in vitro studies on growth and morphogenesis of some ... · out of the 17.500 flowering species...
TRANSCRIPT
In vitro studies on growth and morphogenesis of some potential medicinal plants through
tissue culture
DISSERTATION
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF
iRas^ter of ^fjilosiopbp 31n
^ iBotanp ^
L By
SHEIKH ALTAF HUSSAIN
/ • #
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY
ALIGARH -202002 (INDIA) 2014
2 4 NOV 201i
DS4373
m
f^>^a(pra6le &ra4ferUA
-ccHTK^^O^
i
<Prof. (<Dr.)!MofiamanufAnts PhD(Luck.), FISPM, FISG, FBS
Former Chairman & Program Coordinator DST-FISTII (2011-16) UGC-DRSI (2009-14) OBT-HRD (2009-14 DST-FISTI (2006-10) Visiting Professor, KSU, Riyadh P. Maheshwari Medal Awardee
Plant Biotechnlogy Lab., Department of Botany Aljgarh Muslim University Aligarh-202002, U.P. (India) E-mail: [email protected]
Cell: +91-9837305566
Dated: . (Set.6A,2oA^
Certificate
Certified that the dissertation entitled "In vitro studies on growth and morphogenesis
of some potential medicinal plants through tissue culture" submitted to the Aligarh
Muslim University, Aligarh, in fulfillment of the requirement for the award of Master of
Philosophy, embodies the original research work carried out by Mr. Sheikh Altaf Hussain
under my guidance and supervision. The work has not been submitted elsewhere in part
or full for the award of any other degree of this or any other university.
(ProfessorJAoham^^FAms) ^ -"^^^--"^ap^fvfsor ^ X ) 4
Tel. Off: 0571-27020]6: 2700920, 21, 22 (Ext. 3308). Home: 07. Rainbow Roofs-I. Anoopshahar Road. Aligarh -202 122. UP (India)
CONTENTS
Acknowledgements
Abbreviations
CHAPTER 1: 1.1. 1.2. 1.3. 1.3.1 1.3.2 1.3.3 1.3.4 1.4. 1.4.1 1.4.2 1.4.3 1.5
CHAPTER 2:
2.1
2.2
2.3
2.4
2.5
2.6
2.7
INTRODUCTION 1-10 Micropropagation Micropropagation ofR. tetraphylla and R.serpentina Classification of ^. tetraphylla Distribution Botanical description Characteristics and constituents Medicinal importance Classification of/?, serpentina Distribution Botanical description Medicinal importance Objectives
REVIEW OF LITERATURE Historical background Micropropagation Explant type Plant growth regulators Subculturing Rooting Hardening and Acclimatization
11-18
CHAPTER 3: 3.1 3.2 3.3 3.4 3.5 3.6
3.7
3.8
3.9 3.10 3.11 3.12
MATERIALS AND METHODS 19-25 Plant material and explant sources Surface sterilization of explants Establishment and Preparation of aseptic explants Culture media Preparation of stock solution Growth regulator and carbon source Adjustment of pH and gelling of medium Sterilization of glasswares and instruments Sterilization of laminar airflow hood Inoculation Incubation Subculturing
3.13 3.14 3.15 3.16
Rooting Hardening and Acclimatization Chemical and glasswares Statistical analysis
CHAPTER 4: 4.1 4.1.1. 4.1.2. 4.1.3. 4.1.4. 4.1.5. 4.1.6. 4.1.7. 4.2 4.2.1. 4.2.2. 4.2.3.
RESULTS (with Tables & Figures) Direct shoot regeneration of/?, tetraphylla Effect of Cytokinins Combined effect of Cytokinins and auxin Effect of thidiazuron (TDZ) Combined effect of TDZ and BA Effect of metatopolin Rooting of regenerated shoots Acclimatization Direct shoot regeneration in R. serpentina Combined effect of cytokinin and auxin Rooting Acclimatization
26-36
CHAPTERS: 5.1. 5.2. 5.3. 5.4 5.5 CHAPTER 6:
DISCUSSION 37-42 Direct shoot regeneration in R. tetraphylla In vitro Rooting in regenerated shoots of R. tetraphylla Direct shoot regeneration in R. serpentina. In vitro Rooting in regenerated shoots of R. serpentina Hardening and Acclimatization SUMMARY AND CONCLUSIONS 43-44
REFERENCES 45-60
(praise 6e to JiClali, tfie cHerisHer ancf tHe sustainer of the worCd, 'Who Bestowed on
me the divine guidance, enough courage and patience to successfuCCy compete thu
worH^
It 6rings me coCossaC pCeasure while placing on record my sincere gratitude and
reverence to my esteemed supervisor (prof. ((Dr.) 'Mohammad J/lnis for his unparaCleC
and sincere guidance, ^en interest, incessant encouragement, strong motivation,
constructive criticism and idea oriented discussion, which has enabled me to
accomplish this wor^
It is my proud privilege to express my sense of than^ulness to (prof. Tiroz
Mohammad, Chairman (Department of (Botany, JUMV for providing necessary
infrastructure and technical facilities during my wor^
I deem it a privilege to ey^ress my sincere gratitude to (Dr. Jlnwar Shazad(Jlssistant
(professor) and (Dr. aseem Jlhmad ('Young scientist 0)31) for all time support,
encouragement, motivation and suggestions all through my wor^
I am highly appreciative of the support, encouragement and fruitful suggestions
expended all through the course 6y my seniors and laB-mates especially (Dr. J^n({ita
varsheny (Young scientist (DST), r. S^igar Tatima, Ms. jAfshan Jiaaz, Ms. (Rjuphi
'Xaz, Saad (Bin Javid, Md (Rafique Jlhmed, Ms. Jifsheen Shaid, J^aushad jAlam,
Jlness Jlhmad and Ms. Jltjumund Shaeen. JL to^n of appreciation will always
remain for (Dr. JVaseem JLhmadfor his support cooperation in creating a congenial,
amicadle and friendly atmosphere in the la6.
M this inexplicahk moment of joy, I deem it a privilege to thanl^ all my friends
especially Manzoor (Raiees %han, Jiddil(Rashid, Imran Mustafa Mali^ Jl6. Latif
"Wani, Sheilih Mohammad jAsif Kjianday Tanveer, Mir (Rayees, (Pervaiz J4hmad,
SheiUJi (Rameez Jlli and(Bilaljlhmadfor their incessant motivation, encouragement
and support throughout the wor^ I extend my special than^ to (Dr Manzoor
J^hmadfor his sincere help, advice and support during the wor^
I feeC -pCeasure to pay su6Cime oSeisance ancCgratitude to my reverecf ancC Bethvecf
parents, ivdose e^cjuisite Benedictions, unconditionaC Cove and affection, unfCincHing
care and support, prayers and 6(essings right from tHe cradCe ignited my confidence
to exceC academicaCCy andfufiCC my wisfies. (Dedicating this wor^ to my deCoved
parents, I apohgize to them for Searing with aCC the faiCures in commitment on my
part, arising out of Susy worli^schedufe during my studies. 94.y heartfeCt devotion is
due to my sisters who extended uCtimate cooperation and every possiSCe support aCC
through my studies. I may 6e ungratefuC without than^ng aCCmy relatives for their
affection, support and encouragement throughout my studies.
Last 5ut not the [east, I wish to offer my than^ to aCCthose names which couhdnot
6e incCudedhut wiCCaCways hefondCy rememhered.
(Sheikh ACtaf^Hussain)
11
ABBREVIATIONS
2-iP 2-isopentyl adenine
2, 4-D 2, 4-dichlorophenyoxy acetic acid
BA 6-benzyladenine
CH Casein hydrolysate
cm Centimeter
DDW Double distilled water
EDTA Ethylene diamine tetra acetic acid
g Gram
GA3 Gibberellic acid
CM Coconut milk
h Hour
HCl Hydrochloric acid
lAA Indole-3-acetic acid
IBA Indole-3-butyric acid
Kin Kinetin
mT metatopolin
|iM Micromolar
mg Milligram
mg 1 • Milligram per liter
min Minute
ml Milliliter
mM Millimolar
MS Murashige and Skoog's medium
N Normality
NAA a-napthalene acetic acid
NaOH Sodium hydroxide
PABA Para- amino benzoic acid
PGRs Plant growth regulators
TDZ Thidiazuron
UV Ultraviolet
v/v Volume by volume
w/v Weight by volume
% Percentage
C Degree celsius
^/udftet^ - /
1^
Introuction
Introduction
Biodiversity is the store house of species richness and serves as the cushion against
the potentially dangerous environmental changes and economic reforms (Yadav et ui.
2012). It encompasses ail the biological entities that occurs due to their dynamic
interactions among different levels of integration within the living world, in an
ecosystem and in their habitat. Biodiversity of plants is collectively known as plant
genetic resources, and serves as the major biological basis of the world food securit>
and in all means they support the livelihoods of every life form on earth (Alexander ei
ai. 2011). India with wide spectrum of phytogeographic and agro-climatic zones is
blessed with rich diversity of plants. Plants form the key element in maintaining the
biodiversity on this planet Earth. Plants are endowed with the unique capacity to
convert solar energy into chemical energy and synthesize thousands of chemical
substances which are very important to our lives.
India has very rich plant diversity and is one of the 12 mega biodiversity hot spot
centers of the world with 4" rank in Asia. India represents about 12% of the world s
recorded flora with 49,000 plant species, among them 20,000 species belongs to
higher plants, one third of it being endemic and 1000 species being medicinally
important (Krishnan et al., 2011; Singh et ai, 2003). At least 40% of the world
economy and 80% of the needs of the poor people are derived from biological
resources, in addition, the richer the diversity of life, greater the opportunity for
medicinal discoveries, economic development and adaptive responses to such new
challenges as climate change.
Since the beginning of human civilization, medicinal plants have been used virtual!)
in all social cultures for its therapeutic value. Nature has been a source of medicinal
assets from prehistoric times and an impressive number of modem and valuable drugs
have been isolated from these natural sources. Many of these isolations were based on
the uses of agents in herbal medicine. The plant-based traditional medicine system
continues to play an essential role in health care, with about 80% of the world s
inhabitants relying mainly on traditional, largely on herbal medicine for their primar)
health care. The ancient texts like Rig veda, and Atharva Veda mention the use of
several plants as medicine. The books on ayurvedic medicine such as Charaka
Samhita and Susruta Samhita refer to the use of more than 700 herbs (Jain, 1968).
Introtiction
According to WHO, 80% of world population and 65% of Indian rural population
uses traditional form of medicine to meet their primary health care needs (Jain and
Singh. 2010), because they are easily available, non narcotic and also having no side
effects.
India is called the botanical garden of the world because it houses the largest number
of the medicinal plants and acts as the gold mine, serves as the well recorded and
most practiced knowledge of herbal medicine. India officially recognizes about 3000
plants for their medicinal value. It is generally estimated that over 6000 plants in India
are in use. in traditional, folk and herbal medicine representing about 75% of the
medicinal needs of the third world. Medicinal herbs are moving from fringe to
mainstream use, with a greater seeking remedies and health approaches free from side
effects as caused by synthetic chemicals.
Out of the 17.500 flowering species found in India, 1600 are used in traditional
medicine system, and more than 1000 plants are used in indigenous system of
medicine like Ayurveda, Sidha and Unani. thus gaining wide currency and
acceptability for the purpose of financial gain. Market demand has lead to an
increased pressure on these natural resources. The demand of the majority of the
people in developing countries for medicinal plants have been met by indiscriminate
harvesting of spontaneous flora include those in forests, over exploitation of these
wild sources had led many species being extinct, threatened, or endangered.
The big concern is that these medicinal plants are collected from their natural habitat
and involves the destructive harvesting when they are collected. Sometimes leafs,
flowers, seeds, stems, bark, roots, wood or whole plant are used (Anis et ai, 2009).
Due to their multipurpose use they are highly exploited for their market value. The
increase in demand and ruthless collection of these plants are the key factors for their
extinction. Other challenging factors for their existence are population explosion,
rapid industrialization, urbanization, agricultural encroachment; forest degradation,
unscientific harvesting etc. have lead to extinction of more than 150 plants.
It is therefore necessary that systematic cultivation, maintance and assignment for
future use of these medicinal plants be introduced in order to conserve biodiversity
and politic threatened species. Conservation of these medicinal plants is possible
either through in situ or ex situ or by both. The major problems and challenges in in
Introuction
situ conservation of these plants include slow regeneration rates, over exploitation and
destruction of their natural habitat. Plant improvement for agriculture, forestry and
ecological conservation by traditional methods has been a slow process, limited b>
slow growth and life cycles.
Plant biotechnology has provided a large number of tools and techniques which arc
more precise and highly reproducible for rapid multiplication and in generating no\ el
genetic variability, enhancement and transformation. Biotechnology has
revolutionized the fields like basic biology, agriculture, horticulture and forestry.
Biotechnology revolution is the best option for solving the problems of food securitv.
poverty reduction, ecological and environmental conservation in developing countries
(Serageldin 6 /a/., 1999).
Plant tissue culture is one of the important techniques of biotechnology. It involves
the in vitro aseptic culture of cells, tissues, organs and their components in an
artificially prepared medium under defined physical and chemical conditions. It is an
important tool in both basic and applied studies as well as in commercial application.
It was the availability of these techniques that led to the application of tissue culture
to five broad areas i.e. cell behavior, plant modification and improvement, pathogen
free plants and germplasm storage, clonal propagation and product formation. Plant
tissue culture involves the following types of aseptic cultures.
Embryo culture
Cell culture ••-
Callus culture
Seed culture
Plant Tissue Culture
Bud culture
Meristem culture
•Protoplast culture
Organ culture.
Types of cultures
Introuction
Tissue culture propagation also known as micropropagation is a way of dividing cells
and reproducing clones of the mother plant. It also helps to study the mechanism by
which cell differentiates, thus providing an experimental approach to link the genotype
with the phenotype. It is clear that without tissue culture, plant biotechnology is
immature and totally incomplete because it cements together its various aspects; to a
large extent revolution in tissue culture has occurred because of the needs of the new
biotechnology (Varshney and Anis, 2014).
The main advantage of the clonal propagation is the production of large number of
plants that are clones to each other. Desirable stock plants can be chosen and
thousands of high quality identical clones can be produced. Also helps in germplasm
conservation, providing viable methods of regenerating genetically modified cells and
to offer customer - tailored improved material. Tissue culture often promotes genetic
disturbances which resulted in somaclonal variation, thus greatly extending the range
of useful variation available to the breeder (Rout et al, 2000, Kukreja and Dawan
2000 and Sevon and Oksman caldentry 2002).
1.1 Micropropagation
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 vilro 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
varieties 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
medicinaly important plants ranging from a herb to a big tree, both in angiosperms
and gymnosperms. through enhanced axillary bud formation, organogenesis or
somaticembryogenesis. 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
Introuction
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, Ravuolfia tetraphyllu and
R. serpentina has been selected for their large scale propagation and conservation
purposes through tissue culture techniques.
1.2 Micropropagation of Rauvolfia tetraphylla and Rauvolfia serpentina
Earlier reports on micropropagation of R. tetraphylla and R. serpentina using different
explants have been depicted in Table 1 and 2.
Table. 1. Current status oiRauvolfia tetraphylla in tissue culture:
Explant
Nodal
Nodal
Nodal
Nodal
Shoot and nodal
explants
Leaf
Nodal
Nodal
Nodal
Nodal
Response
Multiple shoot, plant
regeneration
Multiple shoot, plant
regeneration
Multiple shoot. plant
regeneration
Multiple shoot and plant
regeneration
in vitro flowering
Effect of NaCI on Callus
induction and its growth
Syn. seed. plant
regeneration
Clonal propagation
Clonal multiplication and evaluation of genetic stability by using DNA markers
Syn. seed and evaluation of genetic fidelity by using RAPD/ISSR Markers
PGR used
BAP and Kin
BAP, Kin and
NAA
BA and NAA
TDZ
BAP and GA3
lAA, NAA, IBA
and 2,4-D
BA, NAA and
IBA
KIN, BA and
NAA
BA and NAA/ RAPD-DNA marker
BA and NAA
References
Patil and Janyanti
(1997)
Sarma et al.,
(1999)
Faisal and Anis
(2002)
Faisal et at.,
(2005b)
Anitha and
Raj itha (2006)
Anitha and Raj itha
(2006)
Faisal et al..
(2006a)
Harisaranraj.
(2009)
Faisal et al.. (2012)
Altar et al, (2012)
Introiiction
Table. 2. Current status of Rauvol/ia serpentina in tissue culture.
Explant
Leaf and stem segments
Auxiliary meristem Nodal
Shoot tips and Nodal segments Nodal
Nodal
Shoot tips, leaves and Nodal segments Nodal
Leaf
Leaf
Leaf and stem
Leaf and stem
Shoot tips and Nodal segments Nodal
Nodal
Nodal
Shoot tip
Mature and immature seeds
Response
Induction and growth of callus
Clonal propagation
Multiple shoot regeneration Multiple shoot regeneration Callus morphogenesis and shoot proliferation Callus and multiple shoot formation
Callus induction and direct shoot regeneration
Callus and multiple shoot regeneration
Callus culture and multiple shoot formation Direct root regeneration
Callus induction and multiple shoot regeneration Callus induction and multiple shoot regeneration Multiple shoot regeneration Multiple shoot regeneration Multiple shoot regeneration Multiple shoot regeneration Multiple shoot regeneration Induction of callus and hairy root formation
PGR used
2,4-D, NAA, IBA, Kin, CM and CH BA and NAA
BA and NAA
BAP and IBA
NAA and BAP 2,4-D, NAA, lAA, BAP and Kin 2,4-D, BAP and IBA
NAA and BAP BAP and lAA
PABA, NAA and IBA 2,4-D, BAP and NAA
2,4-D, BAP and NAA
BAP and NAA BAP, Kin and GA3 BA, Kin and 2ip BAP and NAA BA and NAA
2,4-D and BAP
Refrences
Parveen and Ilahi (1978)
Roja and Heble (1996) Sarker et ah, (1996) Jain et al., (2003) Panday et al., (2007)
Bhandra et al, (2008)
Bhatt et al, (2008)
Pant et al., (2008) Singh et al., (2009) Pandey et al., (2010) Pan war et al., (2011)
Mallick et al, (2012)
Ranjusha et al., (2012) Singh and Dash (2012) Alatar et al, (2012) Pan et al., (2013) Susila et al., (2013) Shetty et al, (2014)
Introiiction
1.3 Rauvolfia tetraphylla L.
Classification
Kingdom : Plantae
Division : Angiosperms
Class : Rudicots
Series: Asterids
Order: Gentianales
Family: Apocynaceae
Genus: Rauvolfia
Species : tetraphylla
Binomial Name: Rauvolfia tetraphylla
Synonyms: Rauvolfia canescens L., R. heterophylla Roem & Schult., R. hirsuta Taeq., R.latifolia var.minor Mull.Arg., R. odontophora van Heurck & Mull. Arg.. R. suhpuhescens L, R. tomentosa Jacq.
Common Names:
English : Devil pepper. Be-still, Milk bush,etc.
Hindi : Barachadrika.
1.3.1. Distribution
It is native to Tropical America and also distributed in Tropical Asia, and Africa. In
India it is found as an escape from cultivation in various states like Uttar Pradesh.
Bihar. Orissa, Madhya Pradesh, Andhra Pradesh, West Bengal and Kerala
(Anonymous, 2003). It is abundantly found in moist and warm regions of West
Bengal, particularly in 24 Parganas and Howrah and Kerala (Jyothi et al., 2012). It is
also grown as ornamental plant in house gardens, despite of the fact that it is
medicinally very important.
1.3.2. Botanical description
It is an evergreen woody, perennial shrub, growing upto the height of 1.5 meter.
Leaves are found in whorls, with four unequal leaves at each node and hence the name
Rauvolfia tetraphylla. Leaves are dark green above and pale green below. Roots are
very bitter and odourless. Bark is pale brown, corky, irregular and having longitudinal
fissures. Flowers are creamy white and fruits are drupes (Anonymous, 2003).
Introiiction
1.3.3 Characteristics and constituents
It is rich source of phytochemical alkaloids and about 30 indole alkaloids have been
reported from it and among them, reserpine is the major and most potent alkaloid
which is pharmacologically most important. The other main alkaloids present in this
plant are ajmaline, alistonine, canescine, corynathene, desperdine, isoreserpiline,
isoreserpine. serpagine, serpentine, tetraphyllicine, yohimbine and pseudoyohimbine
(Anonymous. 2003. Anitha and Rajitha 2004, Jyothi et ai, 2012).
1.3.4. Medicinal importance
In India, it is used as a medicine since from the prehistoric times to cure and relief
the diseases like central nervous system disorders. Reserpine is the potent alkaloid
and pharmacologically most important that depresses the central nervous system;
produces sedation and lowers blood pressure. The root extract of this plant is used
to cure the diseases like diarrhea, dysentery and other intestinal infections, also used
as anthelmithic (for the expulsion of intestinal worms). The plant is known in folk
medicine as a treatment of snake poisoning and for external application for skin
aliments. The roots of this plant are also used to stimulate uterine contraction and
are recommended in difficult child birth cases (Faisal et al., 2006)
Rauvolfia serpentina (L).
1.4, Classification
Kingdom: Plantae
Division: Angiosperms
Class: Rudicots
Series: Asterids
Order: Gentianales
Family: Apocynaceae
Genus: Rauvolfia
Species: serpentina
Binomial Name: Rauvolfia serpentina L.
Synonyms: Rauvolfia serpentina (L.) Ex Kurz., Rauvolfia trifoliata
(Gaertn.) Baill..Iava devil pepper (Engl.). Ophioxylon serpentimm L.
Rauwolfia (Engl.)
8
Introuction
Common names:
English : Serpentine wood, Snakeroot.
Hindi : Sarpagandha, Chota cliand etc.
1.4.1. Distribution
It is found in Bangladesh, Bhutan, China, Indonesia, India, Malayasia, Mynamar,
Nepal. Pakistan, Srilanka and Vietnam. In India it is reported in the Himalayas,
Assam, Java, and the Malaya peninsula.
1.4.2. Botanical description
It is an evergreen small, woody, perennial and erect shrub grows upto the height of
60cm. Roots are tuberous with pale brown cork. Leaves are in whorls of three, elliptic
to lanceolate or obovate, bright green above and pale green below. Petioles are long.
Flowers are white, clustered in axillary cymes, with slender corolla tube and ^
spreading lobes. Fruit is a small drupe (Anonymous, 2003).
1.4.3. Medicinal importance
Rauvolfia serpentina (L.) Benth, is an important medicinal shrub of faniil\
Apocynaceae. Its roots contain about 50 indole alkaloids, including the
therapeutically important reserpine, ajmalicine, rescinnamine and yohimbine. Phc
herbal plant is used as medicine for high blood pressure, insomnia, anxiety and other
disorders of the central nervous system and epilepsy (Ghani 1998). This species has
been listed as an endangered in the red-data book because of its fast disappearance
and indiscriminate collection from the wild and depletion from the natural habitat due
to poor seed germination.
Micropropagation is specifically used for the species whose clonal propagation is
needed. In the present study attempt has been made to propagate the plant by multiple
shoot regeneration and ultimately production of complete plantlets using different
cvtokinins and auxins alone or in combination of different ratio.
Introuction
1.5. Objectives
The present experimental work was undertaken with an aim of following objectives:
1. To establish and proliferate axenic cultures from juvenile and mature nodal
explants.
2. To formulate culture conditions for regeneration, multiplication in somatic
tissues and regenerant differentiation.
3. To evaluate the optimal medium for direct regeneration.
4. To optimize the media for in vitro rooting.
5. To standardize the hardening and acclimatization procedure for in vitro raised plants
^Rfvieiv of Literature
REVIEW OF LITERATURE
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
(totipotentcy). 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 Eicchornia crassipes on Knop's salt solution with sucrose and observed ob\ ious
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 endeavor.
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 root and shoot tips in vitro. White
(1934) had established successfully indefinite culture of tomato root fips, using
1(fview of Literature
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 coworkers 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 cytokinins.
Skoog and Miller (1957) putforth 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 composifion 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 coworkers 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 superficial cell layers have been used
12
^Rfview of Literature
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) Inifiation of asepfic culture - from explants
ii) Shoot multiplication of the explants
iii) Rooting of the in vitro regenerated shoots.
iv) Transplantation - Transfer of the acclimafized plants to green house or field
conditions.
13
1(fview of Literature
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 muUiplication gained momentum in the 1970s.
Furthermore, in vitro growth and development of a plant is determined by a number
of complex factors.
i) The genetic makeup of the plant.
ii) Nutrients; water, macro and micro elements and sugars.
iii) Physical growth factors, light, temperature, pH, O2 and CO2 concentrations.
iv) 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
tetraphylla (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, cytokinins and their different
14
([(fvie-w of Literature
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 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).
Cytokinins 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). Cytokinins 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 cytokinins 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
cytokinins 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 tetraphyllu (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
tetraphyllu (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 BA. SimilarU nodal
segment of various leguminous tree species showed good axillary bud proliferation on
MS medium supplemented with BA including Albizia chinensis ( Sinha et ai. 2000).
Pterocarpous marsupium (Chand and Singh 2004) and Albizia lehbek (Per\een et ai.
2012). Maximum shoot multiplication in Terminalia bellerica (Metha et ai. 2012).
15
^Rfview 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 Cardiospermum halicacabum (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 MS
medium supplemented with TDZ. A good effect of TDZ was investigated on in vitro
shoot proliferation from nodal segments of Rauvolfia tetraphylla (Faisal et al, 2005).
Metatopolin (niT), is used in the plant tissue culture either alone or in combination
with the BA. 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 arhorescens, (Amoo et al. 2012), studied the evalvated roles of
different aromatic cytokinins types (mT, mTR, MemT and BAR) on direct
organogenesis in vitro and anti oxidant 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 cytokinins in combination played a significant role
in the multiplication of many plant species. Among the different tested combinations
of different cytokinins (BA and Kin) with auxins (NAA, lAA and IBA), BA 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
<l(fview of Literature
and for better uptake of nutrients. Subculturing has a significant effect on shool
proliferation and multiplication in many woody plants and is mainly done after 3 to 4
weeks.
In Picrorhiza kurro (Upadhyay et al., 1989) shoot number increased as the number of
subcultures were increased. 2200 plantlets were obtained from single shoot explants
in Thymus vulgaris (Bajaj et al., 1998), grown in vitro for four months followed b>
four subculturings. 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 BA (12.0 \xM) and NAA (3.0 \xM). Mass multiplication of
shoots was reported in Cunila galiodes (Fracaro and Echeverrigaray 2001), without
any decline in shoot number up to eight months and were subcultured after every four
weeks. Significant shoot improvement was observed in Leptadenia reticulata (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.
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 Anemopaegma arvense (Pereira
et al.. 2003), Pongamiapinnata (Sugla et al., 2007), Ocimum hasilicum (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).
Ruta graveolens (Ahmad et al, 2012). Also, good results for root induction were
reported on half strength MS medium in Albizia lebheck (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.
17
^ffview 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 tetraphylla (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
Aclimatization of the obtained micropropagated 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 difficuh 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 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 transplantational 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. Acclimatizafion period of micropropagated
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 condhions.
Materials and Methods
Materials and Methods
3.1. Plant material and explants sources:
Nodal explants were collected in the month of June 2013 and April 2014 from a 5
year old Rauvolfia tetraphylla and 1 year old R. serpentina respectively, from the
field grown plants, maintained in the Botanical Garden of the Aligarh Muslim
University, Aligarh.
3.2. Surface sterilization of explants
The young nodal explants of size 1.5cm were collected in glass beaker, washed
carefully under the running tap water for 30 minutes, treated with 5% (v/v) savlon (a
liquid disinfectant) for 3 minutes, followed by soaking in 5% (v/v) labolenc
(Qualigens, Mumbai, India) for 8 min., followed by thorough washing under tap
water. The explants were surface sterilized with freshly prepared 0.1% (w/v) mercuric
chloride (HgCb) for 3 min. followed by repeated rinsing with sterilized single
distilled water 5 times till all the traces of HgCb were removed. The sterilized
explants were inoculated onto the freshly prepared culture medium for shoot bud
induction, proliferation and multiplication.
3.3. Establishment and preparation of aseptic explants
The aseptic or disinfected Nodal segment explants were inoculated on MS (Murashige
& Skoog, 1962) basal media alone or supplemented with different concentrations t)f
6-bezyladenine (BA) 1.0, 2.5, 5.0, 7.5 and 10|iM, kinetin (Kin) 1.0, 2.5, 5.0, 7.5 and
lO^M. thidiazuron (TDZ) 0.2, 0.4, 0.6, 0.8 andl.O^M and metatopolin (mT) 5.0, 7.5.
10. 12.5 and 15|J.M for shoot regeneration. The explants used were of 1.5 cm in
length.
3.4. Culture media
In vitro growth, development and morphogenesis of explants is mainly governed by the composition and modification of the culture media. In the present work MS (Murashige & Skoog, 1962) basal medium was used throughout the experimental work. The chemical composition of various constituents of the MS basal media along with their concentration used, are listed in table 2.
3.5. Preparation of stock solution
MS stock solution was prepared in a series of four stocks- stock I- Macro nutrients
(20X). stock II - Micronutrients (200X), stock III Iron salt (lOOX) and stock IV
MateriaCs amfMetfiocCs
Vitamins (lOOX) except sucrose. All the stocks were prepared separately by
dissolving the solute in required amount of double distilled water. MS III stock
solution being very photosensitive was immediately wrapped with black paper or
aluminum foil to avoid photo oxidation. To prepare one liter of medium, 50 ml of
stock 1, 5 ml of stock II, 10 ml each of stock III and IV are required (composition is
given in table 3). The stock solutions were stored in a refrigerator at A^C and were
regularly checked for any kind of visible or bacterial contamination.
For each plant growth regulator, separate stock solutions were prepared by dissolving
them in a few drops of appropriate solvent (IN NaOH for cytokinins and IN absolute
alcohol for auxins) and then required amount of DDW was added to make the overall
solution of Ijj-M concentration. In the present study, different concentrations of plant
growth regulators were prepared from the stock solutions by using the formula:
S iVrS .V.
Here
Si = Strength of stock solution
Vi= Volume of the stock solution required
S2= Strength of the desired solution
Vi = Volume of the desired solution
20
MateriaCs andMetHocfs
Table.3. Nutritional components of MS basal media
Components
Macronutrients
MgS04.7H20
KH2PO4
KNO3
NH4NO3
CaCl2.2H20
Micron utrients
H3BO3
MnS04.7H20
ZnS04.7H20
Na2Mo04.2H20
CUSO4.5H2O
C0CI2.6H2O
KI
FeS04.7H20
Na2EDTA.2H20
Organic supplements (Vitamins)
Thiamine HCl
Pyridoxine HCl
Nicotinic acid
Myo-inositol
Others
Glycine
Sucrose (g)
Amount (mg/1)
370
170
1900
1650
440
6.2
22.3
8.6
0.25
0.025
0.025
0.83
27.8
37.3
0.5
0.5
0.5
100
2.0
30
21
^Materials atufMetfiods Table. 4. Stock solutions for MS basal medium.
Constituents Amount (mg/1)
Stock solution I
Macronutrients (20X)
MgS04.7H20
KH2PO4
KNO3
NH4NO3
CaCl2.2H20
Stock solution II
Micronutrients(200X)
H3BO3
MnS04.7H20
ZnS04.7H20
Na2MoO4.2H20
CUSO4.5H2O
C0CI2.6H2O
KI
Stock solution III
Iron Salt (lOOX)
FeS04.7H20
Na2EDTA.2H20
Stock solution IV
Vitamins(lOOX)
Thiamine HCL
Pyridoxine HCl
Nicotinic acid
Myo-inositol
Glycene
7400
3400
38000
33000
8800
1240
4460
1720
50
05
05
166
2780
3730
50
50
50
10000
200
22
MateriaCs andMetHods 3.6. Growth regulators and carbon source
Depending upon the need of experimental plan, MS medium was differently
supplemented with plant growth regulators like 6-benzyladenine (BA), Kinetin (kin).
Thidiazuron (TDZ) and Metatopolin (mT) either singly or in combination with
different levels (O.I, 05, 1.0, 1.5 and 2.0) (iM of auxins a-naphthalene acetic acid
(NAA) and indole-3-butaric acid (IBA), as mentioned in the results. Sucrose (3"o)
was used as the key carbon source in all the experiments.
3.7. Adjustment of pH and gelling of medium
pH 5.8 was maintained by using IN NaOH or IN HCl with the aid of pH meter
(Toshcon Industries Pvt. Ltd. Hardwar India) of all the media before adding the
gelling agent. The medium was solidified by adding 0.8% (w/v) agar (Thermo Fisher
Scientific Qualigens Pvt Ltd., Mumbai India), dissolved in the microwave oven until
the clear gel was formed. The medium was then poured in 25 x 150 mm culture tubes
and 100 ml Erlenmeyer culture flasks (Borosil, India), each containing 20 ml and 50
ml medium respectively. The medium was also dispensed in the jam bottles with
plastic caps. The culture tubes and culture flasks were closed with the cotton plugs.
wrapped with butter paper and followed by the sterilization of medium in an
autoclave at 1.06 kgcm'' or 15 psi pressure at a temperature of 121 *C for 18 minutes.
3.8. Sterilization of glasswares and instruments
All the glassware and instruments (wrapped with the aluminum foil) and distilled
water were sterilized by autoclaving at 1.06 kgcm" for 20 minutes. The forceps,
scalpel etc made of stainless steel was re-sterilized by dipping them in 70% ethanol
followed by flame sterilization and cooling before each inoculation made.
3.9. Sterilization of laminar air flow hood
Laminar air flow hood (NSW, New Delhi) was sterilized by switching on the
ultraviolet (UV-B) lamp for 20 minutes prior to the inoculation and then wiping the
working surface area with 70% alcohol (ethanol) before the initiation of ever\
operation inside the cabinet.
3.10. Inoculation
All the inoculations were carried out under the aseptic conditions of laminar air flow
hood. Every time before starting the inoculation process, the hands were rinsed with
23
Materials and Methods 70% (v/v) ethanol. The instruments used for inoculation were resterlized time to time
by dipping them in rectified spirit followed by flaming and cooling. Expalnts were
transferred to sterilized petridishes and then inoculated into the culture vials with the
aid of sterilized forceps followed by immediate plugging infront of the spirit lamp to
avoid any kind of physical or biological contamination.
3.11. Incubation
All the cultures were labeled and placed inside the culture room at 24 ± 2°C, having
the relative humidity of 60 - 65 %, photoperiod of 16 h, photosynthetic photon flux
density (PPFD) of 20 (im/m^/sec provided by cool white fluorescent tube lights (40W,
Phillips, India).
3.12. Subculturing
For further shoot multiplication and proliferation, the inoculated explants were
transferred onto the same fresh medium after every three weeks.
3.13. Rooting
The proliferated microshoots of 4-6 cm length were transferred to the freshly prepared
MS liquid media, supplemented with the root inducing hormones NAA and IBA with
different concentrations. Individual shoots were employed for rooting by two step
culture procedure with the aid of filter paper bridge (Whatman No.l). After 5 weeks
rooted plantlets were transferred to plastic pots containing sterilized soilrite, and then
covered by polybags to retain the proper moisture for ex-vitro conditions. After 2
weeks of exposure to soilrite these plantlets were transferred to the pots containing
normal garden soil and manure in the ratio of 3:1. Data on percentage of rooting mean
number of roots and root length per shoot were recorded after 4 weeks of inoculation
i.e. before their transplantation to the sterilized soilrite.
3. 14. Hardening and acclimatization
Plantlets with well developed shoots and roots were transferred from the culture
medium with the help of forcep, washed carefully under the running tapwater and then
transferred to the plastic cups containing sterilized soilrite ( Keltech Energies Ltd.,
Banagalore) under diffused light conditions (16/8h photoperiod). Potted plantlets were
covered with the transparent polythene bags to ensure and retain high humidity inside
the polybag and were watered with half strength MS salt solution without the vitamins,
24
Materials and Methods
for 2 weeks on respective alternate days. The poly-bags were removed after 2 weeks,
in order to acclimatize the plantlets to ex-vitro conditions and after 5 weeks, the
plantlets were potted in garden soil and were maintained in the green house.
3.15. Chemicals and Glasswares
Plant growth regulators like BA, Kin. TDZ, NAA, IBA and metatopolin were
obtained from Sigma- Aldrich (St. Louis. USA). Macro, micro salts, agar, sucrose,
and mercuric chloride were purchased from Qualigens, Merck; India. Glasswares,
such as culture tubes (25 x 150mm), petridishes (17 x 100 mm), and wide mouth
Erlenmeyer flasks (100 ml and 250 ml) were from Borosil India.
3.16. Statistical analysis
In all the experiments, ten replicates per treatment were used and all the experiments
were repeated thrice. The effect of different treatments was quantified and data was
analyzed statistically using SPSS Ver-10 (SPSS Inc, Chicago,USA). The significance
of differences among means were carried out using Duncans Multiple Range Test at
P=0.05. The results are expressed as the means ± SE.
25
^suCts
RESULTS
Rauvolfia teiraphylla L.
4.1. Direct shoot regeneration
4.1.1. Effect of Cytokinins
The nodal explants were obtained from the plants maintained at Botanical garden o^i
the AMU, Aligarh. The explants cultured on hormone free MS media did not show
any growth and eventually necrosed. However, when cultured on MS medium
supplemented with BA (1.0, 2.5, 5.0, 7.5 or lO^M) and Kin (1.0. 2.5, 5.0. 7.5 or
lOfiM). a differential response to shoot induction and proliferation was observed.
Multiple shoot buds were observed after two weeks of incubation. BA was found
more effective than Kin, for shoot bud induction, multiplication and subsequent
proliferation. The maximum regeneration frequency (73%) was observed on MS
medium supplemented with BA 5.0 |iM, which also induced the highest mean number
(21.6 ± 0.66) of shoots, with the maximum shoot length of (5.5 ± 0.81 cm) after 8
weeks of inoculation. Among all the tested concentrations of Kin, highest shoot
regeneration frequency (66%), highest shoot number (7.6 ± 0.33) and maximum shoot
length (5.5 ± 0.81cm) were observed on MS medium supplemented with 2.5 i M Kin.
Regeneration frequencies and number of shoots per explant first increased up to the
optimal level (BA 5.0|aM) and in case of Kin (2.5^M). On increasing the
concentration above the optimal one, the regeneration frequencies and number of
shoots got decreased as mentioned in the table 5.
4.1.2. Combined effect of Cytokinin and Auxin
The synergetic effect of the optimal concentration of cytokinins viz. BA (5.0|iM) and
Kin (2.5|aM) with different concentrations of NAA was also evaluated. Nodal
explants cultured on MS medium supplemented with BA (5.0 |iM) + NAA (0.1, 0.5,
1.0. 1.5 or 2.0 |uM) and Kin (2.5 [M) + NAA (0.1, 0.5, 1.0, 1.5 or 2.0 ^M) separately,
showed emergence of multiple shoots after two weeks of inoculation. Presence of
high cytokinin and low auxin positively influenced the regeneration frequencies of
shoot number and multiplication. The combination of BA with NAA gave
significantly more number of shoots than Kin with NAA. Among the various tested
combinations BA5.0)aM + NAAO.SjiM, yielded highest regeneration frequency
(85%), maximum shoot number (26.3 ± 0.66) with maximum shoots length (6.1 ±
26
(RfsuCts
0.33cm). Among all the tested combinations of Kin 2.5 ^M with NAA (0.1. 0.5, 1.0,
1.5 or 2.0 (JM), highest shoot number (11.0 ± 1.00) with maximum height (5.7 ±
0.39cm) was observed at Kin (2.5|aM) + NAA (0.5|aM) Table 6. The explants
cultured on MS medium supplemented with (BA + NAA) were having large and
condensed nodes as compared to the (Kin + NAA), which were having larger
internodes.
4.1.3. Effect of TDZ
Nodal explants when cultured on the growth regulator free MS medium failed to
respond morphologically but when cultured on MS medium supplemented with
different concentrations (0.2. 0.4. 0.6. 0.8 or 1.0 |iM) of TDZ; multiple shoot
induction was observed after three weeks of inoculation. The explants were
transferred onto the same fresh medium after every three weeks. Positive effect of
TDZ on shoot induction, multiplication and proliferation was observed upto the four
weeks of inoculation. The highest shoot regeneration frequencies (65%) with
maximum shoot number (7.6 ± 0.33) and shoot length (4.6 ± 0.19cm) were observed
on MS media supplemented with TDZ 0.4 |iM as compared to the other tested
concentrations table.7. Shoot number and shoot proliferation increased upto the four
weeks. After four weeks the regenerated shoots became hyperhydric, desiccated and
distorted when cultured on the same fresh medium.
4.1.4. Combined effect of TDZ and BA
To overcome ill effects of prolonged TDZ exposure, a combined effect of TDZ (0.2,
0.4. 0.6. 0.8 or 1.0 jiM) with BA (l.OjiM) was evaluated. Among all the tested
concentrations of TDZ (0.2, 0.4, 0.6. 0.8 and 1.0 \iM) with BA (1.0 |aM), TDZ (0.4
|iiM) + BA (1.0 |iM) was most effective and produced highest shoot number (8.6 ±
0.33) and maximum shoot length (5.2 ± 0.25cm). There was normal shoot
development and proliferation after four weeks of inoculation. Shoot number and
proliferation first increased up to optimal concentration (0.4jiM TDZ + 1.0 jaM BA)
and then decreased on increasing the concentrations of TDZ (0.6, 0.8 and 1.0 iM)
with BA 1.0 |iM as is depicted well in the table 8.
4.1.5. Effect of metatopolin
Nodal explants failed to grow and did not responded morphologically to the growth
regulator free MS media. But when the nodal explants were inoculated on the MS
27
^suCts
medium supplemented with different concentrations (5.0. 7.5, 10, 12.5 or 15|.iM) of
mT, emergence of multiple shoot buds was observed after two weeks of inoculation.
The number and frequency of shoot bud regeneration and proliferation with regard to
the concentration used also varied. On moving from lower (5.0 |j.M) to higher
concentration (12.5|aM), an increase in shoot number (4.6 ± 0.17 to 15.6 ± 0.33) and
proliferation was observed, however, beyond optimal concentration (12.5|iM m\).
shoot number and proliferation decreased as depicted in table (9). Among all the
tested concentrations (5.0, 7.5, 10, 12.5 or 15|iM) of mT, maximum regeneration
frequency (80%), and highest shoot number (15.6 ± 0.33) with maximum height (5.7
± 0.25cm) per explant was observed at mT (12.5|iM) after eight weeks.
4.1.6. Rooting of regenerated shoots
For rooting, the in vitro regenerated shoots (4-5cm) were excised and transferred to
the MS liquid medium, supplemented with different concentrations of IBA and NAA
(0.1. 0.3, 0.5, 0.8 or 1.0 \xM) separately. The basal cut portion of the shoots supported
by the Whatman filter paper number 1, resulted in the root formation after two weeks
of culture and no rooting was observed on growth regulator free MS liquid medium.
The maximum frequency of root formation 90%, number of roots (9.3 ± 1.45) and
maximum root length (3.3 ± 0.33 cm) per shootlet was achieved on medium
containing IBA (0.3|j,M) after four weeks of incubation. MS medium supplemented
with NAA also induced roots on varying degree and gave the optimal rooting at NAA
(0.8|iM). Of the two auxins used, IBA was found to be more effective than NAA for
root induction as depicted well in the table 10.
4.1.7. Acclimatization
Plantlets having 4-6 fully expanded leaves with well developed roots were
successfully hardened off inside the culture room in Soilrite^'^ for 4 weeks and
eventually established in garden soil. The percentage survival of the in vitro raised
plants was (80%) in Soilrite and about 80% plantlets survived well when transferred
to the mixture of garden soil and manure in the ratio of 3:1 (fig. C). The
micropropagated plants appeared morphologically similar with normal leaf, shape and
growth patterns, without any detectable variation.
28
(l^suCts
Table. 5. Effect of cytokinins (BA and Kin), on shoot regeneration from nodal segments of
R. tetraphylla in MS medium after eight weeks of culture.
Growth]
BA
0.0
1.0
2.5
5.0
7.5
10
0.0
0.0
0.0
0.0
0.0
regulators |iiM)
Kin
0.0
0.0
0.0
0.0
0.0
0.0
1.0
2.5
5.0
7.5
10
o/ /o
Response
00
10
40
90
55
49
30
76
53
49
38
Mean number
of shoots/ explant
0.0 ± 0.00'
9.6 ± 1.45''
12.6± 3.71'
21.6 ± 0.66'
16.6 ± 0.66"
13.3 ± 0.33"'
5.0 ± 0.57'
7.6 ± 0.33"'
6.3 ± 0.88"'
3.6 ± 0.33"
4.0 ± 0.00'
Mean shoot
length (cm)
0.0 ± 0.00*
3.6 ± 0.50"'
5.5 ± 0.73'
5.5 ± 0.81"
4.6 ± 0.24'"
3.9 ± 0.32"'
4.6 ± 0.16'"
4.9 ± 0.86'"
4.6 ± 0.28'"
3.7 ± 0.35"'
3.1 ± 0.33'
Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.
29
(I(fsuCts
Table.6. Combined effect of cytokinins (BA and Kin) with NAA on in vitro shoot regeneration in R. tetraphylla after eight weeks of culture.
Growth
BA
5.0
5.0
5.0
5.0
5.0
0.0
0.0
0.0
0.0
0.0
regulators
Kin
0.0
0.0
0.0
0.0
0.0
2.5
2.5
2.5
2.5
2.5
;(HM)
NAA
0.1
0.5
1.0
1.5
2.0
0.1
0.5
1.0
1.5
2.0
%
Response
40
85
72
66
55
30
80
72
65
48
Mean number
of shoots/ explant
16.0
26.3
20.0
16.3
14.3
6.6
11.0
9.6
8.0
6.6
±
±
±
±
±
±
±
±
±
±
1.52
0.66 *
1.00^
0.33'
0.88'
0.88'
l.OO"
0.88"'
0.57'
1.20'
Mean
Lengt
5.0
6.1
4.9
4.8
4.6
4.9
5.7
5.3
4.5
4.6
±
±
±
±
±
±
±
±
±
Shoot
h (cm).
0.35'''
0.33"
0.31'"
0.24* '
0.14'''
0.45"'
0.39'''
0.28"^'
0.38'
±0.41*'^
Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.
30
<I(fsu[ts
Table. 7. Effect of TDZ on shoot regeneration in R. tetraphylla after four weeks of culture.
TDZ(^M)
0.0 0.02 0.04 0.06 0.08 1.0
%
Response 0 30 65 60 40 10
Mean number of Shoots/ explants
0.0 ± 0.00" 4.6 ± 0.33'' 7.6 ± 0.33' 5.3 ± 0.33" 5.0 ± 0.57'' 2.6 ± 0.33'
Mean Shoot Length (cm)
0.0 ±0.00' 4.1 ±0.26"'' 4.6 ±0.19' 3.9 ±0.28'" 3.7 ±0.37'' 3.6 ± 0.29"
Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.
Table. 8. Combined effect of TDZ and BA on shoot regeneration in R. tetraphylla after four weeks of culture.
Growth regulators
TDZ 0.2
0.4
0.6
0.8
1.0
BA 1.0
1.0
1.0
1.0
1.0
o/ /o
Rsponse 50
76
63
44
25
Mean number ofshoots/ explant
5.3 ±0.33'
8.6 ±0.33'
6.6 ±0.33"
5.6 ± 0.66"'
4.6 ±0.33'
Mean shoot length (cm)
4.5 ± 0.25'"
5.2 ±0.25'
4.5 ±0.31'"
4.0 ±0.35"
4.0 ±0.35"
Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.
1(fsu[ts
Table. 9. Effect of mT on shoot regeneration in R. tetraphylla after eight weeks of culture.
metatopolin (^M) % Mean number Mean Shoot Response of Shoots/explants Length(cni)
_ _
5.0
7.5
10.0
12.5
15.0
Values represent means ± SE. Means followed by the same letter are not significantl\ different using Duncan's multiple range test.
00
16
33
61
75
55
0.0 ± 0.00'
5.0 ± 0.57"
8.3 ±0.33'
11.3 ±0.33"
15.6 ±0.33'
12.0 ± l.OO"
0.0 ± 0.00'
4.6 ±0.17"
4.7 ± 0.09"
5.0 ±0.15"
5.7 ±0.25'
4.8 ±0.35"
32
(RfsuCts
Table. 10. Effect of auxins on root induction from in vitro raised microshoots of R. tetraphylla after four weeks of culture.
Auxins (^M) % Mean number Mean root IBA NAA Rooting of roots / shoot length(cm)
"(To oo (xo 0.0 ± 0.0 ' ooToW
0.1 0.0 30 6.6 ± 0.88"'' 1.4 ±0.19'^
0.3 0.0 80 9.3 ±1.45' 3.3 ±0.33'
0.5 0.0 68 8.0 ±1.15'"= 1.6 ± 0.0.32*''
0.8 0.0 42 6.3 ±0.88''"' 1.7 ±0.23*''
1.0 0.0 39 4.3 ±0.88"*^ 1.3 ±0.25'
0.0 0.1 15 3.0 ±0.57' 1.2 ±0.24'
0.0 0.3 35 4.6±0.66''' 1.8 ±0.38"'
0.0 0.5 60 5.6 ±0.33'" 2.0 ±0.51"'
0.0 0.8 42 8.6 ±0.88'" 2.6 ±0.38'"
0.0 1.0 36 5.3 ±0.33''' 1.6 ±0.39"'
Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.
33
Explanation of figure 1 (A-D)
Multiple shoot induction in R. tetraphylla from mature nodal explants on MS + BA
(5.0 ^M)
A. after two weeks of culture.
B. after four weeks of culture.
Inset in figure A- mature nodal explant.
Multiple shoot induction from mature nodal explants of R. tetraphylla on MS + Kin
(2.5 \M)
C. after two weeks of culture.
D. after four weeks of culture.
Explanation of figure 2 (A-D)
Multiple shoot induction and proliferation in R. tetraphylla from nodal explants on
MS + BA (5.0 ^M) + NAA (0.5 |aM)
A. after four weeks of culture.
B. after eight weeks of culture.
Multiple shoot induction and proliferation from nodal explants on MS + Kin (2.5
|iM) + NAA (0.5 \iU)
C. after four weeks of culture.
D. after eight weeks of culture.
Explanation of figure 3 (A-D)
Multiple shoot induction from mature nodal explants of i?. tetraphylla on MS + TDZ
(0.4 (iM),
A. after two weeks of culture
B. after four weeks of culture.
Multiple shoot induction and proliferation from nodal explants of R. tetraphylla on
MS + TDZ (0.4 |iM) + BA (1.0 ^M)
C. after four weeks of culture.
D. after six weeks of culture.
Explanation of figure 4 (A-B)
Multiple shoot induction, multiplication and proliferation from nodal explants on
MS+ mT(12.5^M).
A. after four weeks of culture.
B. after eight weeks of culture.
Explanation of figure 5 (A-C)
A. In vitro rooting of R. tetraphylla on MS + IBA (0.3 \xM) employing filter
paper bridge technique after four weeks of incubation.
B. Plantlets with well developed roots and shoots before transplantation in
soilrite.
C. Hardened and acclimatized plantlets of R. tetraphylla, after four weeks of
transplantation to soilrite.
^suCts
Rauvolfia serpentina L.
4.2. Direct shoot regeneration of Rauvolfia serpentina.
4.2.1. Combined effect of cytokinin and auxin.
Nodal explants from the field grown one year old plants of R. serpentina were
cultured on MS medium supplemented with various concentrations of BA and NAA
in combination (table 11). Initiation of multiple shoot regeneration and elongation of
shoot primodia started after two weeks of culture. The best and rapid multiple shoot
production was observed on MS medium supplemented with BA (8.0|iM) + NAA
(2.0|^M), in which highest shoot percentage (85%) with (7.6 ± 0.70) multipleshoots
per culture, within 4 weeks was recorded. An average of 20 cultures showed 35
shoots per flask when subcultured on the same fresh medium every eight weeks.
4.2.2. Rooting
For root induction, individual regenerated (5-6cm) healthy shoots were excised and
transferred to half strength MS medium supplemented with (2.0, 4.0 or 8.0|aM) IBA
or NAA). Good rooting was attained in a medium containing NAA (2.0)aM) and
sucrose (30g/l) within 10-15 days (table 12).
4.2.3 Acclimatization
The plantlets with well developed roots and shoots were transfersd into plastic pots
containing a mixture of soil and sand (2:1) and then acclimatized gradually to the
natural sunlight. Almost 75% of the regenerated plants survived and showed a
vigorous growth without any morphological variation.
34
(RfsuCts
Table. 11. Combined effect of cytokinin (BA) and auxin NAA on multiple shoot
formation in Rauvolfia serpentina after four weeks of incubation.
Growth reguIators(|iiM) % Mean number Mean shoot Response of shoots/explant Length (cm)
BA NAA
T o ZO 55 2.2 ± 0.07" 1.6^=0.05"
4.0 2.0 60 3.2 ±1.00' 1.5 ±0.05"
6.0 2.0 75 4.6 ±0.70" 2.6 ±0.07"
8.0 2.0 85 7.6 ±0.70' 4.2 ±0.09'
10.0 2.0 50 2.2 ±0.09" 1.6 ±0.05'
Values represent means ± SE. Means followed by the same letter are not significantly
different using Duncan's multiple range test.
^suCts
Table.l2. Effect of different auxins in half strength MS media on rooting of miroshoots of Rauvolfia serpentina, after four weeks of incubation.
Auxins
NAA
0.0
2.0
4.0
8.0
10.0
0.0
0.0
0.0
0.0
itiM)
IBA
0.0
0.0
0.0
0.0
0.0
2.0
4.0
8.0
10.0
%
Rooting
00
95
70
55
50
80
62
48
40
Mean number of
roots/shoot
0.0 ± O.OO''
3.6 ±0.05'
2.7 ±0.05"
1.7±0.01'
1.5 ±0.01 '
2.8 ± 0.05"
1.9 ±0.10"
1.7 ±0.05"
1.2 ±0.10'
Mean root
length(cm)
0.0 ± O-O'
3.0 ±0.07'
2.2 ±0.37'"
2.0 ± 0.30'"
1.1 ± 0.09"'
2.4 ±0.41'
2.2 ± 0.30'
1.8 ±0.30'"
1.2±0.10"
Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.
36
Explanation of figure 6 (A-C)
Induction of multiple shoots and from mature nodal explants in R. serpentina on MS
+ BA (8.0 |JM) + NAA (2.0^M)
A. after two weeks of culture.
B. after four weeks of culture.
C. Shoot elongation and proliferation in R. serpentina after eight weeks of culture.
Explanation of figure 7 (A-C)
A. In vitro rooting of R. serpentina on MS + NAA (2.0 i M) employing filter
paper bridge technique after four weeks of incubation.
B. Plantlet with well developed roots and shoots before transplantation in
soilrite.
C. Hardened and acclimatized plantlets of R. serpentina, after four weeks of
transplantation to soilrite.
p m
^ki^ter^ -S
m. ^i6euA6ian/
(Discussion
DISCUSSION
Increasing world population and decreasing aerable lands have made it a challenge to
produce food, medicine and energy that would suffice. One of the most neglected
biological entities are the medicinal plants growing in the wild and are diminishing
quickly around the globe because of ruthless overexploitation and even more b> the
environmental degradation. The need for herbal medicine in recent years has
highlighted the importance for conservation and propagation of many prized
medicinal plants. An efficient and most suited alternative solution to the problems
faced by the phytopharmaceutical industry is the development of in vitro regeneration
system for important medicinal species. Plant tissue culture serves as a savior for
many prized medicinal plants and the process can be exploited for continuous supply
of plant materials to various pharmaceutical companies as well as for generating large
number of cloned plants for their rehabilitation in natural habitat, conservation and
sustainable utilization.
During the last 10 years, a number of woody plants have been successfully propagated
in vitro using juvenile and mature nodal explants for direct shoot regeneration Ahmad
et al, (2006), Faisal and Anis (2007), Khan et al, (2011), and Parveen et al, (2013).
Micropropagation offers means for rapid and mass multiplication of the existing stock
of germplasm for biomass production and conservation of the rare and threatened
species (Hussain and Anis 2004, Anis et al, 2005 and Parveen et al, 2010) and
Ahmad et al., 2013). Keeping in mind the need for micropropagation, the results
obtained during the study in R. tetraphylla and R. serpentina have been discussed in
the light of existing literature on the subject.
5.1. Direct shoot regeneration in R. tetraphylla.
Microproagation of R.tetraphylla has been achieved through rapid proliferation of
nodal segments on MS medium, supplemented with different cytokinins like BA, Kin,
TDZ and mT singly or in combination with auxin (NAA). The nodal segments failed
to respond morphogenetically when cultured on growth regulator free MS media. All
the cytokinins used in the present study facilitated axillary bud induction but the
maximum shoot bud induction and proliferation was recorded on MS medium
supplemented with 5.0^M BA. The edge of BA over other cytokinins has also been
reported in several medicinal and aromatic plants viz. Tylophora indica (Faisal and
37
(Discussion
Anis, 2003), Pterocarpous marsupium (Anis et al, 2005), Mucuna pruriens (Faisal et
a/., 2006). Cassia siamea (Sreelatha et al, 2007), Rauvolfia tetraphylla (Harisararaj et
al. 2009), Terminalia bellehca (Metha et al. 2012) and Albizia lebbeck ( Perveen et
al, 2013).
Variation in shoot growth parameters in terms of shoot number and shoot length was
found when minor differences were made in the respective concentrations. Increase in
concentration of BA beyond the optimal level had a negative effect and exhibited
reduction in regeneration frequencies and shoot number from each explant. Similar
investigations have been reported in many plant species Albizia chinensis (Sinha
et al, 2000) and Pterocarpous marsupium (Anis et al, 2005). The combined effect of
auxins and cytokinins on shoot induction and multiplication of various medicinal
plants have been reported (Jahan et al, 2011, Perveen et al, 2011 and Varshney and
Anis, 2012).
Cytokinin is required, in optimal concentration for shoot induction and proliferation in
many genotypes however, inclusion of auxin along with cytokinin enhanced the shoot
regeneration (Rout et al, 1999, Ahmad et al, 2013). Highest shoot number and
maximum shoot length was obtained when optimal cytokinin BA (5.0^M) was
combined with lower concentration of an auxin NAA (0.5|iM). The synergistic effect
of BA and NAA has been reported in many medicinal plant species such as Vitex
nigundo (Ahmad et al, 2013), Rauvolfia tetraphylla (Faisal et al, 2012), thus,
individual and interactive effect of BA and NAA could ensure better in vitro
regeneration and their synergism in proper concentration was extremely favourable
for shoot multiplication. In accordance with these reports, the present study also
demonstrated the positive modification of shoot bud induction efficacy, obtained by
employing low concentration of auxin in addition with cytokinins.
Among the different tested combinations of cytokinins with auxin, BA with NAA was
found optimal for regeneration of shoots from nodal segments. Similar observations
have been reported in various plant species like Albizia lebbeck (Perveen and Anis
2012), Withania somnifera (Fatima and Anis 2012), Acacia ehrenbergiania (Javed et
al, 2013) and Euphorbia cotinifolia (Perveeen et al, 2013). These results are further
supported, by the earlier findings of several workers, who reported the addition of low
level auxin with cytokinin promoted shoot induction and proliferation as in Wrightia
tinctoria (Purohit and Kukda, 1994, Acacia catechu (Kaur et al, 1998), Psoralea
38
(Discussion
corylifolia (Anis and Faisal 2005), Rauvolfia tetraphylla (Faisal et al, 2005).
Terminalia arjuna (Pandey et a/., 2006)., R. serpentina (Baksha et al., 2007) and
R. serpentina (Panwar et al., 2011).
Thiazuron (N-phenyl-N-1, 2, 3-thidiazol-5-yl urea) has cytokinin like activity and has
been effective in low concentrations to stimulate shoot formation (Sankhla et al,
1996. Binzel et al, 1996, Murthy et al, 1998). Use of TDZ for woody plant
regeneration has been excellently reviewed by Huetteman and Preece (1993). fhis
potent cytokinin has been used for most of the plant species including Psoralea
corylifolia (Faisal and Anis 2006), Pterocarpous marsupium (Hussain et al, 2007)
and Vitex negundo (Ahmad and Anis 2007).
In addition to the other cytokinins nodal explants were also cultured on MS medium
supplemented with TDZ. Among all the tested concentrations (0.2, 0.4, 0.6, 0.8 or 1.0
\iM) of TDZ, 0.4^M was more effective for maximum number of shoot and shoot
length after four weeks of culture. The shoots became hyperhyric, distorted and
fasciated when subcultured on the same fresh medium after four weeks. Prolonged
exposure to TDZ had also caused hyperhydric, stunted, distorted and fasciated shoots
as reported in Rhododendron (Preece and Imel 1991), Dalbergia sisso (Pradhan et al.
1998), Rauvolfia tetraphylla (Faisal et al, 2005) and Cassia angustifolia (Siddique
and Anis 2007). To overcome these ill effects, the nodal explants were cultured on the
MS medium supplemented with BA and TDZ. Among all the tested combinations of
(TDZ and BA), TDZ (0.4 f M) + BA (1.0 |aM) proved to be optimal in regenerating
normal shoots.
Metatopolin (mT), is used in the plant tissue culture either alone or in combination
with the BA and has a direct effect on shoot length enhancement and increase in
secondary metabolite production (Randall et al, 2011). In sea oats Uniola paniculala
L. genotypes. Carmen valego et al, 2009 reported the positive effect of mT over BA
for shoot multiplication. In Aloe arborescens, Amoo et al, (2012) evaluated the role
of different aromatic cytokinin types and concentrations on direct organogenesis and
anti-oxidant activity of regenerated shoots, and secondary metabolite production and
reported improvement in every aspect. In the present study, nodal segments of R.
tetraphylla were cultured on MS medium supplemented with different concentrations
of mT and 12.5|LIM mT was found to be optimal for maximum shoot regeneration and
proliferation. Also, Steven et al, (2007) reported that, when nodal segments of
39
(Discussion
Juglam nigra were cultured on MS medium supplemented with mT, significant
increase in shoot number, elongation and number of nodes per micro shoot was
observed.
5,2. In vitro rooting in regenerated shoots oiR. tetraphylla
Rooting of in vitro regenerated microshoots and transplantation of the plantlets to the
field is the most important, crucial and essential step, but a difficult task in tissue
culture of woody plants. The microshoots failed to develop roots on growth regulator
free MS medium. The regenerated well developed shoots (4-5 cm) were isolated
from the proliferated cultures and were transferred to full strength MS medium
supplemented with different concentrafions (0.1, 0.3, 0.5, 0.8 and 1.0 fxM ) of NAA
and IBA separately. Among all the tested concentrations of auxins NAA and IBA,
0.3|iM IBA was most effective and proved to be optimal for in vitro rooting of R.
tetraphylla, on which 80% of the regenerated shoots developed roots with an average
root number (9.3 ± 1.45) and length (3.3 ± 0.33cm) per shoot after four weeks of
incubation. Out of the two auxins tested, IBA was found more effective than NAA.
Effectiveness of IBA for in vitro rooting has been reported for several medicinal
plants including Gymnema sylvestre (Komalavalli and Rao 2002), Tylophora indica
(Faisal and Anis 2003), Pesudarthria viscida (Baskar et al., 2006), Clitoria ternatea
(Shazad et al., 2007) and Abrus precatorius (Perveen at al, 2013). The edge of IBA
over NAA in root development may be due to several factors such as its preferential
uptake, transport and stability over other auxins and subsequent gene activation
(Ludwig -Mller, 2000).
5.3. Direct shoot regeneration in R. serpentina
Micropropagation of R. serpentina has been achieved through rapid proliferation of
nodal segments on MS medium supplemented with different concentratios (2.0 -
10|iM) of BA with NAA (2.0|iM). All the combined concentrations of cytokinin (BA)
with auxin (NAA) facilitated axillary bud induction and shoot proliferation but BA
(8.0 laM) along with NAA (2.0^M) proved to be optimal for the highest shoot
regeneration frequency (85%) with maximum shoot number (7.6 ± 0.70) and shoot
length (4.2 ± 0.09)cm, after four weeks of culture. The synergisfic effect of higher
concentration of cytokinin (BA) with low concentration of auxin (NAA) has proved to
be optimal for best shoot regeneration, proliferafion and mulfiplication in many
40
(Discussion
medicinal plants {Mucuna pruriens Faisal et al., 2005, Rauvolfia tetraphylla Faisal et
ai. 2012, Vitex trifolia Ahmad et a/.. 2013 and Acacia ehrenbergiana Javed et al.
2013).
5.4, In vitro rooting in regenerated stioots of R. serpentina
The regenerated well developed microshoots (4-5cm) were isolated from the
proliferated cultures and were transferred to the half strength MS medium
supplemented with different concentrations (2.0-10.0 \iM) of auxin NAA and IBA
separately. Among all the tested concentration half strength MS medium supplemented
with NAA (2 .0 | JM) proved to be optimal for the highest rooting regeneration
frequency (95%) with maximum root number (3.6 ± 0.05) and root length (3.0 ± 0.07
cm). Similar findings have been reported in Vitex trifolia (Ahmed and Anis 2012) and
Withania somnifera (Fatima and Anis 2012). Further work is in progress.
Hardening and Acclimatization
Hardening and acclimatization are the most important and crucial steps for achieving
success in micropropagation program. Micropropagated plantlets when transferred
from in vitro to ex vitro conditions like green house or field show low survival or
reduced growth rate due to sudden changes in the environment (Eliasson et al.. 1994
and Pospisilova et al, 1999). The special conditions during in vitro culture results in
the formation of plantlets of abnormal morphology, anatomy and physiology, when
such in vitro raised plantlets are transferred to a relatively less humid external
environment they undergo desiccation, water loss and finally death (Selvapandiyan et
a/., 1988). Thus, an acclimatization period of 2-3 weeks to overcome these
abnormalities is required.
In the present study, the rooted plantlets of R. tetraphylla and R. serpentina were
successfully hardened off inside the growth room in a selected planting substrate
(soilrite). Type of potting mixture used to acclimatize plants under ex vitro conditions
is one of the important factors in determining the survival percentage of the plants
during acclimatization process.
Similarly various hardening substrates like garden soil, vermiculite, soilrite etc, were
used by several workers, Faisal et al., (2006), Tiwari et al., (2001) and Varshney and
Anis (2012). Soilrite being more porous substrate holds more water than vermiculite
and garden soil and thus promote better growth of tender roots of tissue culture raised
41
(Discussion
plants during hardening. The plantlets were covered with transparent polythene bags
to maintain high humidity and also to prevent desiccation with the control
temperature, photoperiod and irradiance. These were then transferred to the garden
soil after four weeks.
42
Summary and ConcCusion
SUMMARY AND CONCLUSION
Plant tissue culture technique provides a viable alternative for managing the
valuable assets (phytodiversity) in a sustainable manner. Micropropagation
provides an efficient method for ex situ conservation of plant biodiversity and
mass multiplication of various plant species from a very small piece of available
plant material.
Plant species and cultivars are genetically specific to their nutrient requirements
hence no universal medium has been devised for in vitro cultures. An
understanding of optimal nutrient concentration could lead to increased growth
and evoke morphogenesis in vitro more efficiently. Axillary shoot bud
proliferation is known to be a reliable method for clonal propagation of many
economic plants.
Micropropagation is the only aspect of plant tissue culture that has been
convincingly documented with regard to its feasibility for mass scale propagation
commercially. Success of in vitro regeneration depends on the control of
morphogenesis, which is influenced by several factors namely genetic back
ground, kinds of tissue or explants, nutritional components, growth regulators and
culture environment.
The findings of my experimental investigation lead to the following conclusions;
1. Direct multiple shoot regeneration has been obtained from nodal explants
using different cytokinins (BA, Kin, mT) either singly or in combination
with auxins (IBA and NAA) in R. tetraphylla.
2. Among different cytokinins tested, MS medium supplemented with BA (5.0
^M) was found suitable for multiple shoot regeneration in R. tetraphylla.
3. Maximum shoot numbers 26.3 ± 0.33 with 5.0 ± 0.33 cm shoot length was
achieved on MS medium containing BA (5.0|iM) + NAA (0.5|iM) in nodal
explants of 7?. tetraphylla, after 8 weeks of culture.
4. Of the different concentrations of mT tested, 12.5 |aM was found best for
the maximum shoot regeneration where 15.6 ± 0.33 shoots whh 5.7 ± 0.25
cm shoot length was obtained after eight weeks in R. tetraphylla.
43
Summary and ConcCusion
5. A combination of TDZ (0.4|iM) and BA (1.0 |aM) was found optimal for
(76%) regeneration, 8.6 ± 0.33 shoots and 5.2 ± 0.25 cm shoot length after
four weeks of culture in R. tetraphylla.
6. MS medium supplemented with 0.3 |aM IB A gave maximum (80%) rooting
after four weeks of culture in R. tetraphylla.
7. A combination of BA and NAA proved effective for the direct shoot
regeneration in R. serpentina. MS medium supplemented with BA
(8.0|iM) + NAA (2.0|am) proved to be optimal for maximum shoot
regeneration.
8. Vi MS medium supplemented with NAA (2.0|iM) showed best Rooting
in R. serpentina, after four weeks of incubation.
9. The in vitro raised plantlets were successfully hardened off in Soilrite
followed by their transfer to garden soil.
44
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