influence of iaa on in vitro shoot elongation of eucalyptus

Available online at www.ijntps.org | ISSN: 2277 – 2782
INTERNATIONAL JOURNAL OF NOVEL TRENDS IN PHARMACEUTICAL SCIENCES
RESEARCH ARTICLE
INFLUENCE OF IAA ON IN VITRO SHOOT ELONGATION OF
EUCALYPTUS TERETICORNIS-103 ; A POTENCIAL PULP
WOOD CLONE
*
Logeswari N and Kanagavalli J
Department of Biotechnology,Vivekanandha College For Women, Tiruchengode, TN, India
Article Info
Article history
Received 02 Feb 2014
Revised 01 Feb 2014
Accepted 09 Feb 2014
Available online 28Feb 2014
Keywords
Eucalyptus
tereticarnis,
astringent,
thrmogenic,
antiseptic, deodorant.
Abstract
Paper is an essential commodity required for communication. Literacy pursuits,
packaging and a variety of the applications. Wood is the principle source of
cellulosic fiber for pulp and paper manufacture. At present, wood provides about
93% of word’s virgin fiber requirement, whole non-wood sources; mainly Bagasse,
straws, and bamboo provide the reminder. Eucalyptus tereticarnis is a fast growing
tree can reach 30 to 45 meters in hight and 1 to 2 meter in diameter. Eucalyptus oil
is acrid and bitter and is a ruptured, astringent, thrmogenic, antiseptic, deodorant,
stimulant, carminative, and digestive, cardio phonic, diuretic, expectorant, insect
repellent, rubefacient and antipyretic. It is useful in healing flatulence; halptosis
worm intestation cardiac debility, tuberculosis. Chronic cough, asthma, bronchitis,
pyorrhea, burns, dyspepsia and skin diseases. The present study indicate that the in
vitro culture of Eucalyptus tereticarnis clone 103 using WPM medium along 0.4mg/L
auxin,produced superior quality and yield with higher micro shoot, when compare
with the other MS ,Nicth media having various concentration of IAA. The result
confirms 0.4mg/L auxin in WPM medim improved shoot elongation of micro shoot
length and number of shoot that can improve the productivity. It deals with the
influence of auxins in the in vitro shoot elongation of Eucalyptus tereticarnis
clone103. The results of this study can be utilized for further research programs. This
may lead to increased production of the Eucalyptus plants in the shoot period of
time, because the Eucalyptus tree which is useful in medicinal, economical aspects.
INTRODUCTION
Paper is an essential commodity required for
communication.literacy pursuits, packaging and
avariety of the applications.The significance of
paper and paper products in modern life is obvious
number of manufacture product plays a more
meaningful role in human activity.Paper provides a
means of recording storage and dissemination of
information.The world wide production of paper
and paper board is around 300million tones of
which 45% is used for printing and writing 40% for
packaging and 15% for hygienic and health
products.
Chronology of Technology Development
Paper derives its name from the reedy plant,
“Papyrus” [1][2][3][4][5][6][7][8][9][10][11]and [12].
The ancient Egyptians produced the world’s first
VOLUME 4 | NUMBER 1 | FEB | 2014
fibrous writing material by beating and pressing
together thin layer of the plant stem. However
complete defibering of true paper making was
absent. After period of several centuries the art of
paper making extended into Middle East and Later
reached Europe where cotton and linen rags
became the main raw materials. By 1680 the first
paper mill was set up serapore in West Bengal.
Characterstics of Wood
Wood is the principle source of cellulosic fiber for
pulp and paper manufacture. At present ,wood
provides about 93% of word’s virgin fiber
To whom correspondence should be addressed:
Logeswari N
Email: [email protected]
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Logeswari N and Kanagavalli J, Influence of IAA on in vitro shoot elongation of Eucalyptus tereticornis-103; A
potencial pulp wood clone
requirement, whole non-wood sources; mainly
Bagasse, straws and bamboo provide the
remainder.
Botanically woods are classified into two major
groups
Software or conifer
Hardwood or deciduos
The yield of pulp is directly related to density
Hardwoods tend to have higher densities man
softwood.There contain a larger proportion of holo
cellulose and les lingnin as compared to softwoods
but a greater percentage of extractives.
Eucalyptus
Euclyptus belongs to predominantly southern
hemisphere family, Myrtacease (Johnson and Briggs
1984) [13]. The genus Euclyptus consist of about
700 species. They are exotic fast growing tress
introduced to india from Australia.
Classification of Eucalyptus teretricornis
Kingdom
:Plantae
Sub
:Tracheiobiota
Super Divison
:Spermatophyta
Divison
:Magnolioplryta
Class
:Magndioprida
Sub Class
:rosidae
Orders
:myrtales
Family
:myrtaceae
Genus
:Eucalyptus
Species
:tereticornis
Characterstics of Euclyptus teretricornis
Eucalyptus teretricornis is a fast growing tree that
can reach 30 to 45 meters in height and 1to 2 meter
in diameter.They are used to plant poor sites as
they come up in poorer site and give fuel and pulp
wood at a quick rate. Eucalptus species and highly
medicinal due to the oil in their leaves rotation age
followed for Eucalyptus is 8 years and two coppice
crops are taken.The replanting is done Eucalyptus
species for suitable pulp production.
Uses
Wood is used as source for pulp manufacture.there
has been a rapid increase in world production of
chemical. Eucalyptus pulp from about 40,000 tones
per year is early 1985 for continous pulp
wood.Supplies it is important to have plantation of
uniform bole size and quality.Henace it is desirable
to develop reliable methods of propogation to
reduce the upstream process variation.
VOLUME 4 | NUMBER 1 | FEB | 2014
Medicinal Value of Eucalyptus
Eucalyptus oil is a crid and bitter and is a ruptured
astringent, thrmogenic,
antiseptic, Deodorant,
stimulant, carminative, and digestive, cardio phonic,
diuretic, expectorant, insect repellant, Rubefacient
and antipyretic.It is in healing flatulence;halptosis
worm intestation cardiac debility , tuberculosis,
chronic cough, asthma, bronchitis, pyorrhea, burns,
and dyspepsia and skin diseases.Any internal
consumption of the oil in excess will cause cardiac
deabily, vomiting and diarrhea.
In Vitro Propagation
Practices of rapidly multifying stock plant material
to produce a large number of progeny plants using
modern plant tissue culture methods.
Initiation.
Multiplication.
Elogatrion
Establishment
Begin with the selection of material to be
propagared clean stock material that are free from
viruses and fungi are important in the production of
the healthy paints .
Initiation
Plant material in chosen for culture of explants
begins and is dependent on the type of tissue used.
The explants are then surfaces sterilizerd. This
position of plant on growth medium containing
sucrose and plant regulators Some are grown on
simple media while other required complex media
containing vitamin , amino acids etc.
Multiplication
Mulitiplication is taking tissue samples produced
during first initiation and increasing their number
following the successful introduced and growth of
plant tissue through repeated cycles of this process,
single explants can be increased from one too
hundreads.If the tissue is grown as small parts
called plantlets, hormones are often added that
cause the plantlets to produce many small off
shoots can be removed and recultured. Depending
on the type of tissue grown, multiplication can
involve different methos and media.
Elongation
In vitro shoots were elongated with the help of
Elongation media and were incubated in the dark at
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Logeswari N and Kanagavalli J, Influence of IAA on in vitro shoot elongation of Eucalyptus tereticornis-103; A
potencial pulp wood clone
24-26 C.Regenerated shoots were obtained after 5
weeks.
Pretrnsplant
This stage involves treating the plantlets/shoots
produced to encoverage root growth and
hardening.It is performed in vitro or in sterile test
tube environment.
Root growth does not always occur in the earlier
stages in plant cell culture and is of course a require
of succefful plant growth after initial stages.It is
often performed in vitro by transferring the
plantlets to a growth medium, containing auxin
which stimulates root intiation.Some plant is micro
propagated and grown in culture and normal
cuttings are mode that is then rooted ex vitro.
Micro propagation is an essential component of
plant biotechnology.In offers a solution in which
several hundre’s of identical plant cann be
produced from the same amount of materials
required to produce single new plant by traditional
methods.
Hardening refers to the preparation of the plantlets
for a natural growth environment.Until this stage,
the plantlets have been grown in ideal condition
designed to encourage rapid plant growth. Due to
lack of necessity. The plants are likely to be highly
susceptible to disease and often do not have fully
functional dermal coverings and will be in efficient
in their use of water and energy,
In vitro conditions are humidity and plats grown
under there condition do not form a working cuticle
and stomata that keep the plant from drying our.
When taken out of the culture plants need time to
adjust to natural conditions. This is done by moving
the plants to a location high in humidity, such as
green house with regular.
Advantages
• Produce disease free clones.
• Rapid clonal multiplication of selected
varities
• Preservation of valuable germ plasm
• Expedition of plant material from one
country to another
PLANT TISSUE CULTURE MEDIA
Medium
A Medium is defined as a formulation of organic
salts and organic compounds (a part from major
carbohydrates
sources
and
plant
growth
regulators), used for nutrition of plant cultures.
VOLUME 4 | NUMBER 1 | FEB | 2014
Lioyd And Mc Cown: Wood Plant Medium
(WPM)
Woody plant medium was developed for the shoot
culture of tress and shrubs as Betula, Kalmia, Rosa
and Rhododendron in 1980 . It has same NO₃⁻ and
NH₄⁺ concentrations as Anderson’s medium more
potassium and a high level of sulpahte ions; but the
MS level of phosphate and CT, WPM medium is
widely used for the propagation of ornamental
shrubs and tress in commercial laboratories.
Murashige And Skoog Medium (MS Medium)
Murashige and skoog in 1962 [14], Originally set
out to final an as yet discovered growth hormones
present in tobacco juice. No such compounds was
discovered instead, analysis of juiced tobacco and
tobacco revealed higher concentrations of specific
minerals in plant tissues than were previously
known. A series of experiment demonstrated that
varying the levels of these nutrients enhanced
growth substantially over exiting formulations.
Nitsch Medium
The medium was developed by Nitsch and Nitsch in
year of 1956, and frequently used for anther culture.
Its concentration level between the WPM and MS
were the tried in the experinment for the elongation
of Euclyptus tereticorins.
PLANT GROWTH REGULATORS
The growth regulators required in shoot tip
obtained synthetically or through fermentation
process and added to plant tissue culture medium.
They are termed as plant growth regulators. They
growth regulators are
• Auxin
• Cytokinin
• Gibberellinus
• Ethylene
• Abcisic acid
Auxin
Auxin is a compound that positively influence cell
enlargement, bud formation and root initiation.
They also promote the production of other
hormones and in conjuction with cytokinins they
control the growth of stems, root, flowers and fruits.
Indole 3-Acetic Acid (IAA)
The Indole 3-acetic in tissue culture auxins have
been used for cell division and root differentiation.
The auxins are commonly used in tissue culture are
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Logeswari N and Kanagavalli J, Influence of IAA on in vitro shoot elongation of Eucalyptus tereticornis-103; A
potencial pulp wood clone
IAA.IAA has also been used with other regulators to
induce direct morphogenesis and for meristerm and
shoot cultures. Auxins are capable ofinitiating cell
division and are involved in the origin of meristems
giving rise to either unorganized tissue or defined
organs. Auxins are used for meristems or single
node culture and then at very low rates in
combination with a cytokinin. The rate of IAA is
more rapid in the light and is accelerated by the
presence of MS salts.
AIM OF THE STUDY
The object of the work is to study the
influence of Auxin on shoot elongation of
Eucalyptus tereticornis clone-103.
To study the effect of various concentration
of IAA in the three different media such as
MS, Nitsch and Woody plant medium
Monitoring the impact of auxin (IAA) on
shoot elongation,
shoot lengths and
number of shoots
MATEIALS AND METHODS
Plant Material
Nodul cutting Eucalyptus tereticornis clone-103
used for experiments, a number of myrtaceae
family, The nodul cutting were collected from clonal
multiplication area(CMA) maintained in Tamilnadu
newsprint and paper limited(TNPL),Karur .Initially
stock plants were cut at one o food above ground
level after 2 years of growth to stimulate coppice
growth.
CULTURE MEDIUMMURASHIGE AND SKOOG
(MS) MEDIUM (MSA)
MACRONUTRIENTS Mg/L
KNO₃
1900
NH₄NO₃
1650
CaCl₂
332
MgSO₄.7H₂O
370
KH₂PO₄
170
MICRONUTRIENTS Mg/L
MnSO₄H₂O
17
ZnSOP₄7H₂O
8.6
H₃BO₄
6.2
KI
0.83
CuSO₄
0.16
Na₂.Mo.O₄.2H₂O
0.25
CoCl₂.6H₂O
0.25
Na2.EDTA.2H₂O
37.25
FeSO₄.7H₂O
27.85
VOLUME 4 | NUMBER 1 | FEB | 2014
Both Macronutrients and Micronutrients chemical
was dissolved separately in beaker and then upto 1
litre with distilled water (which is used as a stock
solution)
MSB
VITAMINS AND
ORGANICS
Inosital
Thiamine HCL
Nicotinic acid
Pyridoxine HCL
Glycine
D-Biotin
Calcium Pantothenate
Sugrose
IAA
Mg/L
100
0.3
5.0
0.5
2.0
0.05
0.05
(20g/l)
(0.1-0.5 mg/l)
Pᴴ was adjusted to 5.6-5.8 with NaoH or HCl
Nitsh Medium (NMA)
MACRONUTRIENT
KNO₃
NH₄NO₃
CaCl₂.2H₂O
MgSO₄.7H₂O
KH₂PO₄
MICRONUTRIENT
MnSO₄H₂O
ZnSO₄7H₂O
H₃BO₄
CuSO₄ .5 H₂O
Na₂.Mo.O₄.2H₂O
Na2.EDTA.2H₂O
FeSO₄.7H₂O
NMC
Inosital
Thiamine HCL
Nicotinic acid
Pyridoxine HCL
Folic acid
D-Biotin
Glycine
Sugrose
IAA
Mg/L
950
720
166
185
68
25
10
10
0.025
25
37.3
27.8
100
0.5
5.0
0.5
0.5
0.05
2.0
(20g/l)
(0.1-0.5 mg/l)
WOODY PLANT MEDIUM WPA
MACRONUTTRIENT
Mg/L
NH₄NO₃
400
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Logeswari N and Kanagavalli J, Influence of IAA on in vitro shoot elongation of Eucalyptus tereticornis-103; A
potencial pulp wood clone
CaCl₂
MgSO₄.7H₂O
KH₂PO₄
CaNO₃.4H₂O
K₂SO₄
MICRONUTTRIENT
MnSO₄H₂O
ZnSO₄7H₂O
H₃BO₃
CuSO₄ .
Na₂.Mo.O₄.2H₂O
Na2.EDTA.2H₂O
FeSO₄.7H₂O
96
370
170
556
990
Mg/L
29.43
8.6
6.2
0.25
0.25
37.3
27.8
WPC
Inosital
Thiamine HCL
Nicotinic acid
Pyridoxine HCL
Cap
Biotin
Riboflavin
Glycine
Sugrose
IAA
100
1.0
0.5
0.5
0.1
0.1
0.1
5.0
2.0
(20g/l)
Hormone
Indole- 3 acetic acid (IAA)
(0.1-0.5 mg/l)
Elongation
MS medium
MSA
MSB
Sucrose
Ribiflovin
Pᴴ
Agar
40 ml
10 ml
20 g
2 mg
6.2
6g
Nitsh Medium
NMA
NMB
NMC
Sucrose
Pᴴ
Agar
100 ml
100 ml
100 ml
20 g
6.2
6g
WPM MEDIUM
WPA
WPB
WPC
50 ml
10 ml
10 ml
VOLUME 4 | NUMBER 1 | FEB | 2014
Sucrose
Pᴴ
Agar
20 g
6.2
6g
APPARATUS REQUIRED
• 100ml culture bottle with media
• Surgical Knifes
• Surgical forceps
• Sterile cotton and filter paper
All the above apparatus has to be autoclaved at
121ºC for 15 minutes
STOCK PREPARATION
Preparation of MS Stock
The basal medium is prepared by mixing
MSA constitute macro and micro amd MSB
constitute Vitamins and organic nutrients together
called
as
Murashige
and
skoog
(MS
medium.1962).The stock concentration of MS salts
is 25x while organic is 50x and it is stored in brown
bottle in the refrigerator to avoid variation in the
concentration of the solution and to prevent
microbial growth.
Preparation of Nitsch Medium
The basal medium is prepared by mixing
NMA constitute macro and micro amd NMB
constitute Vitamins and organic nutrients together
called
as
Nitsch
and
Nitsch(Nitsch
medium.1969)[15]. The stock concentration of NM
salts is 10x while organic is 10x and it is stored in
brown bottle in the refrigerator to avoid variation in
the concentration of the solution and to prevent
microbial growth.
Preparation of WPM Medium
The basal medium is prepared by mixing
WPA
constitute
macro
nutrients
WPB
micronutrients and WPC constitute Vitamins and
organic nutrients together called as WP (LLOYD, G
AND MC COWN medium.1981)[16]. The stock
concentration of WP salts is 20x while organic is
100x and it is stored in brown bottle in the
refrigerator to avoid variation in the concentration
of the solution and to prevent microbial growth.
Preparation of Hormone Stock
The Hormone concentration was used in
mg/L for this 10mg of hormone is taken and made
up to 100ml in a standarding flask with distilled
water eg: IAA
METHODS
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Logeswari N and Kanagavalli J, Influence of IAA on in vitro shoot elongation of Eucalyptus tereticornis-103; A
potencial pulp wood clone
Initial Steps In Tissue Culture of Eucalyptus
tereticornis Clone: 103
Sprouted branches of Eucalyptus clone103
with performed axillary buds were harvested and
act into explants bearing nodel buds.Explant with
their nodel buds is about 2 cm length, and it is kept
under running tap water,alcohol and mercury
chloride followed by washing 3-5 times with sterile
distilled water.
The media was prepared with various
concentration of hormones and the buds were
inoculated.The sprouts are produced after a perioid
of 40 days and placed in multiplication medium
(MS+0.2mg BAP) and after 4-5 subcultures.The
clones were used for elongation experiments.The
elongation medium prepared.
The Pᴴ of t h media was adjusted to 6.2
using sodium hydroxide(or) hydrochloric acid .Then
the media is boiled in a Kettie and the Metton
media were dispensed in culture bottles upto the
mark and were tightly capped.Then the culture
media were sterilized by autoclaving at 121ºC for 15
minutes.Then the bottles are cooled and stored in a
sterile media room to avoid contamination .Under
sterile condition the well sprouted branches or buds
were taken and the shoots were transferred into the
culture bottles with various concentration opf IAA
0.1,0.2,0.3,0.4and 0.5mg/L respectively.
Elongation of Shoots
Clusters of regenerated shoots attached to
the explants were separated and transferred to MS,
Nitsch, and WPM medium respectively containing
various concentration of IAA for further elongation
of shoots.
Maintainance Culture Medium
The culture were maintained in the culture
room at 25ºC under the light intensity of 1350lux
illumination(30-40um2S1) with a photoperiod of
16:18 hours, by cool white flurosent tubes for visible
range. The in vitro studies were carried out for a
period of four weeks and data’s were collected and
results were tabulated.
Statistical Analysis
The percentage of response, number of
shoots and number of shoot length were monitored
as growth parameters. Data were recorded after 1
week of culture.
RESULT AND DISCUSSION
The present study was carried out to find
out the impact of auxin concentration on invitro
shoot elongation of Eucalyptus tereticornis clone
103. Micro shoots were sub cultured on three
different media such as MS, Nitsch,WPM and
medium having various concentration of IAA
ranging from 0.0 to 0.5mg/ L. The culture were
maintained in the plant growth room at 25± 1ºC
with 16/8 photoperiod through out the
experiments.
The study was monitored weekly once and
measurement was done with respect to number of
shoots and shoot length After four weeks WPM
medium was found to be effective for Eucalyptus
tereticornis clone 103 with 0.4mg/L IAA ,when
compared to all the medium tested (Nitsch & MS),
as well as all the concentration tested (0.0 to 0.5).
The optimum growth response of Eucalyptus
tereticornis clone 103 were observed in WPM
medium with 0.04mg/L IAA and the lowest growth
response of Eucalyptus tereticornis clone103 was
observed in MS & Nitsch at 0.4mg/L IAA. The
control plants that were grown in WPM, Nitsch &
MS with out IAA showed minimal growth when
compared to plants grown in the medium with IAA.
Fig 1. Shoot elongation of Eucalyptus tereticornis clone 103 in nitsch medium
Fig 2. Shoot elongation of Eucalyptus tereticornis clone 103 in murashige and skoog medium
VOLUME 4 | NUMBER 1 | FEB | 2014
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Logeswari N and Kanagavalli J, Influence of IAA on in vitro shoot elongation of Eucalyptus tereticornis-103; A
potencial pulp wood clone
Fig 3. Shoot elongation of Eucalyptus tereticornis clone 103 in woody plant medium
Impact of Auxin (IAA) on Eucalyptus tereticornis (c103) in
different medium on the formation of number of shoot under
invitro condition
S.No
Concentration of
IAA mg/L
1
2
3
4
5
6
CONTROL
0.1
0.2
0.3
0.4
0.5
Number of Micro Shoots
NITSCH
5
7
9
10
10
8
MS
7
8
10
11
13
9
WPM
9
10
11
12
16
13
1
0.9
0.8
0.7
0.6
NITSCH
0.5
0.4
MS
0.3
WPM
0.2
0.1
0
CONTROL
VOLUME 4 | NUMBER 1 | FEB | 2014
0.1
0.2
0.3
0.4
0.5
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Logeswari N and Kanagavalli J, Influence of IAA on in vitro shoot elongation of Eucalyptus tereticornis-103; A
potencial pulp wood clone
Impact of Auxin (IAA) on Eucalyptus tereticornis (c103) in different
medium on the formation of micro shoot length under invitro condition
S.No
Concentration of
IAA mg/L
1
2
3
4
5
6
CONTROL
0.1
0.2
0.3
0.4
0.5
Micro – Shoots Length(cm)
NITSCH
0.1
0.2
0.4
0.5
0.5
0.6
MS
0.3
0.4
0.5
0.6
0.7
0.7
WPM
0.5
0.5
0.7
0.8
0.9
0.8
1
0.9
0.8
0.7
0.6
NITSCH
0.5
MS
0.4
WPM
0.3
0.2
0.1
0
CONTROL
0.1
0.2
CONCLUSION
The present study indicate that the in vitro culture
of Eucalyptus tereticarnis clone 103 using WPM
medium along 0.4mg/L auxin, produced superior
quality and yield with higher micro shoot, when
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