●
0.5 ●
10 ●
2 ●
3 ● 4 ●
3 ●
1.6 ●
30%
●
1.5 ●
10 ●
1 ●
3 ● 3 ●
2.33 ●
1.16 ●
23.33%
●
2.0 ●
10 ●
3 ●
4 ● 6 ●
4.33 ●
2.16 ●
4.33%
●
2.5 ●
10 ●
3 ●
4 ● 5 ●
4 ●
1.8 ●
40%
●
3.0 ●
10 ●
2 ●
3 ● 3 ●
2.66 ●
1.33 ●
26.66%
●
3.5 ●
10 ●
1 ●
2 ● 4 ●
2.33 ●
1.16 ●
23.33%
●
4.0 ●
10 ●
2 ●
4 ● 3 ●
3 ●
1.5 ●
30%
●
4.5 ●
10 ●
1 ●
2 ● 3 ●
1.33 ●
0.66 ●
13.33%
●
5.0 ●
10 ●
1 ●
2 ● 2 ●
1.66 ●
0.83 ●
16.66%

Chart Title

4 4 4 4 4

3 3 3 3 3 3 3 3 3

2 2 2 2 2 2 2

1 1 1 1
0 0 0

CONTROL CONC 2 CONC 3 CONC 4 CONC 5 CONC 6 CONC 7 CONC 8 CONC 9 CONC 10

Rep 1 Rep 2 Rep 3

Graphical Representation On Root Induction

45
Table.No 9 : Effect of different hardening mixture on R.Serpentina

●
Test ●
Mixture ●
No.of ●
Response ●
Averag ●
% of
●
Expla of e ●
surviv
nt ●
3 repeats ●
no.of al

survi
val
●
T1 ●
Soil : cocopeat: ●
10 ●
6 ●
5 ●
7 ●
6.0 ●
60
●
vermiculite
(2:1:1)
●
T2 ●
Soil : cocopeat: ●
10 ●
4 ● 5 ● 7 ● 5.33 ●
53.33
●
perlite (2:1:1)
●
T3 ●
Soil : cocopeat ●
10 ●
5 ● 6 ● 8 ● 6.33 ●
60.33
●
(2:1)
●
T4 ●
Soil :vermiculite ●
10 ●
7 ● 8 ● 9 ● 8.0 ●
80
:
●
perlite (2:1)
●
T5 ●
cocopeat ●
10 ●
4 ● 6 ● 7 ● 5.66 ●
56.66
●
T6 ●
cocopeat: ●
10 ●
6 ● 6 ● 7 ● 6.33 ●
63.33
●
vermiculite (1:1)
●
T7 ●
Soil : cocopeat: ●
10 ●
6 ● 7 ● 8 ● 7.0 ●
70
●
vermiculite :
perlite (2:1:1:1)
●
T8 ●
cocopeat: ●
10 ●
5 ● 5 ● 6 ● 5.33 ●
53.33
●
vermiculite :
perlite (1:1:1:)

46
 Chart Title

9
8 8 8
7 7 7 7 7 7
6 6 6 6 6 6 6
5 5 5 5
4 4

T1 T2 T3 T4 T5 T6 T7 T8

Rep 1 Rep 2 Rep 3

Graphical Representation On Hardening

47
48
2. Result :

1. Effect of cytokinin on shoot induction :

Micropropagation of R.Serpentina was successfully carried out. MS medium with standard

concentration of stock solution was used for micropropagation. Nodes are used as explant.
standardization of different concentration of growth regulators was carried out among which
5.5 mg/l of KIN. Shows best result for shoot induction.

4.2.2 Effect of auxin on root formation :

4.3 Discussion :

The most frequently used micro propagation method for commercial production utilizes
enhanced axillary shoot proliferation cultured medium.

Micropropagation process supports in achieving mass production of healthy plants in low or

minimum space requirement.

On the other hand, micro propagation process comprises of high labour costs, danger of
variation and loss by contamination.

Multiplication rates are tend to be slow at first, but later, if the cultural conditions are
satisfactory (temp: 25 ± 2; humidity 55 ± 5% ; photoperiod – light/dark 16hrs/8 hrs), a rapid
multiplication can be achieved.

Generally, the plant taken for micro propagation are the ones which are high in demand in
terms of number , are the demand round the year and have to be multiplied irrespective of
season , are amongst the endangered ones, have to be preserved for their special qualities.

49
CONCLUSION

50
CHAPTER NO.5

CONCLUSION
Plant tissue culture represents most promising areas of application and giving
an outlook into the future . While tissue – culture offers tremendous benefits, it could be a
bane if undertaken improperly. It could be the fastest means of spreading disease in plant.
If the source of material to be tissue cultured is disease – infected , the resulting tissue-
cultured plantlets will carry the disease . Since tissue culture could produce thousands if not
millions of plantlets tremendously fast, the disease could spread super fast. Thus conducting
tissue culture we must be sure that the parent is free from infection or disease .

51
REFERENCE

52
 REFERENCE

 Akter S, Banu TA, Habib A, Afrin S, Khatun A, Khan S and Islam S (2013), In vitro
clonal multiplication of Aegle marmelos (L.) Corr. through cotyledonary node
culture, Bangladesh J. Sci. Ind. Res. 48(1): 13-18. DOI: 10.3329/bjsir.v48i1.15408
 Alatar AA, Faisal M, Hegazy AK and Hend AA(2012), High frequency shoots
regeneration and plant establishment of Rauvolfia serpentina: An endangered
medicinal plant, J. Med. Plants Res. 6(17): 3324-3329.
 Anitha S and Kumari BDR (2006), Stimulation of reserpine biosynthesis in the callus
of Rauvolfia tetraphyla L. by precursor feeding, Afr. J. Biotechnol. 5: 659-661.
 Baksha R, Jahan M ,Khatun R and Munshi JL(2007), In vitro Rapid Clonal
Propagation of Rauvolfia serpentina (Linn.) Benth, Bangladesh J. Sci. Ind. Res.
42(1): 37-44. DOI: 10.3329/bjsir.v42i1.353
 Bhatara VS, Sharma JN, Gupta S and Gupta YK (1997), Images in psychiatry
Rauvolfia serpentina: The first antipsychotic, Am. J. Psychiatry. 154:
894-894. DOI: 10.1176/ajp.154.7.894
 Bhatt R, Arif M, Gaur A K and Rao PB (2008), Rauwolfia serpentina: Protocol
optimization for in vitro propagation. Afr. J. Biotechnol. 7(23): 4265-4268.
 Gupta R (1988), Genetic resources of medicinal plant. Indian J. Plant Genet. Res. 1:
98-102. DOI: 10.3329/ bjsir.v47i3.13058
 Islam S, Zahan M, Akter S, Banu TA, Habib A, Khan S and Jahana MAA (2010),
Mass propagation of Feronia limonia L. through tissue culture. Bangladesh J. Sci.
Ind. Res. 45 (1): 75-78. DOI: 10.1021/np058007n 9
 Itoh A, Kumashiro T, Yamaguchi M, Nagakura N, Mizushina Y, Nishi Tand
Tanahashi T (2005), Indole alkaloids and other constituents of Rauvolfia
serpentina, J. Nat. Prod. 68: 848-852.
 Jaheduzzaman , Habib A, Banu TA, Rahman RB, Akter S, Banu TA, Khan S and
Islam S (2012), In-vitro Regeneration of an Important Medicinal Plant Centella
asiatica (L.) Urban, Bangladesh J. Sci. Ind. Res. 47(3): 269-272. DOI: 10.1055/s-
2006-957903
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  Jain V, Singh D, Swarnalata S and Saraf S (2003), In-vitro micro propagation
of Rauvolfia serpentina through multiple shoot generation, Anc Sci Life.
XXIII(1): 44-49. DOI: 10.3923/ijb.2011.249.254
 Janarthanam B and Sumathi E (2011), High Frequency Shoot Regeneration from
Inter nodal Explants of Santalum album, L. Int. J. Bot. 7: 249-254.
 Kirillova NV, Smirnova MG, Komov VP (2001), Sequential isolation of
superoxide dismutase and ajmaline from tissue culture of Rauvolfia
serpentina Benth, Appl Biochem Microbiol 37: 181-185. DOI:
10.3923/ajps.2010.285.298
 Murashige T and Skoog F (1962), A revised medium for rapid growth and
bioassays with tobacco tissue cultures, Physiol. Plant. 15: 443-477. DOI:
10.1111/j.1399- 3054.1962.tb08052.x
 Nadeem M, Palni LMS, Purohit AN, Pandey H and Nandi SK (2000), Propagation
and conservation of Podophyllum hexamdrum Royle: an important medicinal
herb, Biol. Conserv. 92: 121–129. DOI: 10.1016/ S0006-3207(99)00059-2
 Nathan, Kline NS (1954), Use of Rauvolfia serpentina Benth in
neuropsychiatric conditions. Ann N Y Acad. Sci. 59: 107–32. DOI: :
10.1111 /j.1749- 6632. 1954.tb45922.x
 Pandiyan P and Selvaraj T (2012), In vitro multiplication of Bacopa monnieri
(L.) Pennell from shoot tip and nodal explants, Int J Environ Agric Res. 8(3):
1099-1108.
 Panwar GS, Attitalla IH and Guru SK (2011), An Efficient in vitro Clonal
Propagation and Estimation of Reserpine Content in Different Plant Parts of
Rauwolfia serpentina L., Am.-Eurasian J. Sci. Res. 6(4): 217-222.
 Rani A, Kumar M and Kumar S (2014), Effect of growth regulators
on micropropagation of Rauvolfia serpentina (L.) Benth, Journal of Applied
and Natural Science 6(2): 507-511. DOI: 10.31018/jans.v6i2.490

Rashmi R and Trivedi MP (2016), Rapid in vitro regeneration of an endangered

medicinal plant sarpagandha (Rauvolfia serpentina L.), Eur J Pharm Med Res. 3(5): 276-284.
DOI: 10.4236/ajps.2012.34053

54
55
 GLOSSARY

 Adventitious: A rising from other than the usual place e.g. shoots- bud formed from
callus tissue.
 Agar: A polysaccharide bearing gel forming capacity obtained mainly from certain
species of red algae.
 Amino acid: An organic compound containing an amino (-NH2) and acid growth (-
COOH). Amino acids are building blocks (subunits) that are polymerase to form
proteins.
 Ammonium Nitrate: Influences regeneration potential of shoot tips.
 Apical Meristem: Meristematic cell at the apex of shoot or root.
 Aseptic culture: Culture of various explants under aseptic condition.
 Aseptic: With the reference to in- vitro producers it main free from all micro-organism.
 Auxins: A group of plant growth regulators which are involved in call elongation.
E.g. Indole acetic acid.
 Bavistin: Avoid fungal contamination.
 Boric Acid: Higher rate in cell wall synthesis.
 Bud: A largely meristematic under developed shoot usually protected by scale leaves.
 Calcium Chloride: Formation of cell walls, root and shoot development.
 Callus Culture: A mass unorganized cells, capable of growth on a nutrient medium.
 Callus: A mass of unorganized cell resulting either as a consequence of wounding in
plants or in tissue culture.
 Cell: The smallest leaving unit of biological structure and function capable of self
reproduction.
 Clone: A group of identical organism, cells or molecules descended from a single
ancestral organism, cells or molecules.
 Cobalt Chloride: The rate of callus proliferation is higher and greatest formation of
chlorophylls.
 Copper Sulphate: Activates some enzymes in plants in lignin synthesis and assists in
plant metabolism.
 Cultivar: A variety obtained by selective breeding and propagated as pure line.

56
 Cytokines: A class of plant growth regulators chemically and functionally related to
the natural plant hormone, zeatin.
 EDTA: (Ethylene diamine tetracetic acid) A Chelating agent, used to check iron from
precipitating in solution.
 Ethanol: It helps in removal of disinfectants.
 Explants: An excised portion of plant, organ or tissue used for initiation of an invitro
culture.
 Ferrous Sulphate: It plays vital role in plant metabolism. Glycine: Serve as the
source
of amino acids.
 Hormone: A natural chemical that exerts strong controlling effect on growth
development and metabolism at very low concentration and usually at sites other than
that of its synthesis.
 IAA: It have been used to induced embryogenesis.
 Internodes: The portion of a stem between two successive nodes.
 In-vitro: Literally means “ in-glass” , e.g: A tissue tube now applied to growth of
tissue in any type of culture container.
 Kinetin: Ability to induced cell division and inducing formation of callus to
regenerated shoot tissue.
 Macronutrients: An essential elements required by plant in relatively large quantity.
 Magnase Sulphate: It helps increasing root cell elongation and resistance to root
pathogens.
 Magnesium Sulphate: Involve in photosynthetic and respiration system.
 Mercuric Chloride: It is phototoxic which is used to remove all trace from plant.
Meristem: Undifferentiated but mitotically active cell tissue of plants.
 Micronutrients: An essential elements required by plant in relatively small quantity.
 Micropropagation: Another term for in-vitro propagation. Myo-inositol: It important
for normal plant growth and development.
 Nicotinic Acid: Act as enzymatic co-factors in universal pathways including glycolysis
and TCA.
 Organ: A part of plant body adopted for specific function.
 PH: It is the negative logarithm of the hydrogen ion concentration of any solution or
measure for acidity or alkalinity.

57
 Photo hormone: The generic name for all classes of hormone produces by plants
especially those that elevate plant growth.
 Photoperiod: The length of light and dark period of daily illumination of a plant.
 Potassium dihydrogen Orthophosphate: It servers as source of phosphate and it is
essential component of Nucleic acid phospholipids, enzyme cofactor.
 Potassium Iodide: Increasing secretion of respiratory fluids resulting in decreased
mucus viscosity.
 Potassium Nitrate: Essential in cell division, stomatal regulation and protein synthesis.
Pyridoxines
 HCL: It is resulted from reduced cell division and elongation.
 Regeneration: The development and formation of new organs.
 Sodium EDTA: It presence develops the shoot buds and elongation.
 Sodium Molybdate: Main role are convert inorganic phosphorus into organic forms in
plants.
 Somatic embryogenesis: Formation of embryos from asexual cells.
 Sucrose: Disaccharide CHO, made up of a molecule of glucose linked to a molecule of
fructose.
 Thiamine HCL: It important for primary metabolism in plants and enzyme involved
in the synthesis of amino acid.
 Tissue: A group of cells that perform collective function
 Vitamins: Naturally occurring organic substances necessary in small amount for the
normal metabolism of plants.
 Zinc Sulphate: It increasing growth of tissue was stimulated by auxin be required for
a variety of metabolic processes in plants.

58
 ANNEXURE

CHEMICAL COMPOSITION OF MS MEDIA

●
Sr.No ●
Chemical Name ●
Quantity

●
1 ●
Macronutrients Stock A ( 200X) ●
50 ml/l

●
2 ●
Calcium Stock B (200X) ●
50 ml/l

●
3 ●
Micronutrients C (200X) ●
5 ml/l

●
4 ●
Vitamin Stock D (200X) ●
5ml/l

●
5 ●
Iron Stock E (200X) ●
5ml/l

●
6 ●
Myoinositol ●
0.1 g/l

●
7 ●
Sucrose ●
30 g/l

●
8 ●
Agar ●
8 g/l

●
9 ●
Growth Regulator (BAP) ●
0.025 g/l

59