dk carnation

8/16/2019 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.

&ethod! 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 &ETH'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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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!;

'/


48/56


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


50/56


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


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


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

+$


53/56


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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55/56


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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/

dk carnation

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