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Transcript of Dk Carnation
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Deependra Kumar Singh Hh Biotech, Agra
INTRODUCTION
PLANT TISSUE CULTURE
Plant tissue culture offers the ability to produce large number of
identical plants without the need for large amounts of space (as would be
needed in an outdoor environment). This is achieved by placing small
cuttings of actively growing plant parts onto a sterile plant tissue culture
media. Plant tissue culture media consists of all the required nutrients and
vitamins, sugar, hormones and a gelling agent to solidify the media. Most
commonly shoot tips are micro propagated by manipulating the hormone
balance of the plant part so that more shoots are produced. These shoots are
then separated and can be made to grow roots by again manipulating the
hormone balance. In this way, one plant of a superior genotype can be
cloned to produce large number of identical plants.
Appication! o" pant ce Cuture #
. Plant tissue culture is used widely in plant science! it also has a number
of commercial applications. "pplications include#
$. Micropropagation is widely used in forestry and in floriculture.
Micropropagation can also be used to conserve rare or endangered plant
species.
%. " plant breeder may use tissue culture to screen cells rather than plants
for advantageous characters, e.g. herbicide resistance&tolerance.
'. argescale growth of plant cells in liquid culture inside bioreactors as a
source of secondary products, li*e recombinant proteins used as
biopharmaceuticals.
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+. To cross distantly related species by protoplast fusion and regeneration of
the novel hybrid.
. To crosspollinate distantly related species and then tissue culture the
resulting embryo which would otherwise normally die (-mbryo escue)./. 0or production of doubled monoploid plants from haploid cultures to
achieve homo1ygous lines more rapidly in breeding programmes, usually
by treatment with colchicine which causes doubling of the chromosome
number.
2. "s a tissue for transformation, followed by either shortterm testing of
genetic constructs or regeneration of transgenic plants.
3. 4ertain techniques such as meristem tip culture may be employed that
can be used to produce clean plant material from virused stoc*, such as
potatoes and many species of soft fruit.
5.It can become versatile tool for Mass multiplication of elite clones,
-limination of disease in planting material, 4reation of super genotypes
of agricultural crops, which either was not possible through conventional
plant breeding methods.
.Tissue culture propagation can greatly enhance our ability to produce
consistently uniform superior planting material for e6port and domestic
mar*et.
$
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AIM
7orticulture and floriculture presents huge opportunity for
commercial production of fruits, vegetables, ornamental plants etc. and
offers lucrative investment opportunity for Indian farmer and agro
entrepreneurs. There is a high demand for these products in the domestic and
international mar*et. India, with its rich agriculture heritage can successfully
position itself as a *ey supplier of various agro, horticulture and other crops
of commercial importance.
4ultivation of floriculture has been identified as *ey activity that can
generate significant revenues and growth opportunities for farmers.
The benefits of plant tissue culture are e6tensive in the agricultural world.
Micropropagation is favourable to traditional crop breeding methods in
many respects, the first being that it allows for the production of huge
numbers of plants in a very short period of time.
4arnation has gained popularity in the past few years in many
countries of the world and it is in great demand in the floral industry as cut
flower as well as potted plant due to its beauty, colour, long vase life, and
ability to rehydrate after long transportation. 4arnation is one of the leading
cut flowers and ran*s among the top ten cut flowers of the world. It is an
important commercial flower grown throughout the world in a wide range of
climatic conditions.
'
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INTRODUCTION ABOUT PLANT
4arnation is a term that is used for plants in the Dianthus caryophyllus
group. The name caryophyllus, is from the 9ree* karyon (a nut) > phyllon (aleaf) ie nut leaved! the term comes from the old name of the Indian clove tree
( Eugenia caryophyllata) and was transferred to the carnation because the
flower was so strongly scented of cloves.
4arnations are also commonly referred to by their scientific name, ?@ianthus?,
the name given by the 9ree* botanist Theopharastus. 4arnations got the name
@ianthus from two 9ree* Aords ?dios?, referring to the god Beus, and
?anthos?, meaning flower. 4arnations are thus ?The 0lowers of 9od? or
=@ivine flowerC thus giving us dianthus D the horticultural name for this plant.
"nd while it may be a plant of the 9ods, it is also a plant of great fol*lore.
4oronae the atin word for EcoronationsF gave us carnations D the flowers that
were used in the chaplets. The name carnation is derived from the atin term
“Carnatio” meaning fleshness.
"nother common name =;weet AilliamC comes to us from the atin
ocellus or EeyeF referring to the lighter color patch in the middle of the flower.
The atin became oeillet in 0rench and was corrupted and slurred by the
-nglish to willy D then to Ailliam. The ;weet part come from the fragrance or
perhaps from an old ballad named =0air Margaret and ;weet AilliamC from the
/55Fs when the plant was first grown in
gardens. It is probably native to the
Mediterranean region but its e6act range
is un*nown due to e6tensive cultivation
for the last $,555 years. It is the wild
ancestor of the garden 4arnation.
+
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;cientific :ame # Dianthus Caryophyllus
0amily # 4aryophyllaceae
4ommon names # 4arnation, @ivine flower,
4love pin*, 9illy 0lower
4olour # Garious
Scientifc classifcation
Hingdom # Plantae
@ivision # Magnoliophyta
4lass # Magnoliopsida
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'rder#
• 4aryophyllales is an order of "o%ering pant! that includes the cacti,
carnation!, amaranth!, ice pant!, and most carni(orou! pant!. Many
members are !uccuent, having fleshy !tem! or ea(e!.
• The 4aryophyllales includes about J of dicot !pecie!. This order is
considered a core eudicot, which is also referred to as the core
tricolpates.
• 4urrently, the 4aryophyllales contains %% families, 3$ genera and
,++ species.
)ami*#
• The 4aryophyllaceae, commonly called the pin* family or carnation
family, is a family of flowering plants.
• The species are dicotyledons included in the order 4aryophyllales. It is a
large family, with 22 genera and some $,555 species.
• This cosmopolitan family of herbaceous plants is best represented in
temperate climates, with a few species growing on tropical mountains.
• ;ome of the more commonly *nown members include pin*s and
carnations (@ianthus), and 0irepin* and campions (ychnis and ;ilene).
• Many species are grown as ornamental plants, and some species are
widespread weeds.
• Most species grow in the Mediterranean and bordering regions of -urope
and "sia.
• The number of genera and species in the southern hemisphere is rather
small, although the family does contain "ntarctic Pearlwort
(Colobanthus quitensis), the worldKs southernmost dicot, which is one of
only two flowering plants found in "ntarctica.
/
http://en.wikipedia.org/wiki/Order_(biology)http://en.wikipedia.org/wiki/Flowering_planthttp://en.wikipedia.org/wiki/Cactushttp://en.wikipedia.org/wiki/Dianthus_caryophyllushttp://en.wikipedia.org/wiki/Amaranthhttp://en.wikipedia.org/wiki/Ice_planthttp://en.wikipedia.org/wiki/Carnivorous_planthttp://en.wikipedia.org/wiki/Succulent_planthttp://en.wikipedia.org/wiki/Plant_stemhttp://en.wikipedia.org/wiki/Leafhttp://en.wikipedia.org/wiki/Dicothttp://en.wikipedia.org/wiki/Specieshttp://en.wikipedia.org/wiki/Eudicothttp://en.wikipedia.org/wiki/Family_(biology)http://en.wikipedia.org/wiki/Flowering_planthttp://en.wikipedia.org/wiki/Dicotyledonhttp://en.wikipedia.org/wiki/Caryophyllaleshttp://en.wikipedia.org/wiki/Genushttp://en.wikipedia.org/wiki/Specieshttp://en.wikipedia.org/wiki/Cosmopolitan_familyhttp://en.wikipedia.org/wiki/Herbaceous_planthttp://en.wikipedia.org/wiki/Dianthushttp://en.wikipedia.org/wiki/Lychnishttp://en.wikipedia.org/wiki/Silenehttp://en.wikipedia.org/wiki/Specieshttp://en.wikipedia.org/wiki/Ornamental_planthttp://en.wikipedia.org/wiki/Weedhttp://en.wikipedia.org/wiki/Mediterraneanhttp://en.wikipedia.org/wiki/Europehttp://en.wikipedia.org/wiki/Asiahttp://en.wikipedia.org/wiki/Southern_hemispherehttp://en.wikipedia.org/wiki/Antarctic_Pearlworthttp://en.wikipedia.org/wiki/The_world's_southernmosthttp://en.wikipedia.org/wiki/Antarcticahttp://en.wikipedia.org/wiki/Order_(biology)http://en.wikipedia.org/wiki/Flowering_planthttp://en.wikipedia.org/wiki/Cactushttp://en.wikipedia.org/wiki/Dianthus_caryophyllushttp://en.wikipedia.org/wiki/Amaranthhttp://en.wikipedia.org/wiki/Ice_planthttp://en.wikipedia.org/wiki/Carnivorous_planthttp://en.wikipedia.org/wiki/Succulent_planthttp://en.wikipedia.org/wiki/Plant_stemhttp://en.wikipedia.org/wiki/Leafhttp://en.wikipedia.org/wiki/Dicothttp://en.wikipedia.org/wiki/Specieshttp://en.wikipedia.org/wiki/Eudicothttp://en.wikipedia.org/wiki/Family_(biology)http://en.wikipedia.org/wiki/Flowering_planthttp://en.wikipedia.org/wiki/Dicotyledonhttp://en.wikipedia.org/wiki/Caryophyllaleshttp://en.wikipedia.org/wiki/Genushttp://en.wikipedia.org/wiki/Specieshttp://en.wikipedia.org/wiki/Cosmopolitan_familyhttp://en.wikipedia.org/wiki/Herbaceous_planthttp://en.wikipedia.org/wiki/Dianthushttp://en.wikipedia.org/wiki/Lychnishttp://en.wikipedia.org/wiki/Silenehttp://en.wikipedia.org/wiki/Specieshttp://en.wikipedia.org/wiki/Ornamental_planthttp://en.wikipedia.org/wiki/Weedhttp://en.wikipedia.org/wiki/Mediterraneanhttp://en.wikipedia.org/wiki/Europehttp://en.wikipedia.org/wiki/Asiahttp://en.wikipedia.org/wiki/Southern_hemispherehttp://en.wikipedia.org/wiki/Antarctic_Pearlworthttp://en.wikipedia.org/wiki/The_world's_southernmosthttp://en.wikipedia.org/wiki/Antarctica
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+enu!#
• Dianthus i s a genu! of about %55 !pecie! of "o%ering pant! in the
family Car*oph*aceae.
• The species are mostly perennial herbs, a few are annual or biennial, and
some are low !u$!hru$! with woody basal stems.
• The ea(e! are opposite, simple, mostly linear and often strongly
glaucous greygreen to bluegreen.
• The "o%er! have five petals, typically with a frilled or pinked margin,
and are (in almost all species) pale to dar* pin*.
•
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Mor!olo"#$
Loung outdoor plants send up between one to five stems that can
each produce up to si6 flowers. ;tems are woody at the base but
have herbaceous branches. eaves are opposite, linear, flat and
soft in te6ture and their colour varies from green to grey blue
or purple. They have conspicuous sheaths. The flowering stems
are often swollen and brittle at the nodes.
It is a her$aceou! perennia pant growing to 25 cm tall. The ea(e!
are glaucous greyish green to bluegreen, slender, up to + cm long. The
"o%er! are produced singly or up to five together in a c*me! they are %D+cm diameter, and sweetly scented! the original natural flower colour is bright
pin*ishpurple, but cuti(ar! of other colours, including red, white, yellow
and green, have been developed gro%ing Carnation!
4arnation flowers have five petals, and they may have single or
double blooms which vary in color from white to pin*. ;pecial cultivars in
shades li*e red, orange, and dar* purple have also been produced, along with
variegated blooms. The petals typically have ragged edges, and carnations
have a distinctive slightly spicy smell which some people find very pleasant.
The single flowers of the 4arnations species, @ianthus caryophyllus
have + petals and vary from white to pin* to purple in color. order
4arnation cultivars may have double flowers with as many as '5 petals.
Ahen grown in gardens, 4arnations grow to between and 2.+ cm in
diameter. Petals on 4arnations are generally clawed or serrated. 4arnations
are bise6ual flowers and bloom simply or in a branched or for*ed cluster.
The stamens on 4arnations can occur in one or two whorls, in equal number
or twice the number of the petals.
3
http://en.wikipedia.org/wiki/Herbaceoushttp://en.wikipedia.org/wiki/Perennial_planthttp://en.wikipedia.org/wiki/Leafhttp://en.wikipedia.org/wiki/Flowerhttp://en.wikipedia.org/wiki/Cymehttp://en.wikipedia.org/wiki/Cultivarhttp://en.wikipedia.org/wiki/Herbaceoushttp://en.wikipedia.org/wiki/Perennial_planthttp://en.wikipedia.org/wiki/Leafhttp://en.wikipedia.org/wiki/Flowerhttp://en.wikipedia.org/wiki/Cymehttp://en.wikipedia.org/wiki/Cultivar
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T*pe! o" Carnation! #
4arnation cultivars are mainly of three types#
4arnation cultivars are mainly of three types#
• arge flowered 4arnations one large flower per stem.
• ;pray 4arnations (Mini 4arnations) with lots of smaller flowers .
• @warf flowered 4arnations several small flowers on one stem.
I%ortance o& carnation$'
• 4arnations are used as ornamental plants in gardens and in the cut
flower industry. Moderncutflower varieties of carnation have been
selected for flower si1e, petal number, stem lengthand disease resistance.
In the 3th century, commercial growing was e6tensive in 0rance
andincluded both field production and glasshouse production. "fter
germplasm was transferred tothe ;", carnation breeding and growing
for the cutflower mar*et became very popular in the ;".
• In addition to their use as cut flowers, carnations have been, and are still,
used for culinary purposes. The flower petals have a strong smell of
cloves and can be crystallised or used as agarnish in salads or for
flavouring fruit, fruit salads, butter lemonade, vinegars, conserves and
syrups etc. (see eg 0acciola 335! 7ughes 33%). The ;paniards and
omans used carnation flowers as a spicy flavouring in wine and it isclaimed (eg 4ornett 332) that this culinary useled the -nglish to call
carnations ?sopsinwine? during the time of 4haucer. 7owever, it
isli*ely that this term was actually referring to the culinary clove as it is
arguable that carnations grew in -ngland in the 'th century (Mc9eorge
5
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N 7ammett $55$) although it would appear that flowers of carnations
were added to wine at some stage and were calledsopsinwine.
4arnation petals are one of the ingredients that has been used to ma*e
the 0rench liqueur, green 4hartreuse, since the /th century.• -ssential oil is present in small amounts in petals of the carnation. "bout
+55*g of flowers are required to produce 55g of oil which can then be
used in perfumes. Modern perfumes containing carnation oil include
Lves ;aint aurent
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• It is the national flower of ;pain, and the provincial flower of the
autonomous community of the alearic Islands. It is also the symbol of
the Portuguese 4arnation evolution. The state flower of
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Media Roo%$ The washing area in the media room should be provided
with the brushes of various si1es and shape, a washing machine, a large sin*
and running hot and cold tap water. It should also have steel or plastic
buc*ers to soa* the lab wares to be washed, oven or a hot air cabinet to dry
the washed lab wares and a dust proof cupboard to store them.
*as!in"$ "n area with large sin*s, the brushes of various si1es and
shape, a washing machine, and running hot and cold tap water. " draining
area is necessary since the conventional method for cleaning laboratory glass
ware involves acid soa* followed by subsequent rinsing with distill water.
9lassware should be soa*ed in a $J detergent cleaner for hour followed
by washing with 5/55 4 hot tap water and distill water. The glassware is
first air dried and then *ept in an oven for two hours, at 5O4.
It should also have steel or plastic buc*ers to soa* the lab wares to be
washed, oven or a hot air cabinet to dry the washed lab wares and a dust
proof cupboard to store them.
C+lt+re Roo%$ "ll types of plant tissues are incubated under
conditions of wellcontrolled temperature, humidity, illumination and air
circulation. sually, airconditioners and heaters are used to maintain the
temperature around $+ $5 4. 4ultures are generally grown in diffused light.
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temperature and light are also installed in a culture room. Incubators, large
plant growth chambers, are readily available in the mar*et. They occupy less
space and have the range of control and fle6ibility desirable for growth of
tissues under in vitro conditions.
Re,+ire%ents o& c+lt+re roo%$
. Temperature control (/$/Q4).
$. -lectricity supply essential for lighting, cooling and heating.
%. ;helves for culture rac*s.
'. 0luorescent tubes for lighting.
+. Timer for regulating daylength.
. ac*s for culture vials.
/. otary sha*er.
O-ser.ation or Data Collection Area$
The growth and development of tissues cultured in vitro is generally
monitored by observing cultures at regular intervals, in the culture room.
Plants regenerated from in vitro tissue cultures are transplanted to soil in
pots. The potted plants are ultimately transferred to green houses or growth
cabinets and maintained for further observations under controlled conditions
of light, temperature and humidity.
E,+i%ent +sed in tiss+e c+lt+re la-orator#$
• "nalytical balance (for weighing nutrients for media)• 9radual cylinders and pipettes (for measuring stoc* solutions)
• p7 meter (to regulate p7 of media)
• 7ot plate or stove (to heat and dissolve gelling agent)
• 9lass containers (for heating and dissolving media)
• @ispensing devices (to dispense equal quantities of media)
+
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• " still or deioni1er (water needed for media)
• "utoclave or pressure steam sterili1er (for sterili1ing instruments and
media)
• Transfer instruments (forceps, scalpels, spatulas, blades)
• efrigerator (storage of chemicals and stoc* solution)
• ;tereo microscope (use for meristem culture)
• aminar air flow hood (providing a sterile area for transfers during
initiation and subculturing)
Baance!# Manualsingle pan, capacity 55$55g, electronicstop loading
pH meter# $%5G, +571, single phase supply with combined p7 electrode,
range 5'Ph&5'55mv, temp. compensation 555R4.
ertica autoca(e# with safety valves, pressure gauge, steamrelease coc*.
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Laminar air "o% ca$inet or ti!!ue cuture hood# It is designed to prevent
contamination of semiconductor wafers, biological samples, or any particle
sensitive device. "ir is drawn through a 7-P" (high efficiency particulate
air) filter and blown in a very smooth, laminar flow towards the user. The
cabinet is usually made of stainless steel with no gaps or Soints where spores
might collect. aminar flow cabinets may have a G4 germicidal lamp to
sterili1e the shell and contents when not in use.
The other miscellaneous equipment which are required for tissue
culture are air condition arrow head, unsen burner, deep free1er, dissecting
needles, glass wares, forceps, heaters, magnetic stirrer, inoculation cabinet,
metal trays, bowls, tubes, G germicidal lamps, spatula etc.
/
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./ Inorganic#a. &acro0eement!#0
"s is implied by the name, the stoc* solution supplies those elements
required in large amounts for plant growth and development. Nitrogen,
pho!phoru!, pota!!ium, magne!ium, cacium and !uphur (and
carbon, which is added separately) are usually regarded as macro
elements. These elements usually comprise at least 5.J of the dry
weight of plants. "ll are essential for sustained growth in vitro.
b. &icroeement!#0
These elements are required in trace amounts for plant growth and
development, and have many and diverse roles. &angane!e, iodine,
copper, co$at, $oron, mo*$denum, iron and 1inc usually comprise
the microelements, although other elements such as nic2e and
auminium are frequently found in some formulations.
3/ 'rganic Suppement!#A/ itamin!#0
9enerally, thiamine (vitamin ,), pyrido6ine (vitamin 4),
nicotinic acid (vitamin 5) and myoinositol are included, but only
thiamine is considered to be essential. The others have growthenhancing
properties. The concentrations of each can vary significantly between the
different media compositions.
3
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B/ Amino acid!#0
;ome cultured plant cells can synthesi1e all amino acids, none are
considered essential. 7owever, some media do contain certain amino
acids for their growthenhancing properties, e.g., glycine in M; media.
7owever, high concentrations of certain amino acids can prove to6ic.
4rude ammo acid preparations (e.g., casamino acids! 5) can also be
used (e.g., for protoplast culture), but their undefined nature ma*es themless popular. "mino acid provide a source of reducing nitrogen.
C/ Car$on !ource#0
$5
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9enerally, most plantcell cultures are nonautotrophic and are
therefore entirely dependent on an e6ternal source of carbon. In most
cases, this is sucrose as it is readily assimilated and relatively stable, but
occasionally glucose, maltose, galatose or sorbitiol can also be used and
may prove better then sucrose in speciali1ed circumstances.
D/ Ph*tohormone!#0
Plant growth regulators are the typical media components in
determinimg the developmental pathway of the plant cell. The most
commonly used phytohormones for plantcell culture are the au6ins and
cyto*inins. 7owever, for specific applications with certain species,
abscisic acid or gibberellic acid may be also used. 7owever,There are five main classes of plant growth regulator used in
plant cell culture, namely#
. "u6ins,
$. 4yto*inins,
%. 9ibberellins,'. "bscisic acid,
+. -thylene.
./ Au6in!#0
"u6in effect many developmental processes besides cell elongation.
The au6ins can be defined as a compound with the biological activities li*e
it promote both cell division and cell growth. The most important naturally
occurring au6in is IAA (indole%acetic acid), but its use in plant cell culture
media is limited because it is unstable to both heat and light.
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problems associated with the use of I"". It is more common, though, to use
stable chemical analogues of I"" as a source of au6in in plant cell culture
media. $,'@ichloropheno6yacetic acid ($,'@) is the most commonly used
au6in and is e6tremely effective in most circumstances.
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also substitute for au6in in some culture systems.
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plant tissue culture is not widespread. It does, though, present a particular
problem for plant tissue culture. ;ome plant cell cultures produce ethylene,
which, if it builds up sufficiently, can inhibit the growth and development of
the culture. The type of culture vessel used and its means of closure affect
the gaseous e6change between the culture vessel and the outside atmosphere
and thus the levels of ethylene present in the culture.
E/ 'ther!:
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response of cultured plant tissue oth natural products e6tracted from
seaweeds (e g , agar, agarose, and alginate) and their more recently emerged
substitutes (e.g., 9elrite, Phytagel), obtained from microbial fermentation,
can be used. -ach has its advantages and disadvantages, and the choice is
usually determined by the species and the application "gars and agarose
generally produce gels that are stable for prolonged periods and are
considered not to bind media components e6cessively. Products with various
degrees of purity are available, and lowgelling temperature types can even
enable the embedding of sensitive cells, such as protoplasts.
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d; - pH#0
p7 affects absorption of ions and also solidification of gelling
agent. The p7 of the medium is usually adSust between +.5and .5(
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$/
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Media rearation#0
litre media was prepared in sterile glass containers and the procedure of its
preparation is given below#
Ta*e '55 ml of double distil water in 555 ml standard flas*.
"dd %5 gm sucrose and dissolve it completely.
"dd 55 mg of inositol and dissolve it completely.
"dd in sequence the appropriate amount of solutions as follows#
0reshly prepared macronutrient salt solution 55 ml
;toc* solution of micronutrient salt 5 ml
;toc* solution of iron + ml
;toc* solution of vitamins ml
;ha*e well to mi6 up uniformly
"dSust the p7 of the liquid medium +. D +.2 with the aid of 5.: 74
or 5.: :a
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)igure No/ . < 3# Sho% eectronic de(ice u!ed "or the %eighing o"
&edia component! 73nd i! u!ed "or mea!uring !ma =uantitie! >/>>;
$3
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Sterili/ation:
;terili1ation is the freeing of an article from all living organisms, including
bacteria and their spores. ;terili1ation of culture media, e6plants, containers
and instruments is essential in Plant tissue culture.
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./ Red Hot
Inoculating wires, points of forceps and searing spatulas are sterili1ed by
holding them in the flame of unsen burner until they are seen to be redhot.
3/ )aming
This method is used for sterili1ing scalpel, mouth of culture tubes, glass
slides etc. It involves passing of an article through unsen flame without
allowing it to become redhot.
5/ Hot Air '(en
This is the main means of sterili1ation by dry heat. -6posure at atemperature of 554 for hour is generally employed.
ðod! o" Sterii1ation $* &oi!t Heat#
&echani!m o" 2iing $* moi!t heat
Moist heat *ills the organisms by coagulating and denaturing their en1ymes
and structural protein. ;terili1ation by moist heat of the most resistant spores
generally requires $54 for +%5 minutes. Moist heat is used for the
sterili1ation of culture media, and all other materials through which steam
can penetrate. Moist heat is more effective than dry heat. ;terili1ation can be
done at lower temperatures in a given time at a shorter duration at the same
temperature.
Moist heat can be employed at
. Temperature below 5554
$. Temperature of 5554
%. Temperature above 5554
%
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Moist heat below 1000 C:
./ Pa!teuri1ation o" mi2
In Pasteuri1ation of mil* the temperature employed is either %54 for
%5 minutes or /$54 for $5 seconds.
Moist heat above 1000 C
./ Sterii1ation in an autoca(e
"utoclaving is the most reliable method. It is the method most widely
used for sterili1ation of culture media and surgical supplies. Ahen water is
boiled within a closed vessel at an increased pressure, the temperature at
which it boils and the steam it forms will rise above 555
4.This principle isused in the autoclave. :ormally autoclaving is done at + lbs. (pounds per
sq. inch pressure) and $54 for + minutes.
)actor! In"uencing Sterii1ation $* Heat#
. The temperature and time# they are inversely related, shorter time is
sufficient at high temperatures.
$. :umber of microorganisms and spores# The number of survivors
diminished e6ponentially with the duration of heating
%. @epends on the species, strains and spore forming ability of the microbes.
'. Thermal death point is the lowest temperature to give complete *illing in
aqueous suspension within 5 minutes
+. @epends on the nature of material# a high content of organic substances
generally tends to protect spores and vegetative organisms against heat.. Presence of organic or inorganic disinfectants facilitates *illing by heat
/. p7 also plays an important role in the *illing of microorganisms.
%$
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B. Sterilization by Radiation:-
Methods of sterili1ation e6ist using radiation such as electron beams, Urays,
gamma rays, or subatomic particles.
• 9amma rays are very penetrating and are commonly used for
sterili1ation of disposable medical equipment, such as syringes, needles.
9amma radiation requires bul*y shielding for the safety of the operators!
they also require storage of a radioisotope (usually 4obalt5), which
continuously emits gamma rays (it cannot be turned off, and therefore
always presents a ha1ard in the area of the facility).
• Urays, if low energy, are less penetrating than gamma rays and tend
to require longer e6posure times, but require less shielding. They are
generated by an Uray machine that can be turned off for servicing and
when not in use.
• ltraviolet light irradiation (G, from a germicidal lamp) is useful
only for sterili1ation of surfaces and some transparent obSects. Many
obSects that are transparent to visible light absorb G. G irradiation is
routinely used to sterili1e the interiors of biological safety cabinets between uses, but is ineffective in shaded areas, including areas under
dirt (which may become polymeri1ed after prolonged irradiation, so that
it is very difficult to remove). It also damages many plastics, such as
polystyrene foam.
C. Sterilization by Filtration:-
Ahen fluids are passed through bacteria stopping filters, they are made
free from bacteria.
It is useful for ma*ing preparations of soluble products of bacterial
growth such as to6ins
%%
http://en.wikipedia.org/wiki/X-rayshttp://en.wikipedia.org/wiki/Gamma_rayshttp://en.wikipedia.org/wiki/Subatomic_particlehttp://en.wikipedia.org/wiki/Radioisotopehttp://en.wikipedia.org/wiki/Cobalt-60http://en.wikipedia.org/wiki/X-rayshttp://en.wikipedia.org/wiki/X-ray_machinehttp://en.wikipedia.org/wiki/Ultraviolethttp://en.wikipedia.org/wiki/Lighthttp://en.wikipedia.org/wiki/Germicidal_lamphttp://en.wikipedia.org/wiki/Visible_lighthttp://en.wikipedia.org/wiki/Biological_safety_cabinethttp://en.wikipedia.org/wiki/Polystyrenehttp://en.wikipedia.org/wiki/X-rayshttp://en.wikipedia.org/wiki/Gamma_rayshttp://en.wikipedia.org/wiki/Subatomic_particlehttp://en.wikipedia.org/wiki/Radioisotopehttp://en.wikipedia.org/wiki/Cobalt-60http://en.wikipedia.org/wiki/X-rayshttp://en.wikipedia.org/wiki/X-ray_machinehttp://en.wikipedia.org/wiki/Ultraviolethttp://en.wikipedia.org/wiki/Lighthttp://en.wikipedia.org/wiki/Germicidal_lamphttp://en.wikipedia.org/wiki/Visible_lighthttp://en.wikipedia.org/wiki/Biological_safety_cabinethttp://en.wikipedia.org/wiki/Polystyrene
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iquids that would be damaged by heat such as serum and antibiotic
solutions can be sterili1ed by filtration.
II/ CHE&ICAL Ð'DS# 0
Eth*ene o6ide#0
-thylene o6ide gas *ills bacteria (and their endospores), mold, and fungi,
and can therefore be used to sterili1e substances that would be damaged by
sterili1ing techniques such as pasteuri1ation that rely on heat. -thylene o6ide
is widely used to sterili1e medical supplies such as bandages, sutures, and
surgical implements.
'1one#0
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• Hilling of microorganism by chlorine and its compound is also in part
to the direct combination of chlorine with protein of the cell
membrane and en1ymes.
+utaradeh*de and )ormadeh*de#0
• "ccepted liquid sterili1ing agents, provided that the immersion time is
sufficiently long. "lso used as fi6atives. To *ill all spores in a clear
liquid can ta*e up to $ hours with glutaraldehyde and even longer
with formaldehyde.
• ;terili1ation of bloc*s of tissue can ta*e much longer, due to the time
required for the fi6ative to penetrate.
• 9lutaraldehyde and formaldehyde are volatile, and to6ic by both s*in
contact and inhalation.
H*drogen Pero6ide#0
7ydrogen pero6ide is another chemical sterili1ing agent. It is relatively non
to6ic once diluted to low concentrations (although a dangerous o6idi1er at
high concentrations), and leaves no residue.
Pheno and phenoic! compound!#0
• +J aqueous solution of phenol rapidly *ills the vegetative cells of
microorganism, spores are much more resistant.
• Phenolics substances may be either bactericidal or bacteriostatic,
depending upon the concentration used.
• The antimicrobial activity of phenolics is reduced at an al*aline p7
and by organic material.
&ode o" action#0
%+
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@isruption of cell, precipitation of cell proteins, inactivation of en1yme,
lea*age of amino acids from the cell.
Ethano#0
• sed in concentration between +5 D 35J -ffective against vegetative
and nonspore forming cells.
• 0or practical application /5J concentration is used.
• Methyl alcohol is less bactericidal than ethyl alcohol
&ode o" action#0
• "lcohols are protein denaturants They are solvents for lipids and
hence they damage lipid comple6es in cell membrane They are also
dehydrating agents.
Hea(* meta! and their compound!#0
• e.g. mercury, silver, copper
&ode o" action#0
"ct anti microbial by combining with cellular proteins and inactivating
them.
;7 ;
en1yme > 7g4l$ en1yme 7g > $74l
;7 ;
S*nthetic Detergent!#0
;uperior to soaps because they do not form precipitates in al*aline or neither acid water, nor they deposits with minerals found in hard water .
%
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4ationic anionic nonionic
More germicidal less germicidal donFt posses significant antimicrobial property.
Techni=ue &ateria! !terii1ed
Steam
!terii1ation-Autoca(ing
($Q4 at + psi for $5'5 min)
:utrient media, culture vessels, glass waresand plastic wares
Dr* heat (525Q4 for %h) Instruments (scalpel, forceps, needles etc.),glassware, pipettes, tips and other plasticwares
)ame !terii1ation Instruments (scalpel, forceps, needles etc.),mouth of culture vessel
)iter !terii1ation (membranefiltermade of cellulose nitrate or celluloseacetate of 5.'+ 5.$$Vm poresi1e)
Thermo labile substances li*e growthfactors, "mino acids, vitamins anden1ymes.
Acoho !terii1ation Aor*erFs hands, laminar flow cabinet
Sur"ace !terii1ation (;odiumhypochlorite, hydrogen
pero6ide,mercuric chloride etc)
-6plants
%/
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CARNATION TISSUE CULTURE
The plant materials were ta*en from standardi1es bottles provided by the
institute. Plant materials were collected to see their response under in vitro
conditions. -6cised plant tissues will only grow in vitro on a suitable
artificially prepared nutrient medium which is *nown as culture medium.
If the plant material is ta*en from the e6plants, then e6plants were
surface sterili1ed to remove the microorganisms before inoculating them into
the culture medium and the procedure of e6plants sterili1ation as follows#
The e6plants were placed in running tap water for +D%5 minutes. Immersed
in avestein (fungicide) or @ome6 solution (liquid detergent) for $+D%5
minutes, washed with water for %D+ times. The e6plant is now dipped in
@etol solution for +$5 min for removal of fungicide. Then dip the e6plants
in /5J IP" (isopropyl alcohol) for 5 seconds in "0 and wash them with
distill water for % D + times. Transfer the e6plants into an autoclaved conical
flas* and pour 5.J Mercuric 4hloride (7g4l$) and *eep them for %D+
minutes. @uring this period, the bottle is frequently swirled for sha*ing so
that all surfaces of the e6plants come equally in contact with sterilant. Then
again wash the e6plants thoroughly several times with distill distill to
remove all traces of sterilant.
:ow the e6plants are ready for inoculation but before doing so some
care is ta*en so that there is no entry of any microorganisms at the time of
transferring the surface sterili1ed e6plants on the nutrient medium using the
sterili1ed equipments. 0or achieving this certain steps are followed#
Put all the sterili1ed articles (media, instruments, glass goods)
on the table of laminar air flow cabinet. aminar air flow blows bacteria
free air over the wor*ing surface.
%2
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Put on the switch of G lamps of the laminar air flow for
hour before wor*.
Put off the G lamp without opening the cabinet of the laminar
air flow. Put on the switch of the fluorescent lamp and the blower of the
laminar air flow immediately after the G lamp is switched off.
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Laminar "o% 7ti!!ue cuture %or2 in !terii1ed condition!;
Inoc+lation %et!od$'
se the sterile gloves and equipments for all of the steps are mandatory.
The gloves can be sprayed with a /5J alcohol solution and hands
rubbed together to spread the alcohol Sust prior to placing hands into the
chamber. 4arefully open the container with the plant material and ta*e out the
sterili1ed plant material from the container, place on the sterili1e paper. 4ut the plant according to the culture requirement.
Ta*e a prepared section of plant material in sterile forceps and place
into the medium. eplace the cap tightly on the culture bottle.
0inally transfer these bottles to the
environment control room.
)igure! !ho%ing# cutting and inocuation o" e6pant! in to cuture medium in
aminar air "o% ca$inet/
'5
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Tran!"er en(ironmenta contro room#0
-nvironmental factor greatly influence the process of growth and
differentiation of tissues in culture. "ll types of plants are therefore
incubated under well controlled temperature, humidity, illumination,
and circulation.
" typical culture room should have both light and temperature
programmable for $'hr period. sually air conditioned and room
heaters are used to maintain the temperature around $+ $Q 4
ightening is adSusted in terms of quantities and photoperiod duration
by using automatic cloc*s (cultures are generally grown in diffused
light less then *lsO) further the humidity range varies from $5 D 32
J controllable to %Q 4. and a uniform fords air ventilation is
necessary.
;pecially designed shelves made of glass rigid wire mesh or wood are
provided in culture room for storing culture. -ach shelf is illuminated
separately by a separate set of coolwhite fluorescent light placedabout 2 inches above the culture to give a light intensity of %555 lu6.
Photoperiod maintained at hours in light and 2 hours in dar*.
Individual shelf may also be ventilated by fitting a small fan at one
end of the shelf the culture vessel can be placed directly on the
shelves or trays of suitable si1e where as culture tubes require metallic
rac*s to hold them.
" label is provide having the details of the e6periment ( name of the
plant, e6plants , medium, date of culture and other information) is
stuc* on each tray to ensure identity and recording results.
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" generator should be *ept stand by and emergency power points in
the culture rooms, incubators and growth chambers in order to
maintain the necessary light and temperature condition.
The e6plant in the high temperature is li*ely to lead to dissociation of
the culture medium and tissue damage while at very low temperatures
tissue growth is slow. "gain some tissues grow well in dar* while
others need both light and dar* conditions. ow humidity causes the
quic* desiccation of culture medium and high humidity is favorable
for the contamination of culture medium.
A %e e!ta$i!hed cuture room
'$
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Sta"es o& Carnation tiss+e c+lt+re$
Preparation o" Stoc2 pant
The elite plants are selected and maintained under hygienic conditions (by
spraying fungicide, bactericide and insecticide) and then the plant parts are
ta*en for initiation.
Stage I# 7 Initiation !tage;
Ta*ing the plant from in vivo (=lifeC) to in vitro. This simply means that the
e6plant is ta*en from its normal relationships to the other plant parts and is
placed under artificial =testtubeC culture conditions. The innermost tissue of
surface sterili1ed plant in dissected aseptically and put in to the medium of
growth, Medium contains maSor and miner elements, some vitamins, "mino
acids and growth promoting hormones, solidified by agar.
)igure !ho%ing Initiation !tage o" Dianths Caryo!hyllos on &S media/
'%
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Deependra Kumar Singh Hh Biotech, Agra
Stage II# 7&utipication !tage;
The e6plant undergoes rapid tissue or shoot multiplication. This process can
be repeated several times, depending upon how many plants are ultimately
desired. Ahen the tissue starts growth in stage I and forms a shoot it is
transferred to another medium containing growth promoting hormones
(enhancing cell division). The growing shoot multiplies and forms a dump of
%' shoots. Those are transferred to another medium for shooting and
rooting after optimum growth. If a sterile environment was not maintained,
contamination will be obvious within %' days.
)igure No/ . < 3 # Sho%ing mutipe !hoot propagation "rom In (itro gro%n
!hoot! o" Dianths Caryo!hyllos on &S media/
''
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Stage III# 7 Rooting !tage;
" different growth medium is used to induce root formation from ;tage II
plants/ "fter multiplication, the single shoots are separated and placed into a
shooting are rooting medium. "t this stage the hormones may or may not be
required. The shoot elongates and new root came up. ooting ta*es place
within %' wee*s.
)igure No/ . < 3 # Sho%ing mutipe !hoot propagation "rom In (itro
gro%n !hoot! o" Dianths Caryo!hyllos on &S media/
'+
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Stage I# 7Accimati1ation !tage i/e/ hardening;
Transfer of the plants to potting medium. It involves acclimati1ation of
bottle grown plants to the natural environment in 9reen 7ouse. The
plants are ta*en out of the bottle and the media adhering to the root
system in washed fully. "fter wards the plants are graded as per their
si1e and then transferred singly to wells of portrays containing sterile
medium (a mi6ture of peat moss and perlite). The whole portray with
plants is maintained under high humidity conditions for a couple of
wee*s and there after the portrays are *ept in open in the 9reen 7ouse
under controlled temperature and humidity.
This hardening ta*en wee*s and is called primary hardening
egular sprays of plant protection chemicals are sprayed to achieve
good hygenic condition of the plants.
)igure !ho%ing di""erent !tage! o" !hoot induction and gro%th in cuture
media/
'
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7?e0organi1ed
carnation green hou!e!;
'/
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If proper precaution and totalsterile condition does not ta*en duringinoculation and some other steps li*e
transfer in culture room and mediasterili1ation etc., some chance of contamination can occur. If inoculatedmedia become contaminated, fungusand bacteria grow on the medium and
plant growth not occurs.
@ )igure Sho%ing $otte! %ith no
re!pon!e "or !hoot
proi"eration on cuturemedia/
.
)igure No/ . < 3# Sho%ing contaminated $otte! %ith the gro%th o"
'2
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$acteria < "ungi/
'3
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OBSER0ATION TABLE
No.
ofDays
Date ofInoculation
No. ofEx-plant
Date ofObservation Contamination
No. of
Dead ex-plant Result
Fungal acterial
0 24/02/2009 25
1
2
3 27/2/2009 1 0 0
4
5
67 31/2/2009 1 1 1 Yellow
8
9
10
11 4/2/2009 0 0 0 Green
12
13
14 7/2/2009 1 0 1 Yellow
15
16
17
1819 12/2/2009 0 1 0 Yellow
20
21
22
23 16/2/2009 0 0 0 Green
24
25
26
27
28 21/2/2009 0 0 0 Green
29
30
31
+5
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No.ofDays
Date ofInoculation
No. ofEx-plant
Date ofObservation Contamination
No. ofDead ex-plant Result
Fungal acterial
0 24/03/2009 25
1
2
3 27/3/2009 1 0 0
4
5
6
7 31/3/2009 1 1 1 Yellow
8
9
10
11 4/4/2009 0 0 0 Green
12
13
14 7/4/2009 1 0 1 Yellow
15
16
17
18
19 12/4/2009 0 1 0 Yellow
20
21
22
23 16/4/2009 0 0 0 Green
24
25
26
27
28 21/4/2009 0 0 0 Green
29
30
31
+
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No.ofDays
Date ofInoculation
No. ofEx-plant
Date ofObservation Contamination
No. ofDead ex-plant Result
Fungal acterial
0 24/05/2009 25
1
2
3 27/5/2009 1 0 0
4
5
6
7 31/5/2009 1 1 1 Yellow
8
9
10
11 4/6/2009 0 0 0 Green
12
13
14 7/6/2009 1 0 1 Yellow
15
16
17
18
19 12/6/2009 0 1 0 Yellow
20
21
22
23 16/6/2009 0 0 0 Green
24
25
26
27
28 21/6/2009 0 0 0 Green
29
30
31
+$
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RESULTS AND DISCUSSION
• In vitro techniques offer new possibilities in commercial propagation
of plant. The present study was also underta*en to propagate
commercially important cultivar of carnation. 0or shoot formation
both apical and nodal meristem were used.
• In this study the shoot tip e6plant of Dianthus caryophyllus, was
placed on M; medium for a period of wee*s and in this period the
e6plants showed good response of growth and multiplication.
• In the present study it was also observe apical meristem responded
earlier compared to nodal meristem this potential effect of e6plants is
also discussed by resan et al.,(32$).
• ;imilarly, the leaf of e6plants also showed growth responses when
they are not properly remover before inoculation, there is an increase
in the length of the leaf instead of the shoot elongation.
+%
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⇒ ie, S/T/ and Lia%, S/I/,7.;/ Plant regeneration from shoot tips and
callus of papaya. In Vitro. %(3)# +'+2.
⇒ www.accesse6cellence.org , micropropagation in plant tissue culture.
⇒ www.aggiehoticulture.tamu.edutisscult&tcintro.html
⇒ www.agritechpublication.com
⇒ www.biotech.cornell.edu&inde6.cfm&page&scf\inde6&plant\pissie.html
⇒ www.google.co.in, carnation pictures
⇒ www.hostatissueculture.com
⇒ www.phytotechlab.com
⇒ www.win*ipedia.com , basic *nowledge about carnation
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Yie%20ST%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Liaw%20SI%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.accessexcellence.org/http://www.aggie-hoticulture.tamu.edu-tisscult/tcintro.htmlhttp://www.agritechpublication.com/http://www.biotech.cornell.edu/index.cfm/page/scf_index/plant_pissie.htmlhttp://www.google.co.in/http://www.hostatissueculture.com/http://www.phytotechlab.com/http://www.winkipedia.com/http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Yie%20ST%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Liaw%20SI%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlushttp://www.accessexcellence.org/http://www.aggie-hoticulture.tamu.edu-tisscult/tcintro.htmlhttp://www.agritechpublication.com/http://www.biotech.cornell.edu/index.cfm/page/scf_index/plant_pissie.htmlhttp://www.google.co.in/http://www.hostatissueculture.com/http://www.phytotechlab.com/http://www.winkipedia.com/