IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND

SHOOT INDUCTION
ABBREVIATIONS
IAA : Indole 3 Acetic Acid
IBA : Indole 3 Butyric Acid
NAA : Naphthalene Acetic Acid
BAP : Benzyl Aminopurine
2, 4 D : 2, 4 Dichloro phenoxy Acetic Acid
FeS
4
: Ferrou! Sulphate
"A
3
: "i##eralic Acid
$%&l
2
: 'ercuric &hloride
$
3
B
3
: Boric Acid
(n : (inetin )Fur*uryl Amino Purine+
(N
3
: Pota!!ium Nitrate
($
2
P
3
: Pota!!ium $ydro%en Pho!phate
'S : 'ura!hi%e and S,oo%-! 'edium
'yo.ino!itol : 'em#er o* /itamin B.&omplex
N$
4
N
3
: Ammonium Nitrate
N$
4
$
2
P
4
: Ammonium $ydro%en Pho!phate
)N$
4
+
2
S
4
: Ammonium Sulphate
Na$
2
P
4
: Sodium Dihydro%en ortho.Pho!phate

ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 1
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
SL.NO TABLE OF CONTENTS PAGE
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ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 2
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
OVERVIEW OF FOREST RESEARCH INSTITUTE
Fore!t 3e!earch In!titute )F3I+ Dehradun, i! a premier in!titution under the
Indian &ouncil o* Fore!try 3e!earch and 2ducation )I&F32+1 It @a!
e!ta#li!hed a! Imperial Fore!t 3e!earch In!titute in 0:<>, 5he In!tituteA!
hi!tory i! Birtually !ynonymou! @ith the eBolution and deBelopment o*
!cienti*ic *ore!try, not only in India, #ut oBer the entire Indian !u#.continent1 It
i! Set in a lu!h %reen e!tate !pread oBer 47< hectare!, @ith the outer
$imalaya *ormin% it! #ac, drop, the In!tituteA! main #uildin% i! an impre!!iBe
edi*ice, marryin% "reco.3oman and &olonial !tyle! o* architecture, @ith a
plinth area o* 217 eCuipped la#oratorie!, li#rary, her#arium, ar#oreta, printin%
pre!! and experimental *ield area! *or conductin% *ore!try re!earch, Cuite in
,eepin% @ith the #e!t o* it! ,ind any@here in the @orld1 It! mu!eum!, in
addition to #ein% a Balua#le !ource o* !cienti*ic in*ormation, are a maDor
attraction *or touri!t!1
5he in!titute cater!, in particular, to the re!earch need! o* the Indo.
"an%etic plain! o* PunDa#, $aryana, &handi%arh, Delhi and 4e!tern 6ttar
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 3
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
Prade!h, a! 4ell a! the 61P1 $imalaya!1Fore!t 3e!earch In!titute ha! %ot
lar%e!t Fore!try 3e!earch 'anpo@er in South A!ia, It proBide! #a!ic *ore!try
,no@led%e @ith *rontline re!earch1 In!titute ha! %ot the deemed uniBer!ity
!tatu! @ith !tron% !cienti*ic #a!e and !tore hou!e o* ,no@led%e1 5hru!t
area! o* the in!titute are &on!erBation o* #iodiBer!ity, !ocial *ore!tryEa%ro
*ore!try, con!erBation and eco.re!toration o* ecolo%ically *ra%ile and
di!tur#ed area!, plantin% !toc, improBement pro%ramme o* di**erent !pecie!
etc1
Fore!t 3e!earch In!titute, Dehradun i! dedicated to carry out multi
di!ciplinary *ore!try 3FD, 2ducation and 2xten!ion aimed at creation and
di!!emination o* !cienti*ic ,no@led%e *or !u!taina#le deBelopment,
mana%ement and con!erBation1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 4
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
INTRODUCTION
PLANT TISSUE CULTURE.
Plant ti!!ue culture #roadly re*er! to the in vitro cultiBation o* all plant
part!, @hether a !in%le cell, ti!!ue or an or%an under a!eptic condition!1 5he
underlyin% principle! inBolBed in plant ti!!ue culture are Bery !imple1 Firstly,
a plant part from the intact plant & its inter-organ/ inter-tissue is isolated.
Secondly, it is necessary to provide the plant part with an appropriate
environment in which it can express its intrinsic or induced potential 1 5hi!
mean! that a !uita#le culture medium F proper culture condition! mu!t #e
proBided. Finally, the above procedures must be carried out aseptically.
It @a! in 0:<2 that the @ell ,no@n "erman Plant Phy!iolo%i!t, GGottlieb
Haberlandt )0874.0:47+, attempted to cultiBate plant ti!!ue culture cell! in
Bitro1 $e i! re%arded a! the *ather o* ti!!ue culture1 5i!!ue culture relie! on
three *undamental a#ilitie! o* plant!:
Totioten!" i! the potential or inherent capacity o* a plant cell or
ti!!ue to deBelop into an entire plant i* !uita#ly !timulated1 5otipotency
implie! that all the in*ormation nece!!ary *or %ro@th and reproduction
o* the or%ani!m i! contained in the cell1 Althou%h theoretically all plant
cell! are totipotent the meri!tematic cell! are #e!t a#le to expre!! it1
Dedi##erentiation i! the capacity o* mature cell! to return to
meri!tematic condition and deBelopment o* a ne@ %ro@in% point
Co$eten!" de!cri#e! the endo%enou! potential o* a %iBen cell or ti!!ue to
deBelop in a particular @ay1 For example, a! em#ryo%enically competent
cell! are capa#le o* deBelopin% into *ully *unctional em#ryo!1 5he oppo!ite i!
non.competent or morpho%enetically incapa#le1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 5
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
HISTOR% OF PLANT TISSUE CULTURE
0:<2 Fir!t attempt o* plant ti!!ue culture1 "otlie#$a#erlandt
0:<4 2m#ryo culture o* cruci*era $anni%1
0:22 In.Bitro %ermination o* orchid !eed! (nud!on1
0:22 InBitro culture o* root tip!1 3o##in!1
0:27 2m#ryo culture tech1 *or ;inum1 ;ai#ach1
0:34 &ulture o* tomato root!1 4hite1
0:3: 2!ta#li!hment o* callu! culture1 "authert,
No#ecourt ,4hite
0:40 6!e o* coconut mil, in Datura culture1 /an oBer#ee,1
0:44 In Bitro !hoot *ormation in 5o#acco1 S,oo%1
0:72 'eri!tem culture *or Biru! *ree Dahlia1 'orel F 'artin1
0:73 Production o* callu! in "in,%o #ilo#a1 5ulec,i1
0:74 Fir!t plant *rom !in%le cell1 'uir et al1
0:77 Di!coBery o* ,inetin1 'iller et al1
0:7? Auxin:cyto,inin lead! to or%an *ormation1 S,oo%F 'iller1
0:78 In Bitro !omatic em#ryo%ene!i!1
'ahe!h@ariF3an%a!@a
mi1
0:7: Somatic em#ryo%ene!i! D1carota1 3einertFSte@ard1
0:>< 5e!t tu#e *ertilization in P1rhoea!1 (anta1
0:>2 DeBelopment o* 'S media1 'ura!hi%eFS,oo%1
0:>4 Somatic em#ryo%ene!i! in D1innoxia1 "uhaFmahe!h@ari1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 6
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
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0:>? Production o* andro%enic haploid1 Bour%inFNit!ch1
0:?< Fir!t protopla!t *u!ion1 Po@er et al1
0:?2 Protopla!t *u!ion in t@o !p! o* Nicotiana &arl!on1
0:?4 Di!coBery o* 5i pla!mid in A%ro#acterium1 HaenenF;ar#e,e1
0:?8 Production o* Pomato1 'elcher! et al1
0:8< Production o* In!ulin in #acteria1 2li lillyF co1
0:84 5ran!*ormation o* 5o#acco1 De #loc,1
0:8? I!olation o* Bt %ene1 Barton et al1
0::< ;aunch o* $uman %enome proDect
0::7 DNA *in%erprintin% #y AF;P /o! et al1
2<<0 $uman %enome !eCuencin%1 /enter et al
2<<2 Dra*t !eCuence o* 3ice %enome "o** et al
2<<3 Dra*t !eCuence o* 1indica =u et al
2<<7 'ap #a!ed !eCuence o* 3ice %enome
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 7
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
T%PES OF TISSUE CULTURE
&a' Call() !(lt(re*
Callus culture may #e de*ined a! production and maintenance o* an
unor%ani!ed ma!! o* proli*eratiBe cell *rom i!olated plant cell, ti!!ue! or
or%an! #y %ro@in% them on arti*icial nutrient medium in %la!! Bial! under
controlled a!eptic condition!1
&b'Or+an !(lt(re*
5he organ culture re*er! to the in vitro culture and maintenance o* an exci!ed
or%an primordial or @hole part o* an or%an in @ay that may allo@
di**erentiation and pre!erBation o* the architecture andEor *unction1
&!' Sin+le !ell !(lt(re*
Single cell culture i! a method o* %ro@in% i!olated !in%le cell a!eptically on
nutrient medium under controlled condition!1
&d' S()en)ion !(lt(re*
Suspension culture i! a type o* culture in @hich !in%le cell or !mall
a%%re%ate! o* cell multiply @hile !u!pended in a%itated liCuid medium1
&e' E$br"o !(lt(re*
Embryo culture may #e de*ined a! a!eptic i!olation o* em#ryo )o* di**erent
deBelopmental !ta%e!+ *rom the #ul, o* maternal ti!!ue! o* mature !eed or
cap!ule and in vitro culture under a!eptic and controlled phy!ical condition in
%la!! Bial! containin% nutrient !olid or liCuid medium to %ro@ directly into
plantlet
&#' Ant,er !(lt(re*
ndrogenesis i! the in vitro deBelopment o* haploid plant! ori%inatin% *rom
potent pollen %rain! throu%h a !erie! o* cell diBi!ion and di**erentiation1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 8
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
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&+' Pollen !(lt(re*
!ollen culture i! an in vitro techniCue #y @hich the pollen %rain! )pre*era#ly
at the micro!pore !ta%e + are !Cueezed out a!eptically *rom the intact anther
and then cultured on nutrient medium @here the micro!pore! @ithout
producin% male %amete!, deBelop into haploid Embryoids or callu! ti!!ue
that %aBe ri!e to haploid plantlet! #y em#ryo%ene!i! or or%ano%ene!i!1
&,' So$ati! E$br"o+ene)i)*
Somatic embryogenesis i! the proce!! o* a !in%le cell or %roup o* cell!
initiatin% the deBelopmental path@ay that lead! to reproduci#le re%eneration
o* non zy%otic em#ryo! capa#le o* %erminatin% to *orm complete plant!1
&i' Protola)t !(lt(re*
It i! the culture o* i!olated protopla!t! @hich are na,ed plant cell!
!urrounded #y pla!ma mem#rane @hich i! potentially capa#le o* cell @all
re%eneration, cell diBi!ion, %ro@th and plant re%eneration on !uita#le medium
under a!eptic condition!
OVERVIEW OF TISSUE CULTURE
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 9
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
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ADVANTAGES OF TISSUE CULTURE
Plant ti!!ue culture o**er! !eBeral adBanta%e! oBer conBentional propa%ation
method! Be%etatiBe and !exual *or lar%e !cale micropropa%ation1
Shoot multiplication can #e achieBed in !mall !pace1
Propa%ation i! carried out under !terile condition!1 No1 dama%e i! cau!ed
#y in!ect! and di!ea!e!1
In ca!e Biru! *ree material i! u!ed, a lar%e num#er o* Biru! *ree plant! can
#e o#tained1
Plant ti!!ue culture i! carried out under de*ined condition! o*
enBironmental, nutritional and ti!!ue !y!temI there*ore it i! hi%hly
reproductiBe !y!tem under de*ined !et o* condition!1
5he production! i! una**ected #y !ea!onal Bariation1
No care i! reCuired #et@een t@o !u#culture! a! compared to
conBentional method o* Be%etatiBe method o* propa%ation1
Small %la!! hou!e i! reCuired #ecau!e o* !mall miniature !ize plantlet!1
'other plant or %enotype o* !toc, plant can #e !tored and maintained in
vitro @ithout dama%e to enBironment *actor! and !toc, plant!1
It i! po!!i#le to mechanize @hole proce!! o* Be%etatiBe propa%ation *or
lar%e !cale plantation1
5he plant! @hich are di**icult to propa%ate Be%etatiBely #y conBentional
method! can #e propa%ated #y thi! method1
'iniature !tora%e or%an! can #e produced *or %enotype !tora%e and
!u#!eCuent plantation!1
Bein% !terile, tran!port acro!! countrie! i! permi!!i#le @ithout di**icultie!1
Plant ti!!ue culture i! a collection o* techniCue! u!ed to maintain or %ro@
plant cell!, ti!!ue! or or%an! under !terile condition! on a nutrient culture
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 10
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
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medium o* ,no@n compo!ition1 Plant ti!!ue culture i! @idely u!ed to produce
clone! o* a plant in a method ,no@n a! micropropa%ation
-ICROPROPAGATION*
'icropropa%ation i! the practice o* rapidly multiplyin% !toc, plant material to
produce a lar%e num#er o* pro%eny plant!, u!in% modern plant ti!!ue culture
method!1 'icropropa%ation i! u!ed to multiply noBel plant!, !uch a! tho!e
that haBe #een %enetically modi*ied or #red throu%h conBentional plant
#reedin% method!1 It i! al!o u!ed to proBide a !u**icient num#er
o* plantlet! *or plantin% *rom a !toc, plant @hich doe! not produce !eed!, or
doe! not re!pond @ell to Be%etatiBe reproduction1
Frederi!. Ca$ion Ste/ard @a! the per!on @ho di!coBered and
pioneered micropropa%ation and plant ti!!ue culture in the late 0:7<! and
early 0:><!
OB0ECTIVES OF -ICROPROPAGATION
Production o* Biru! *ree !toc,
5o multiply plant! @ho!e multiplication rate i! Bery lo@
5o produce pro%enie! @hich are %enetically identical to their parent!
5o o#tain %enetic Baria#ility
3ecoBery o* di!tant hy#rid!
"ermpla!m con!erBation
"ermpla!m exchan%e
"enetic tran!*ormation J addition or deletion o* %ene
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 11
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
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ADVANTAGES OF -ICROPROPAGATION*
Plant ti!!ue culture o**er! !eBeral adBanta%e! oBer conBentional propa%ation
method! Be%etatiBe and !exual *or lar%e !cale micropropa%ation1
Shoot multiplication can #e achieBed in !mall !pace1
Propa%ation i! carried out under !terile condition!1 No1 dama%e i! cau!ed
#y in!ect! and di!ea!e!1
In ca!e Biru! *ree material i! u!ed, a lar%e num#er o* Biru! *ree plant! can
#e o#tained1
Plant ti!!ue culture i! carried out under de*ined condition! o*
enBironmental, nutritional and ti!!ue !y!temI there*ore it i! hi%hly
reproductiBe !y!tem under de*ined !et o* condition!1
5he production! i! una**ected #y !ea!onal Bariation1
No care i! reCuired #et@een t@o !u#culture! a! compared to
conBentional method o* Be%etatiBe method o* propa%ation1
Small %la!! hou!e i! reCuired #ecau!e o* !mall miniature !ize plantlet!1
'other plant or %enotype o* !toc, plant can #e !tored and maintained in
vitro @ithout dama%e to enBironment *actor! and !toc, plant!1
It i! po!!i#le to mechanize @hole proce!! o* Be%etatiBe propa%ation *or
lar%e !cale plantation1
5he plant! @hich are di**icult to propa%ate Be%etatiBely #y conBentional
method! can #e propa%ated #y thi! method1
'iniature !tora%e or%an! can #e produced *or %enotype !tora%e and
!u#!eCuent plantation!1
Bein% !terile, tran!port acro!! countrie! i! permi!!i#le @ithout di**icultie!1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 12
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
-EDICINAL PLANTS
Durin% the thou!and! o* year! o* early human exi!tence many natural
material @ere identi*ied *or com#atin% human ailment either #y in!tinct or
intuition or trial and error1 5he earlie!t mention o* the medicinal u!e o* plant!
ha! #een *ound in K3i% /eda-, @hich @a! @ritten #et@een 4<<< and 0><<
B1&1 In the GAtharBa /edaL, @e *ind the more Baried u!e o* dru%!1 It i! the
GAyurBedaL, @hich i! con!idered a! an G6pa /edaL that de*inite propertie! o*
dru%! and their u!e! haBe #een %iBen in %reat detailI G&har,a SamhitaL i!
another earlie!t treati!e on GAyurBedaL )><<B1&1+ li!t! a total o* 340 plant!
and plant product! *or u!e in health mana%ement1 GSu!ruta SamhitaL al!o
dealt @ith plant! related to medicine1 Su#!eCuent author! o* later treati!e!
haBe extended the li!t o* AyurBedic !in%le plant dru%! to the !ix hundred
!pecie! o* plant! )9ain S1(1+1
It ha! #een e!timate that out o* a#out 2,<<< dru%! that haBe #een
u!ed in curin% human ailment! in India, only a#out 2<< are o* animal ori%in
and a !imilar num#er are o* mineral ori%in1 5he re!t, i1e1 a#out 0,7<<, are o*
plant ori%in1 5hi! num#er i! not Bery lar%e con!iderin% the Ba!t area o* our
country, and the @ide Bariety o* plant @ealth occurrin% therein1 5he %reat
ran%e o* temperature )a#out 4:M &+, rain*all )*rom 0<< mm to oBer 0<,<<<
mm+ and altitude )!ea.leBel to oBer >,<<<m+ in India account *or the
occurrence o* oBer 07,<<< !pecie! o* hi%her plant!1
In India, the u!e o* di**erent part! o* !eBeral medicinal plant! to cure
!peci*ic ailment! ha! #een in Bo%ue *rom ancient time!1 5he indi%enou!
!y!tem o* medicine namely AyurBedic, !iddha and 6nani haBe #een in
exi!tence *or !eBeral centurie!1 Apart *rom India the!e !y!tem! o* medicine!
are preBalent in (orea, &hina, Sin%apore, 4e!t A!ia and many other
countrie!1 Be!ide! the demand! made #y the!e !y!tem! a! their ra@
material, the demand *or medicinal plant! made #y the modern
pharmaceutical indu!trie! ha! al!o increa!ed mani*old1 5hu! medicinal
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 13
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
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plant! con!titute a %roup o* indu!trially important crop! @hich #rin%
apprecia#le income to the country #y @ay o* export1
India i! a Ba!t country haBin% @ide Bariation! in climate !oil altitude
and latitude @ith Bery rich #otanical @ealth and a lar%e num#er o* diBer!e
type! o* plant! %ro@ @ild indi**erent part! o* the country1 Increa!ed
a@arene!! a#out the potential o* thi! %roup o* intere!tin% and u!e*ul plant!
ha! encoura%ed many innoBatiBe and pro%re!!iBe %ro@er! and
entrepreneur! to ta,e up their cultiBation a! a commercial enterpri!e1 Apart
*rom health care thi! enterpri!e proBide! mean! o* liBelihood to !core! o*
people1
$o@eBer, our ,no@led%e o* medicinal plant! ha! mo!tly #een
inherited traditionally1 6!e o* plant! *or curin% Bariou! ailment! are not
con*ined to the doctor! only #ut i! ,no@n to !eBeral hou!ehold! a! @ell1
In modern medicine al!o plant! occupy a Bery !i%ni*icant place a! ra@
material *or !ome important dru%!, althou%h !ynthetic dru%! and anti#iotic!
#rou%ht a#out a reBolution in controllin% di**erent di!ea!e1 But the!e
!ynthetic dru%! are out o* reach o* million! o* people1 5ho!e @ho liBe in
remote place! depend on traditional healer!, @hom they ,no@ and tru!t1
9udiciou! are o* medicinal her#! can eBen cure deadly di!ea!e! that haBe
lon% de*ied !ynthetic dru%!1

ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 14
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
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BIOTECHNOLOGICAL APPROACHES TOWARDS I-PROVE-ENT OF
-EDICINAL PLANTS*
$er#! are no@ i! %reat demand in #oth deBeloped and deBelopin%
countrie! #ecau!e o* their proBen e**icacy and little or no reported !ide
e**ect!1 Secondary meta#olite!, the actiBe principle! experience to produce
and accumulate are u!ually #io!ynthe!ized in !maller Cualitie!1 5hi! ha!
re!ulted in ruthle!! exploitation o* medicinally important plant! creatin%
im#alance in !upply and demand1 An alternatiBe technolo%y could #e the
application o* in-vitro culture o* de!ira#le medicinal plant! to increa!e the
plantation propa%ate! and enhance the yield! o* !peci*ic dry component!1
Succe!!*ul micro propa%ation protocol! *or Bariou! medicinal plant! haBe
*rom deBeloped and their con!erBation ha! al!o #ecome *ea!i#le throu%h
!ynthetic !eed! and cryopre!eBation techniCue!1 Be!ide! the!e techniCue!,
%enetic en%ineerin% o* medicinal plant! u!in% a%ro#acterium medicinal
tran!*ormation ha! many adBanta%e! that include *a!t %ro@n and hi%h leBel
o* !ta#le production o* !econdary meta#olite! ma,in% thou%h commercially
and economically *ea!i#le1 5i!!ue culture rai!ed plant! can #e a!certained
#y u!in% molecular ma,er!1
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IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 16
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
A Brie# a!!o(nt on Wedelia chinensis
S!ienti#i! !la))i#i!ation
(IN"D': Plantae
)6N3AN(2D+: An%io!perm!
)6N3AN(2D+: 2udicot!
3D23: A!terale!
FA'I;=: A!teraceae
S6BFA'I;=: A!teroideae
"2N6S: "edelia
"edelia chinensis i! a *lo@erin% plant %enu! in the Sun*lo@er *amily
A!teraceae15hey are one o* the %enu! commonly called Gcreepin%
oxeye!L15he name o* the %enu! honour! "erman phy!ician! "eor%e
4ol*%an% 4edel)0>47.0?20'
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 17
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
CO--ON NA-ES
SANS Bhrin%araDa,pita#hrin%i
HINDI Bhanra,#han%ra
BENG Bhimra ,,e!raD
-AR PiBalama,a
GU0 Bhan%aro
TA- Potralai,aindha,eerai
1AN (ae!arDi
-AL A!@a%andhi
De)!rition*Plant! creepin%1 ;eaBe! 2.?cm len%th, >.02mm @idth1
Flo@er! yello@ 2.217cm, *lo@erin% in 9uly to cto#er1 Perennial plant!1
Di)trib(tion*9apan+ $on!hu )IzuPenin!la, (ii Penin!ula+, Shi,o,u,
(yu!hu, ,ina@a)ther nation!+ (orea, &hina, 5ai@an, Philippine!,
/ietnam, ;ao!, &am#odia, 'alay!ia, India1
A procum#ent, perennial her#,@ith li%ht camphor li,e odour,met @ithin @et
place! in 6P,A!!am , AP, and all alon% the coa!tal area!1Stem 47 cm tall,
leaBe! !u#!e!!ile , linear.o#lon%, o#lanceolate, head !olitary, #ri%ht yello@,
on lon% axillary peduncle!, up to 4 cm in diam1
5@o di**erent plant! are mentioned in the literature a! #hringara$a or
#hangara %Eclipta alba and "edeliachinensis#oth o* @hich are u!ed *or
medicinal purpo!e!)&handra,Nuart1D1crude Dru% re!1,0:>:,:,04>0I(arnic,,
ActaPhytother1, Anr!t1, 0:?<,0?,080I@ith INDIA.ra@ material, 02?+
5he her# i! !aid to po!!e!! propertie! !imilar to tho!e o* Ecliptaalba.5he
leaBe! are u!ed *or dyein% %rey hair, and *or promotin% their %ro@thItheir
Duice i! u!ed *or tattooin%, the colour produced #ein% a deep indeli#le #iue
#lac,1 5he root i! pounded and u!ed a! a #lac, dye @ith !alt! o* iron15he
leaBe! are re%arded a! tonic and alternatiBe, u!e*ul in cou%h, cephal%iaetc1A
decoction o* the her# i! u!ed in uterine haemorrha%e)(irt and #a!u , 0:>8+
5he expre!!ed Duice o* her# contain an oil.!olu#le #lac, die, 0012I 5annin,
22<I carotene,0104Ichlorophyll,31?7I!aponin,7<<I phyto!terol,31?7I @axy
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 18
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
compound!, 2:1?I re!in,44I chloro*orm,2?I%um,8<I total !u%ar,0<4<m%El<<%
Duice1An Indian analy!i! o* the her# %aBe ne%atiBe te!t *or al,aloid, #ut the
&hine!e inBe!ti%ation !ho@ed the pre!ence )Bhattacharya 0:70I 'aiti ,
0:>8 I 4illman F ;i,;loydia ,0:?<+ 5he leaBe! contain i!o*laBanoid! and
@edelolactone)<1<7O+15he latter i! the iactone o* 7:>.dihydroxy.2)2:>.
dihydroxy.4 methoxyphenyl+ #enzo*uran.3.car#oxylic acid and i! analo%ou!
in !tructure to cume!terol an e!tro%en *rom cloBer )"oBindachari et al.&0:>0+1
-EDICINAL USES
2 3eputed her#al medicinal 1
3 &ou%h, cephal%ia, liBer, !pleen, !,in di!ea!e!1
4 ;ea* extract u!ed a! hair tonic1
5 ;eaBe! are u!ed in dyein% %rey hair1
6 ;eaBe! are u!ed in promotin% %ro@th o* hair1
7 9uice i! u!ed in !nu** in cephal%ia1
8 Seed!, *lo@er!, leaBe! are u!ed in decoction1
9 5reatment o* o!teoporo!i! o* ,nee1
: AyurBedic preparation1
2; 6!ed in dru% indu!trie!1
5he !pecie! i! economically an important medicinal !pecie! hence there i!
need to ma!! multiply the !pecie! *or %eneration o* #ul, plantin% material *or
pharmaceutical indu!trie!1 4ith thi! Bie@ in mind the pre!ent e**ort @a!
carried out @ith the *ollo@in% o#DectiBe!:.
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 19
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
OB0ECTIVES
5o deBelop !terilization protocol *or e!ta#li!hin% a!ceptic culture o*
"edelia chinensis
5o e!ta#li!h a protocol *or callu! *ormation o* "edelia chinensis *rom
leaBe! and node! a! explant!1
5o e!ta#li!h a protocol *or in Bitro propa%ation o* "edelia chinensis
*rom axillary #ud1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 20
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
REVIEW OF LITERATURE
Ra,$an.-.- and B,adra S.1. &3;;:' !tudied in Bitro propa%ation o*
4edeliachinen!i!15he nodal !e%ment! @ere %ro@n on 'S #a!al media
!upplemented @ith di**erent com#ination and concentration o* cyto,inin )BAP
and (n+ and auxin )IAA and NAA+15he maximum num#er o* multiple !hoot
#ud!Eexplant! diBided *rom nodal !e%ment! culturin% on 'S media
!upplemented @ith 31< m%El BAP and <17 m%El IAA1 Shooted plantlet! @ere
tran!*erred to rootin% media !upplemented @ith 21< m%El IBA1
Ha<(e et al. &3;;:' deBeloped an e**icient and lar%e !cale in Bitro
clonal propa%ation o* Balua#le medicinal plant &hlorophytum#oriBilianum
throu%h callu! culture #y enhanced !hoot cro@n explant and leaBe!1 &ro@n
explant! and leaBe! *rom &hlorophytum#oriBilianum initially treated @ith
BaBi!tin <10O and <10O 2ndo!ulphan and <17O Fluconazole @ere cultured
in !olidi*ied modi*ied 'ura!hi%e and !,oo% )'S+ media, each @ith di**erent
hormonal com#ination *or the e!ta#li!hment o* culture!,callu! induction,
re%eneration o* callu! @ith !omatic em#ryo!, !omatic em#ryo
*ormation1!omatic em#ryo maturation, !omatic em#ryo maturation, !omatic
em#ryo %ermination, multiplication and rootin% o* plant!1 SeBeral plant
%ro@th re%ulator! 2.4 D)2,4.Dichloro phenoxy acetic acid+1
-()ta)i$ -. 1 et al. &3;;8' !tudied a method *or rapid in Bitro
propa%ation #y multiple !hoot induction o* /ernoniaamy%dalina , an
important medicinal plant #elon%in% to compo!itae *amily1 'ultiple !hoot!
@ere induced in Bitro *rom the !tem nodal !e%ment! on 'S media
containin% BAP alone or in com#ination @ith NAA and (inetin alone or in the
com#ination @ith 2,4.D1'aximum num#er o* !hoot! )?12 !hoot! per explant+
@a! o#!erBed on the media containin% BAP )<17 m%El+ in com#ination @ith
NAA )<17 m%El+1 3e%enerated !hoot! @ere rooted on 'S media
!upplemented @ith 2m%El NAA1 5he rooted in Bitro rai!ed plantlet! @ere
acclimati!ed in %ro@th cham#er and and !ucce!!*ully tran!*erred to %reen
hou!e1
Ra,$an=EI> Wa.il H=EI > Said=EI > 1,li#a H.D &3;;9'1 deBeloped
!u**icient and promi!in% protocol *or enhancement and production o*
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 21
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
!copolamine and hyo!cyamine in calli and re%enerate culture! o*
Daturametel @a! e!ta#li!hed1 5he hi%he!t Balue o* calli @a! produced *rom
hypocotyl , lea*, !tem and root explant! re!pectiBely1 '! media
!upplemented @ith 0 m%El o* NAA and BA %aBe the #e!t re!ult! o* calli
production1 $o@eBer, 'S medium containin% 0m%El o* 2,4.d and (in re!ulted
the maximum num#er o* !hoot! *ormation *rom lea* explant! a! compared
@ith other type o* explant!1&alli and Be%etatiBe %ro@th dynamic! @ere
inBe!ti%ated durin% 7 @ee,! o* cultiBation1
Sarit,a > Naid( &3;;8' !tudied In Bitro *lo@erin% , 'n vitro *ruitin% and
e**ectiBe micropropa%ation protocol in 4ithania!omni*era , an anti tumour
medicinal plant u!in% axillary #ud explant!1 5he 'S !upplemented @ith N
>.
#enzyl adenine )BA+ 21< m%El and NAA <10 m%El @a! *ound optimum *or
production o* multiple !hoot!1 5he re%enerated plantlet! @ere *ound to *orm
tiny %reen *ioral #ud! a*ter 4.> @ee,! o* culture in 'S medium !upplemented
@ith ,inetin )<17m%El+ and IAA )<10m%El+1 5hi! paper de!cri#e! In Bitro
*lo@erin% !y!tem to oBercome pro#lem! a!!ociated @ith *lo@er %ro@yh and
deBelopment a! @ell a! *ruit and !eed production in Bitro1
L""a..ann(Si?ane)an and B"o(n+R"o(n+0eon+ &3;;:' deBelop
relia#le protocol *or direct and indirect !hoot re%eneration1 A maximum
num#er o* !hoot! @a! o#tained *rom nodal explant! @hen cultured on 'S
medium !upplemented @ith 01< m%El BAP, <17 m%El IBA and 21< m%El adenine
!ul*ate1 3e%enerated !hoot! @ere rooted #e!t on hal* !tren%th 'S medium
containin% <17 m%El NAA and 3O )@EB+ !ucro!e1 5he re%rnerated plantlet!
@ere acclimatized in the culture room and !ucce!!*ully tran!*erred in !oil1
T.-alli.ade?i> P. Sent,il.($ar> S. Pa(l)a$" !tudied the in Bitro
re%eneration o* Plum#a%ozeylanica and exhi#ited that the callu! @a!
initiated in the #a!al medium containin% BAP, NAA, 2,4.D and IBA1 5he hi%h
amount o* or%anic calli @a! induced in the #a!al medium !upplemented @ith
2,4.D alone at 21< m%El1 In the !u#culture the adBentitiuo! !hoot *ormation
@a! prominiently hi%her )83O+ in the #a!al medium containin% BAP and NAA
at 317 m%El and <13 m%El re!pectiBely1 IAA)01<m%El+ e**ectiBely produced
hi%her percenta%e ):<O+ o* root! and root %ro@th1 A*ter !eCuential
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 22
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
hardenin%, !urBi#ility rate @a! o#!erBed to #e !i%ni*icantly hi%her )8<O+ in
the hardenin% medium containin% %arden !oil, !and and Bermicompo!t in the
ratio o* 0:0:0 #y Bolume under %reen hou!e condition!1
Siddi<(e %a)ir Ha))an et al. &3;;6' !tudied the e**ect o*
Plum#a%ozeylanica extract! a%ain!t the toxic e**ect induced #y 0<
u'ethinyle!tradiol in cultured human peripheral #lood lymphocyte! u!in%
chromo!omal a#erration a! a parameter and in the third in!tar larBae o*
tran!%enic Dro!ophilla melano%a!ter )h!p ?<. lac H+ B%: )<17< and 01< ulEml
o* ethyniyle!tradiol+ u!in% h!p ?< expre!!ion a! a parameter1 5he !elected
do!e! o* P1zeylanica extract i1e 01<?7x0<.4, 21027x0<.4, 3107x0<.4 and
410?x0<.4 %Eml @ere not toxic #ut @ere potent enou%h to reduce the toxic
e**ect induced #y ethynyle!tadiol in cultured human #lood lymphocyte!1
Tila.> 0ai> Ad,i.ari> So($a"a.anti> T,o$a) !tudied the #ene*icial
e**ect!, antioxidant, anti athero%enic, cardiotonic, hypoprotectiBe, and neuro
protectiBe propertie! o* P1zeylanica )&hitra,+1 5hey al!o !tudied that root
#ar, o* P1zeylanica contain! Plum#a%in )in !econdary cortex and medullary
ray cell!+, *ree %luco!e, *ructo!e and enzyme protea!e and inBerta!e1 3oot!
are u!e*ul in !timulatin% di%e!tiBe proce!! and po!!e!! a#oti*acient and
Be!icant propertie!1 In conclu!ion, their !tudy reBeal! that the extract o*
P1zeylanica and it! actiBe in%redient Plum#a%in haBe !i%ni*icant antioxidant
a#ilitie! that may po!!i#ly explain !ome o* the reported therapeutic e**ect!1
-a,ari),i R.P &3;;7' !tudied Fuarium !olani F 3hizoctonia
#ataticola cau!in% !eedlin% mortality in A!h@a%andha, myco*lora a!!ociated
@ith !eed! o* 9a@ahar a!h@a%andha !ho@ed hi%h O o* #oth F1 !olani and 31
#ataticola cau!in% lo@ %ermination o* !eed! and !eedlin% mortality
Fun%icide! and 5richoderma !pp1@a! e**ectiBe in redu!in% !eedlin%
mortality1
Patel 1 V. et al. &2::8=:9' did a *ield experiment to !tandadi!e tech1
to rai!e a a %ood crop o* A!h@a%andha, a*optin% di**1 !eed rate and crop
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 23
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
duration1 3e!ult! indicated that neither the crop duration nor !eed rate!
in*luenced hei%ht, len%th,or %irth o* root1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 24
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
-ATERIALS AND -ETHODOLG%
-ATERIALS*
2' LABORATOR% RE@UIRE-ENTS:
Gla))/are) : 5e!t tu#e!, Petridi!he!, Fla!,!, 'edia #ottle!, &onical
*la!,!, 'ea!urin% cylinder, Bea,er1
E<(i$ent) * &hemical Balance, p$ meter, AutoclaBe, ;aminar *lo@
@ood *or a!eptic tran!*er, pen plat*orm or#ital !ha,er, &on!tant
temperature room or incu#ator, 3e*ri%erator1
3' OTHER RE@UIR-ENTS:
Wa),in+ Area*
5hi! i! Bery important *or a ti!!ue culture la#oratory1 It !hould #e proBided
@ith a lar%e !in,, runnin% hot and cold tap @ater, #ru!he! o* Bariou! !ize!,
deter%ent and a #uc,et o* !in%le di!tilled @ater *or a *inal rin!e o* the
@a!hed %la!! %ood!1
Hot Air O?en*
It i! nece!!ary *or dryin% the @a!hed %la!! %ood!1
Re#ri+erator* It i! e!!ential *or !torin% Bariou! thermo la#ile chemical!
li,e Bitamin!, hormone!, amino acid!, ca!ein hydroly!ate, yea!t extract
coconut mil,,etc1 Stoc, !olution! o* !alt! are al!o ,ept to preBent
contamination1
Di)tillation Plant: A !in%le di!tillation and a dou#le @ater plant are
indi!pen!a#le1 5@o #i% pla!tic container! are reCuired *or !torin% the
di!tilled @ater.
Wei+,in+ balan!e* 5hree type! o* @ei%hin% #alance! Biz1 pan #alanceI
chemical #alance and electric #alance are reCuired *or @ei%hin%
chemical!, !u%ar!, a%ar and other!1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 25
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
Va!(($ P($* It i! reCuired *or *ilterin% liCuid media, !u%ar !olution etc1
throu%h *ilter apparatu! u!in% air !uction1
A(to!la?e* It i! Bery important *or !terilization o* nutrient media, %la!!
%ood! etc
Wor.in+ Table)* 5he!e are nece!!ary *or preparation o* medium1
Wooden Ra!.)* 5he!e are reCuired *or ,eepin% the Bariou! chemical!1

LABORATOR% ORGANISATION*
$o@eBer a !tandard ti!!ue culture la#oratory !hould proBide *acilitie! *or.
4a!hin% and !tora%e o* %la!!@are, pla!tic @are
Preparation, !terilization and !tora%e o* nutrient media1
A!eptic manipulation o* plant material
#!erBation o* culture!1
'aintenance o* culture! under controlled condition!1
Acclimatization o* in Bitro plant! in mi!t cham#er and %reen hou!e1
2' WASHING ROO-*
5he conBentional method *or cleanin% la#oratory %la!!@are inBolBe!
&hromic Acid JSulphuric acid !oa, *ollo@ed #y thorou%h @a!hin% @ith tap
@ater F !u#!eCuent rin!in% @ith di!tilled @ater1 $o@eBer due to corro!iBe
nature o* acid!, it i! recommended that *or routine procedure! %la!!@are
!hould #e !oa,ed in 2Odeter%ent cleaner *or 0> hr! *ollo@ed #y @a!hin%
@ith ><.?< de%ree hot tap @ater, F *inally di!tilled @ater1 5he cleaned
%la!!@are !hould #e then dried at 07<.2<<Pc in a conBection dryin% oBen *or
0.4 hr!1 $i%hly contaminated %la!!@are !hould #e *ir!t treated in a chromic
acid #ath *or 0> hr!1 4a!hin% room !hould #e proBided @ith #ru!he! o*
Bariou! !ize! F !hape! ,a @a!hin% machine ,a lar%e !in, )pre*era#ly lead
lined to re!i!t acid! F al,alie!+ Frunnin% cold Fhot @ater1 5here !hould #e
!teel or pla!tic #uc,et! to !oa, the %la!!@are to #e @a!hed, oBen! or hot air
ca#inet! to dry the @a!hed %la!!@are F a du!t proo* cup#oard to !tore
them1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 26
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
T,e #ollo/in+ re!a(tion) ),o(ld be #ollo/ed*
&ontent! o* container! !hould #e di!carded immediately a*ter completion
o* an experiment1
&ontaminated container! !hould #e autoclaBed, content! di!carded in a
@a!te #ottle or container F then @a!hed1 5he @a!te #ottle containin%
autoclaBed contaminated media !hould not #e emptied in the la#oratory
!in,1
Fla!,! or #ea,er! u!ed *or a%ar #a!ed media !hould #e rin!ed
immediately a*ter di!pen!in% the media in to culture Be!!el! to preBent
dryin% o* the re!idual a%ar in the #ea,er prior to @a!hin%1
3' -EDIA ROO- *
A %ood !upply o* @ater i! a mu!t *or media preparation1 Since tap @ater
can-t #e u!ed *or preparin% medium, proBi!ion mu!t #e made to puri*y
@ater reCuired1 5he u!ual *acilitie! reCuired *or the preparation o* culture
medium include1
A deep *reezer *or !torin% the !toc, !olution!, enzyme i!olation! etc1
A re*ri%erator to !tore Bariou! chemical!, plant material!, !hort.term
!tora%e o* !toc, !olution! etc1
Pla!tic container! *or !torin% di!tilled @ater1
4ei%hin% #alance!1
A hot plate cum ma%netic !tirrer *or di!!olBin% chemical!1
A p$ meter1
An a!pirator or Bocal pump to *acilitate *ilter !terilization1
A !teamer *or nettin% a%ar
An autoclaBe or a dome!tic pre!!ure coo,er *or media !terilization1
An ideal ti!!ue culture la#oratory !hould haBe at lea!t t@o #i% room! and a
!mall room1 ne #i% room i! *or "eneral la#oratory @or, !uch a! @a!hin%,
preparation o* media, autoclaBin%, di!tillation o* @ater etc1 5he other #i%
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 27
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
room i! *or ,eepin% culture! under controlled li%ht temperature and humidity1
5he !mall room i! *or a!eptic 4or, and *or ,eepin% autoclaBed article!1
4' TRANSFER AREA *
Finally it i! Bery e!!ential that all precaution! #e ta,en to preBent the entry
o* any contaminant into the culture Bial @hen it! mouth i! opened either *or
!u#culture or *or inoculatin% *re!h ti!!ue!1 5o achieBe thi!, all tran!*er
operation! are carried out under !trictly a!eptic condition! F ;aminar Air *lo@
ca#inet! are u!ed1 5hi! ha! a !mall motor to #lo@ air that *ir!t pa!!e!
throu%h a coar!e *ilter, @here it lo!e! lar%e particle! F !u#!eCuently throu%h
a *ine *ilter )$2PA *ilter+ hi%h e**iciency per*orated air *ilter @hich ultra clean
the air1 5he Belocity o* the air comin% out o* thi! *ilter i! a#out 2?QR3m min .0
that i! adeCuate *or preBentin% the contamination o* the @or,in% area1
5' CULTURE ROO-*
Plant ti!!ue culture! !hould #e incu#ated under condition! o* @ell controlled
temp1, illumination, photoperiod, humidity F air circulation1 For mo!t culture
condition! temp1 , control !hould #e adeCuate to !tay @ithinQR0de%ree in a
ran%e *rom 0< .32Pc1 5he photoperiod may #e !et accordin% to the !peci*ic
type o* culture1 "la!! te!t tu#e!I conical *la!,! and @ide mouth #ottle! o*
di**erent !ize! are %enerally u!ed a! culture Be!!el!1 For culture tu#e! F
*la!,!, traditional cotton plu%! are u!ed1 5he culture room i! maintained at a
controlled temp1 6!ually air conditioner! and heater! attached to a
temperature controller are u!ed to maintain the temperature around 27QR21
&ulture! are %enerally %ro@n in di**u!ed li%ht )le!! than 0 , lux+1 5he culture
room !hould #e proBided @ith !pecially de!i%ned !helBe! to !tore culture!1
2ach !hel* i! proBided @ith a !eparate !et o* *luore!cent tu#e!1
6' GROWTH CHA-BER*
"ro@th &ham#er! F "reen hou!e o* di**erent ,ind! are reCuired at !eBeral
!ta%e! o* the ti!!ue culture proce!!I
5o let the explant! %ro@ F deBelop in!ide te!t tu#e! F Dar!1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 28
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
5o let the plant! to %ro@ to ordinary !ize a*ter their remoBal *rom te!t
tu#e!1
Gro/t, C,a$ber) For EAlant)*
A cham#er !uited to %ro@th o* explant! !hould #e thermally conditioned !o
that it! air temperature doe! not Bary more thanQR0Pc *rom the optimum
temp1 I* !mall !cale experiment! are per*ormed, illuminated #acteriolo%ical
incu#ator! may #e !u**icient, #ut i* ma!! production i! planned, an arti*icially
illuminated F temp1 controlled room i! e!!ential1 In addition to air
conditionin%, a !y!tem o* internal Bentilation !hould #e deBi!ed to preBent
poc,et! o* heated air #ein% *ormed F remainin% !ta%nant, @hich can #e a
pro#lem in a room *illed @ith illuminated !helBe!1 $eated air can !ta%nate
#et@een the !helBe! near the lamp!, @hich may heat #oth the !urroundin%
air and the !helBe! a#oBe them1 &on!eCuently, a net@or, o* *an! !hould #e
in!talled at the ed%e o* each !hel* depth !hould not exceed 8<.:<cm i* their
acce!! i! limited to one !ide, @hile dou#le that !ize i! accepta#le i* the
acce!! i! *rom t@o !ide!15he %ro@th cham#er it!el* may #e in!talled either in
an internal room o* #uildin% or may #e ea!ily #uilt @ith pre*a#ricated panel!1
In either ca!e it !hould #e @ell in!ulated @ith appropriate material in order to
aBoid lea,a%e to heat1
7' GREEN HOUSE FOR ACCLI-TIBATION*
3ooted plantlet!, %ro@in% in clo!ed Dar, in la#oratory condition! cannot #e
#rou%ht directly into the natural enBironment or eBen into the normal %reen
hou!e condition! #ecau!e the leBel o* humidity i! too lo@1 In!ide the Dar, the
relatiBe humidity i! Bery hi%h, and acclimatization !hould #e %radual1
Furthermore, a*ter the initial rootin% pha!e, the !oil i! only di!in*ected F no
lon%er completely !terilized !o that condition! *aBourin% re.in*ection mu!t #e
aBoided1 5here*ore the Dar! !hould #e opened in a !pecial !ection o* la# *or
the tran!*er o* plantlet! to di!in*ected earth1 Be*ore the tran!*er, all !u%ary
media !hould #e @a!hed o** the root! in order to aBoid the deBelopment o*
mould! F #acteria1 5he !oil may #e di!in*ected #y applyin% &$3Br or #y
autoclaBin%1 It i! then u!ed to *ill !pecially deBi!ed !erial pot! made o* paper,
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 29
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
peat o* #lac, pla!tic *ilm1 4hicheBer type i! cho!en, #ut they can #e handled
!erially in lar%e num#er! F that the plant! may #e ea!ily tran!planted1
nce the plantlet! are planted in the !oil, the tray! @ith the pot!
!hould #e tran!*erred into !pecial %reen hou!e!, F under !pecial tunnel!
made *rom pla!tic *ilm1 6nder the tunnel, *inely atomized @ater i! !prayed to
,eep the humidity Bery hi%h1 5he !pray i! intermittent Fcontrolled #y Bariou!
,ind! o* !en!or!1 In many area!, @ater mu!t #e deminerali!ed #e*ore u!e to
aBoid nozzle o#!truction throu%h lime #uilt up1 5he humidity #ein% !o hi%h,
hy%ienic condition!, initial di!in*ection! o* the %reen hou!e F e**icient
draina%e are o* paramount importance to aBoid the !pread o* di!ea!e!1
nce the plantlet! are acclimatized F haBe oBercome the !hoc, o*
tran!plant, the humidity can #e %radually decrea!ed in order to accu!tom
them to ordinary condition! o* %ro@th, initially in a normal %reen hou!e F
later in the open air1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 30
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
PHOTOPLATE.;2* LABORATOR% E@UIP-ENTS USED IN PLANT
TISSUE CULTURE
HOT AIR
OVEN
VERTICAL
AUTOCLAVE
CULTURE
ROO-
GREEN
HOUSE
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 31
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
-ETHODOLOG%
STERILIBATION
5he contaminant! may %iBe out meta#olic @a!te! @hich are toxic to plant
ti!!ue!1 It i! there*ore e!!ential to maintain a completely a!eptic
enBironment in!ide the culture Be!!el!1 For thi! t@o o#Biou! precaution!
are:
Not to !hare the plant ti!!ue culture @or,in% area @ith micro#iolo%i!t! F
patholo%i!t!1
5o remoBe contaminated culture! *rom the culture area a! !oon a!
detected1
So(r!e) o# Conta$ination o# T,e -edi($*
&ulture Be!!el!
'edium it!el*
5he explant!
5he enBironment o* tran!*er area
In!trument! u!ed to handle plant material durin% inoculation F
!u#culture1
5he enBironment o* the culture room
5he operator
2 1SteriliCation O# Gla))/are D Pla)ti!/are*
"la!! culture Bial! are mo!tly !terilized to%ether @ith the medium1 For pre.
!terilized medium, autoclaBin% or dry heatin% in an oBen at 0><.08<
o
& *or
3 hr! may !terilize the %la!!@are1 Di!adBanta%e! o* dry heat !terilization
are poor circulation F !lo@ penetration1 5here*ore proper loadin% o* the
oBen i! e!!ential1 "la!!@are i! allo@ed to cool #e*ore remoBal *rom the
oBen1 &ertain type! o* pla!tic la# @are can al!o #e heat !terilized1
3. SteriliCation o# In)tr($ent)*
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 32
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
5he in!trument! u!ed *or a!eptic manipulation!, !uch a! *orcep!, !calpel!,
needle! and !patula are normally !terilized #y dippin% in :7O ethanol
*ollo@ed #y *lamin% and coolin%1 5hi! i! done at the !tart o* tran!*er @or, F
!eBeral time! durin% the operation1
4. SteriliCation o# t,e lant -aterial*
Sur*ace o* the plant part carrie! a @ide ran%e o* micro#ial contaminant!1 5o
aBoid the !ource in*ection, the ti!!ue mu!t #e thorou%hly !ur*ace !terilized
#e*ore plantin% it on the nutrient medium1 5o di!in*ect plant ti!!ue!, Bariou!
!terilizin% a%ent! haBe #een u!ed1 <10O $%&l2 i! mo!t e**ectiBe F u!ed to
!ur*ace !terilize! many plant ti!!ue!1
a1 &ulture Be!!el!
#1 'edia it!el*
c1 5he explant!
d1 5he enBironment o* tran!*er area
e1 In!trument! u!ed to handle plant material durin% inoculation F
!u#culture1
*1 5he enBironment o* the culture room
%1 5he operator
5. SteriliCation O# T,e -edi($*
5he micro#ial contaminant i! normally pre!ent in the medium *rom the !tart1
5o de!troy them, the mouth o* the culture Be!!el! !hould #e clo!ed @ith a
!uita#le #acterial proo* clo!ure F the Be!!el! are autoclaBed1 Sterilization
depend! on the temperature and not directly on the pre!!ure1 'edium
!hould #e autoclaBed at 020
<
c F 01<x 0< pa *or 07 minute!1 &are mu!t #e
ta,en @hile coolin% the !olution!1 3apid lo!! o* pre!!ure, exceed the rate o*
reduction in temperature @ill ma,e the liCuid Bi%orou!ly heated1 5he
pre!!ure %au%e o* the autoclaBe !hould #e at zero #e*ore the autoclaBe i!
opened1
STAGES OF -ICROPROPAGATION
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 33
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
'icro propa%ation i! the propa%ation o* the !elected %enotype u!in%
in Bitro techniCue1 5he main adBanta%e o* micropropa%ation i! that thi!
techniCue i! alternatiBe method o* Be%etatiBe propa%ation and i! applied @ith
the o#DectiBe o* enhancin% the rate o* multiplication1 &urrently @orld @ide u!e
o* micropropa%ated plant! i! a#out 7< million per annum1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 34
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
5echniCue o* 'icropropa%ation %enerally inBolBe! 4 !ta%e!.
Sta+e 2. Initiation o* A!eptic culture
Sta+e 3. 'ultiplication
Sta+e 4. 3ootin% o* @hole plant.
2lon%ation o* in vitro *ormed !hoot1
3ootin% o* in vitro *ormed !hoot1
Sta+e 5. 5ran!*er o* plant! to "reen $ou!e1
Deber+, and -aene )0:80+ introduced the !ta%e zero, ma,in%
micropropa%ation a *iBe. !ta%e proce!!1
STAGE ;* PREPARATIVE STAGE
5hi! !ta%e inBolBe! the preparation o* mother plant! to proBide Cuality
explant! *or #etter e!ta#li!hment o* a!eptic culture in !ta%e 01 For thi! t@o
*actor! may #e con!idered1
H"+ieni! Condition. 5o reduce contamination the de!ira#le condition! are J
&a' "ro@ explant in %reen hou!e1
&b' 5emperature !hould #e hi%h 27
<
& and relatiBe humidity !hould #e
lo@ ?<O1
&!' &hemotherapy could #e u!ed *or Biru! eradication1
PH%SIOLOGICAL CONDITIONS
"ro@th o* explant! could #e controlled #y phy!iolo%ical condition!1
&a' 5hrou%h plant %ro@th re%ulator! treatment!
&b' Startin% experiment @ith the material @hich it!el* cultured in vitro
&!' By proBidin% con!tant photoperiod
In !ta%e <, #oth apical and all nodal !e%ment! could #e u!ed a! primary
explant!1 Sta%e < al!o include! expo!in% the !toc, plant! to !uita#le li%ht,
temperature and %ro@th re%ulator treatment! to improBe the Cuality o*
explant!1 In ca!e o* photo!en!itiBe plant!, photoperiod i! controlled in
%la!!hou!e to o#tain !uita#le explant! throu%hout the year1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 35
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
STAGE 2* INITIATION OF ASEPTIC CULTURE
In thi! !ta%e !terilization o* explant! and e!ta#li!hment o* explant! @ere
done1 5he plant or%an u!ed to initiate a culture i! called explant1 5he choice
o* explant depend! on the method o* !hoot multiplication to #e *ollo@ed1 For
mo!t micropropa%ation @or, the explant o* choice i! an apical or axillary #ud1
STAGE 3* -ULTIPLICATION OF SHOOT B% REPEATED SUBCULTURES
ON -ULTIPLICATION -EDIA
5hi! i! the mo!t important !ta%e and the rate o* multiplication determine!
lar%ely the !ucce!! o* micropropa%ation !y!tem1 5hi! can #e achieBed #y.
2nhanced axillary #ranchin%
AdBentitiou! #ud *ormation
5hrou%h callu!in%
2. ENHANCED AEILLAR% BRANCHING
5he axillary #ud pre!ent in the axil o* each lea* either deBelop! in to a !in%le
!hoot or *orm a clu!ter o* !hoot! in the pre!ence o* cyto,inin )BAP 01< m%E l+
in the medium1 5he conBentional method o* Be%etatiBe propa%ation #y !tem
cuttin%! utilize! the a#ility o* axillary #ud! to ta,e oBer the *unction o* the
main !hoot in the a#!ence o* a terminal #ud1 In culture!, the rate o* !hoot
multiplication #y enhanced axillary #ranchin% can #e !u#!tantially enhanced
#y %ro@in% !hoot! in a medium containin% !uita#le cyto,inin! at an
appropriate concentration @ith or @ithout an auxin1 Due to continuou!
aBaila#ility o* cyto,inin the !hoot! *ormed #y the #ud deBelop! axillary #ud!
@hich may %ro@ directly into !hoot!1 5hi! proce!! may #e repeated !eBeral
time! and the initial explant tran!*ormed into a ma!! o* #ranche!1 5hi!
method i! currently mo!t popular approach to clonal propa%ation #ecau!e
cell! o* the !hoot apex are uni*ormly diploid and are lea!t !u!cepti#le to
%enotype chan%e! under culture condition!1

ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 36
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
3. ADVENTITIOUS BUD FOR-ATION
Bud! ari!in% *rom part other than the lea* axil or !hoot apex are called
adBentitiou! #ud!1 In many plant! i! a !tandard horticulture practice1
4. THROUGH CALLUSING
Plant cell! are totipotent di**erentiated cell! commonly ,no@n a! callu!1 5hi!
either %iBe! ri!e to !hoot #ud or #ipolar !tructure re!em#lin% em#ryo
)!omatic em#ryo+1 5hi! method i! u!ed @hen aim i! to induce Baria#ility
e!pecially in !el*. pollinatin% !pecie! @ith narro@ %enetic #a!e1
Di**erentiation o* plant! *rom cultured cell! may occur Bia !hoot.root
*ormation or !omatic em#ryo%ene!i!1 5hi! o*ten called the *a!te!t method o*
multiplication1 A !omatic em#ryo%enic !y!tem once e!ta#li!hed lend! it!el* to
#etter control than or%ano%ene!i!1 Since !omatic em#ryo! are #ipolar
!tructure! @ith de*ined root and !hoot meri!tem!, the rootin% !ta%e reCuired
*or micro !hoot! %et! eliminated1 Al!o, !omatic em#ryo! #ein% !mall uni*orm
and #ipolar are more amena#le to automation at the multiplication !ta%e and
*or *ield plantin% a! !ynthetic !eed!1
STAGE 4* IN VITRO ROOTING OF -ICROSHOOTS
'n- vitro %ro@n !hoot! lac, root !y!tem1 5o o#tain *ull plant!, the !hoot! mu!t
#e tran!*erred to a rootin% medium1 For rootin%, indiBidual !hoot! mea!urin%
a#out 2 cm in len%th are exci!ed and tran!*erred to the 3ootin% 'edium1
STAGE 5* HARDENING D ACCLI-ATIBATION OF TISSUE CULTURE
PLANTLETS
5hi! i! the *inal !ta%e and reCuire! care*ul handlin% o* plant!1 5he
tran!plantation *rom completely controlled condition! !hould #e %radual1 5hi!
proce!! o* %radually preparin% the plant! to !urBiBe in the *ield condition! i!
called acclimatization1 5he plant! produced in ti!!ue culture, althou%h %reen
in colourI do not prepare !u**icient *ood *or their o@n !urBiBal1 Al!o in!ide the
culture Be!!el! humidity i! Bery hi%h and thu! the natural protectiBe coBerin%
o* cuticle i! not *ully deBeloped1 5here*ore immediately a*ter tran!*er plant!
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 37
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
are maintained under hi%h humidity1 ptimum condition! are proBided to
plant! in %reen hou!e1
=oun% re%enerated rooted plantlet! @ill #e tran!*erred to !oil *or proper
hardenin% @hich @ill #e accompli!hed under mi!t hou!e condition!1 A*ter
hardenin% the plantlet! @ill #e ,ept *or acclimatization1 Acclimatization
procedure @ill #e tried @ith !oil and al!o @ith !oil, Bermiculite and *armyard
manure )0:0:0+ *inally the plantlet! @ill #e tran!*erred to *ield1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 38
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
-ETHODOLOG% OF WOR1DONE
Preparatory pha!e
"la!!@are preparation
'edia preparation
'edia !terilization
Sur*ace !terilization o* explant
Inoculation E incu#ation
'ultiplication
3ootin%
$ardenin% and acclimatization
2' PREPRATOR% PHASE F STERILIBATION*
5he mo!t important and rather di**icult a!pect o* the in Bitro techniCue! i!
the reCuirement to carry out Bariou! operation! under a!eptic condition!1
Bacteria and *un%i are the mo!t common contaminant! o#!erBed in
culture!1 5he!e micro#e! are omnipre!ent in our enBironment and on
comin% into contact @ith the culture media, *ine optimal condition! and
%ro@ much *a!ter than the cultured ti!!ue1 &on!eCuently, the ti!!ue i!
,illed due to contamination1 5he contaminant! may al!o %iBe out meta#olic
@a!te! @hich are toxic to plant ti!!ue!1 In Bie@ o* thi!, the maintenance o*
a completely a!eptic enBironment in!ide the culture Bial @here ti!!ue
%ro@! i! a#!olutely e!!ential1
Gla))/are rearation*
"la!! culture Bial!, metal in!trument! and aluminium *oil can all #e
!terilized #y properly @rapped @ith aluminium *oil #e*ore !terilization and
autoclaBin% under !team at a pre!!ure o* 07lpaEin and a temperature o*
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 39
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
02<Sc *or 07 min1 5he in!trument! u!ed *or a!eptic manipulation! are
u!ually made o* metal1 Normally, in!trument! !uch a! *orcep!, !calpel!,
needle! and !patula! are !terilized #y dippin% in :7O ethanol, *ollo@ed
#y *lamin% and coolin%1 5hi! techniCue i! called *lame !terilization1
AutoclaBin% o* metallic in!trument! i! %enerally aBoided a! they may ru!t
and #ecome #lunt1
-edia rearation*
5he mo!t important *actor %oBernin% the %ro@th F morpho%ene!i! o* plant
ti!!ue culture i! the compo!ition o* culture medium1 Nutritional
reCuirement! *or optimal %ro@th o* a ti!!ue in Bitro may Bary @ith the
!pecie!1 2Ben ti!!ue! *rom di**erent part! o* a plant may haBe di**erent
reCuirement! *or !ati!*actory %ro@th1 A! !uch no !in%le medium can #e
!u%%e!ted a! #ein% entirely !ati!*actory *or all type! o* plant ti!!ue! F
or%an! #ut %enerally all ti!!ue culture media are made up o*:
'acronutrient!
'icronutrient!
r%anic !upplement!
"ro@th re%ulator!
&ar#ohydrate! )!ucro!e+
a' -a!ron(trient)*
A! implied #y the name, the !toc, !olution !upplie! macroelement! reCuired
in lar%e amount! *or plant %ro@th and deBelopment1 Nitro%en, pho!phoru!,
pota!!ium, ma%ne!ium, calcium, and !ulphur )and car#on, @hich i! added
!eparately+ are u!ually re%arded a! macroelement!1 5he!e element! u!ually
compri!e at lea!t <10O o* the dry @ei%ht o* plant!1 Nitro%en i! mo!t
commonly !upplied a! a mixture o* nitrate ion! )*rom (N3+ and ammonium
ion! )*rom N$4N3+1 5heoretically, there i! an adBanta%e in !upplyin%
nitro%en in the *orm o* ammonium ion!, a! nitro%en mu!t #e in the reduced
*orm to #e incorporated into macromolecule!1 Nitrate ion! there*ore need to
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 40
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
#e reduced #e*ore incorporation1 $o@eBer, at hi%h concentration!,
ammonium ion! can #e toxic to plant cell culture! and upta,e o* ammonium
ion! *rom the medium cau!e! acidi*ication o* the medium1 For ammonium
ion! to #e u!ed a! the !ole nitro%en !ource, the medium need! to #e
#u**ered1 $i%h concentration! o* ammonium ion! can al!o cau!e culture
pro#lem! #y increa!in% the *reCuency o* nitri*ication )the culture appear!
pale and K%la!!y- and i! u!ually un!uita#le *or *urther culture+1 6!in% a
mixture o* nitrate and ammonium ion! ha! the adBanta%e o* @ea,ly #u**erin%
the medium a! the upta,e o* nitrate ion! cau!e! $T ion! to #e excreted1
Pho!phoru! i! u!ually !upplied a! the pho!phate ion o* ammonium, !odium,
or pota!!ium !alt!1 $i%h concentration! o* pho!phate can lead to the
precipitation o* medium element! a! in!olu#le pho!phate! Function! o*
!ome macro element! are a! *ollo@!:
Nitro+en: &omponent o* protein!, nucleic acid!, and !ome coenzyme!I
element reCuired in the %reate!t amount!1
Pota))i($* 3e%ulate! o!motic potentialI principal inor%anic cation1
Cal!i($* &ell.@all !ynthe!i!, mem#rane *unction, cell !i%nalin%1
-a+ne)i($: 2nzyme co*actor, component o* chlorophyll1
P,o),or()* &omponent o* nucleic acid!I ener%y tran!*erI component o*
intermediate! in re!piration and photo!ynthe!i!1
b' -i!ron(trient)*
'icroelement! are reCuired in trace amount! *or plant %ro@th and
deBelopment, and haBe many and diBer!e role!1 'an%ane!e, iodine, copper,
co#alt, #oron, moly#denum, iron, and zinc u!ually compri!e the
microelement!, althou%h other element! !uch a! nic,el and aluminium are
*ound *reCuently in !ome *ormulation!1 Iron i! u!ually added a! iron
!ulphate, althou%h iron citrate can al!o #e u!ed1 2thylenediaminetetra.acetic
acid )2D5A+ i! u!ually u!ed in conDunction @ith iron !ulphate1 5he 2D5A
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 41
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
complexe! @ith the iron to allo@ the !lo@ and continuou! relea!e o* iron into
the medium 6ncomplexed iron can precipitate out o* the medium a! *erric
oxide1Function! o* !ome microelement! are a! *ollo@!:
S(l,(r * &omponent o* !ome amino acid! )methionine, cy!teine+ and
!ome co*actor!
C,lorine: 3eCuired *or photo!ynthe!i!
Iron: 2lectron tran!*er a! a component o* cytochrome!
-an+ane)e* 2nzyme co*actor
Cobalt* &omponent o* !ome Bitamin!
Coer* 2nzyme co*actorI electron.tran!*er reaction!
Bin!* 2nzyme co*actorI chlorophyll #io!ynthe!i!
-ol"bden($* 2nzyme co*actorI component o* nitrate reducta!e
!' Or+ani! )(le$ent)*
nly t@o Bitamin!, thiamine )Bitamin B0+ and myoino!itol )con!idered a B
Bitamin+, are con!idered e!!ential *or the culture o* plant cell! in Bitro1
$o@eBer, other Bitamin! are o*ten added to plant cell culture media *or
hi!torical rea!on!1 Amino acid! are al!o commonly included in the or%anic
!upplement1 5he mo!t *reCuently u!ed i! %lycine )ar%inine, a!para%ine,
a!partic acid, alanine, %lutamic acid, %lutamine, and proline are al!o u!ed+,
#ut in many ca!e! it! inclu!ion i! not e!!ential1Amino acid! proBide a !ource
o* reduced nitro%en and, li,e ammonium ion!, upta,e cau!e! acidi*ication o*
the medium1 &a!ein hydroly!ate can #e u!ed a! a relatiBely cheap !ource o*
a mix o* amino acid!1 Function! o* !ome or%anic !upplement! are a!
*ollo@!:
Vita$in)*
Plant! can produce their reCuirement! o* Bitamin!1 $o@eBer plant cell
culture! need to #e !upplemented @ith certain Bitamin!1 5hiamine )Bit1B0+,
Niacin )Bit1B>+ ,Pyridoxine )Bit1B>+, 'yo.ino!itol )mem#er o* the Bit1B
complex+1Some other Bitamin! that *ind !peci*ic u!e! in all culture! are
pantothenic acid, Bit1&, Bit1D F Bit121
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 42
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
T,ia$ine = InBolBed in direct #io!ynthe!i! o* certain amino acid! F
e!!ential co.*actor o* car#ohydrate meta#oli!m.
Vit.E Gantioxidant
Vit.C GpreBent #lac,enin% o* explant! durin% i!olation
Vit.D Gha! %ro@th re%ulatory e**ect1
A$ino a!id)*
"lycine ha! little e**ect in the %ro@th o* plant1 5hey may #e directly utilized
#y plant #e proBided a! N2 !ource1
Unde#ined )(le$ent)*
Numerou! complex nutritiBe mixture! o* unde*ined compo!ition, li,e ca!ein
hydroly!ate )&$+,coconut mil,)&'+,corn mil,, malt extract)'2+,tomato
Duice)59+ Fyea!t extract )=2+ haBe #een u!ed to promote the %ro@th o*
certain calli For%an!1
Carbon )o(r!e*
Sucro!e i! cheap, ea!ily aBaila#le, readily a!!imilated, and relatiBely !ta#le,
and i! there*ore the mo!t commonly u!ed car#on !ource1 ther
car#ohydrate! )!uch a! %luco!e, malto!e, %alacto!e, and !or#itol+ can al!o
#e u!ed, and in !pecialized circum!tance! may proBe !uperior to !ucro!e1
Gro/t, Re+(lator)*
Plant %ro@th re%ulator! are the critical media component! in determinin% the
deBelopmental path@ay o* the plant cell!1 5he plant %ro@th re%ulator! u!ed
mo!t commonly are plant hormone! or their !ynthetic analo%ue!1Plant
%ro@th re%ulator! are a! *ollo@!:
Auxin!
&yto,ine!
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 43
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
"i##erellin!
A#!ci!ic acid
2thylene

ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 44
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
A(Ain)*
Auxin! promote #oth cell diBi!ion and cell %ro@th1 5he mo!t important
naturally occurrin% auxin i! indole.3.acetic acid )IAA+, #ut it! u!e in plant cell
culture media i! limited #ecau!e it i! un!ta#le to #oth heat and li%ht1
cca!ionally, amino acid conDu%ate! o* IAA )!uch a! indole.acetyl.l.alanine
and indole.acetyl.l.%lycine+, @hich are more !ta#le, are u!ed to partially
alleBiate the pro#lem! a!!ociated @ith the u!e o* IAA1 It i! more common,
thou%h, to u!e !ta#le chemical analo%ue! o* IAA a! a !ource o* auxin in
plant cell culture media1 2,4.Dichlorophenoxyacetic acid )2,4.D+ i! the mo!t
commonly u!ed auxin and i! extremely e**ectiBe in mo!t circum!tance!1
ther auxin! are al!o aBaila#le and !ome may #e more e**ectiBe or Kpotent-
than 2,4.D in !ome in!tance!1
C"to.inin)*
&yto,inin! promote cell diBi!ion1 Naturally occurrin% cyto,inin! are a lar%e
%roup o* !tructurally related purine deriBatiBe!1 * the naturally occurrin%
cyto,inin!, t@o haBe !ome u!e in plant ti!!ue culture media zeatin and N>.
)2.i!opentyl+ adenine )2iP+1 5heir u!e i! not @ide!pread a! they are
expen!iBe )particularly zeatin+ and relatiBely un!ta#le1 5he !ynthetic
analo%ue! ,inetin and >.#enzylaminopurine )BAP+ are there*ore u!ed more
*reCuently1 Non.purine.#a!ed chemical!, !uch a! !u#!tituted phenylurea!,
are al!o u!ed a! cyto,inin! in plant cell culture media1 5he!e !u#!tituted
phenylurea! can al!o !u#!titute *or auxin in !ome culture !y!tem!1
Gibberellin)*
5here are numerou!, naturally occurrin%, !tructurally related compound!
termed %i##erellin!1 5hey are inBolBed in re%ulatin% cell elon%ation, and are
a%ronomically important in determinin% plant hei%ht and *ruit.!et1 nly a *e@
o* the %i##erellin! are u!ed in plant ti!!ue culture media, "A3 #ein% the
mo!t common1
Ab)!i)i! a!id*
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 45
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
A#!ci!ic acid )ABA+ inhi#it! cell diBi!ion1 It i! mo!t commonly u!ed in plant
ti!!ue culture to promote di!tinct deBelopmental path@ay! !uch a! !omatic
em#ryo%ene!i!1
Et,"lene*
2thylene i! a %a!eou!, naturally occurrin%, plant %ro@th re%ulator mo!t
commonly a!!ociated @ith controllin% *ruit ripenin% in climacteric *ruit!, and
it! u!e in plant ti!!ue culture i! not @ide!pread1 It doe!, thou%h, pre!ent a
particular pro#lem *or plant ti!!ue culture1 Some plant cell culture! produce
ethylene, @hich, i* it #uild! up !u**iciently, can inhi#it the %ro@th and
deBelopment o* the culture1 5he type o* culture Be!!el u!ed and it! mean!
o* clo!ure a**ect the %a!eou! exchan%e #et@een the culture Be!!el and the
out!ide atmo!phere and thu! the leBel! o* ethylene pre!ent in the culture1
GA-BORG -EDIA*
"am#or% et al in 0:>8 deBeloped "am#or% media *or !oya#ean callu!
culture1 It contain! a much %reater proportion o* nitrate compared to
ammonium ion!
Table 2* CO-POSITION OF GA-BORG -EDIA &STOC1 SOLUTION'
-ACRONUTRIENTS ;.6litreF6;;$l

-ICRONUTRIENTS ;.6litreF6;;$l
(I 7ml
$
3
B
3
7ml
'nS
4
1?$
2
7ml
HnS
4
1?$
2
7ml
Na
2
'o
4
7ml
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 46
KNO
3
10 ml
Cacl
2.
2H
2
O 5ml
MgSO
4
.7H
2
0 10 ml
(NH
4
)
2
SO
4
5ml
NaH
2
PO
2
.H
2
0 5ml
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
&uS
4
17$
2
<17ml
&o&l
2
1>$
2
<17ml
FeS
4
1?$
2
0<ml

ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 47
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
VITA-INS ;.6litreF6;;$l
Ino!itol 7ml
Nicotininc acid 7ml
Pyridoxine1$&l <17ml
5hymine1$&l <17ml
Table 3* CO-POSITION OF -S -EDIA &STOC1 SOLUTION'
-ACRONUTRIENTS +$F6;;$l Ta.en &in litre'
(N3 0:1<
7<ml
N$4N3 0>17
($2P4 01?
&a&l212$2 414
'%S41?$2 31?
-ICRONUTRIENTS
'%S4 14$2 0103<
7ml
HnS414$2 <143
$3B3 <130
&uS417$2 <1<<027
&o&l21>$2 <1<<027
Na2'o412$2 <1<027
(I <1<407
ORGANIC
7ml
5hiamine $&l <1<<7
Nicotinic $&l <1<27
Pyrodoxine $&l <1<27
"lycine <10
'yo ino!itol 71<
Fe SALTS
7ml
FeS41?$2< 21?8
Na22D5A12$2 31?3
SUCROSE 3<%m
A+ar ?%m
H 71>
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 48
Sucros 10 g
!gar 3.5 g
"H 5.6
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
Table 4* HOR-ONAL TREAT-ENT GIVEN FOR CALLUS INDUCTION
Table 5* HOR-ONAL TREAT-ENT GIVEN FOR SHOOT INDUCTION
BAP&$+Fl' NAA&$+Fl' EEPLANT
2.; ;.36 Node)
3.; ;.36 Node)
4.; ;.36 Node)
5.; ;.36 Node)
2.; ;.6; Node)
3.; ;.6; Node)
4.; ;.6; Node)
5.; ;.6; Node)
-edia )teriliCation *
5he nutrient media u!ed in ti!!ue culture! are commonly !terilized #y
autoclaBin% and *ilter !terilization1 Di!tilled @ater, micro.and macronutrient!,
and other !ta#le mixture! are autoclaBed, @herea! the !olution! that
contain thermo la#ile compound! are *ilter !terilized1 &ulture media in %la!!
container! !ealed @ith cotton plu%!, aluminium *oil or pla!tic clo!ure! are
autoclaBed at 07 p!i and 020S & *or 07.4< minute! *rom the time the medium
reache! the reCuired temperature1 2xpo!ure time depend! on the Bolume o*
the liCuid to #e !terilized1 5he pre!!ure !hould not exceed 2< p!i a! hi%her
pre!!ure may lead to the decompo!ition o* car#ohydrate! and other
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 49
2,4-D (mg/l) BAP(mg/l) EXPLANT
0.5 0.1 Leaves and nodes
1 0.1 Leaves and nodes
2 0.1 Leaves and nodes
0.1 Leaves and nodes
4 0.1 Leaves and nodes
0.5 0.5 Leaves and nodes
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
component! o* medium1 /itamin!, amino acid!, plant extract!, hormone!
and car#ohydrate! are thermo la#ile and may decompo!e durin%
autoclaBin%1 5he !olution! o* the!e compound! reCuire *ilter !terilization1 In
thi! proce!! the !olution! are pa!!ed throu%h a #acteria proo* mem#rane.
*ilter under po!itiBe pre!!ure1 A 'illipore or Seitz *ilter @ith a pore !ize o* not
more than <12 Um i! %enerally u!ed in *ilter !terilization1 Filter J !terilized
!olution! are then com#ined @ith other nutrient !u#!tance! !terilized in the
autoclaBe to %iBe a complete medium importance1 All ti!!ue culture
experiment! are carried out under a!eptic condition! in @hich !terilization
*orm! a part1
Table 6 * -INI-U- TI-E FOR AUTOCLAVING NUTRIENT -EDIA
Vol($e
&$l'
SteriliCation Ti$e
&$in'
0 J 2<< 07
2<< J 0<<< 3<
0<<<J 2<<< 4<
3' SURFACE STERILIBATION OF EEPLANT
Sele!tion o# eAlant * =oun% and DuBenile nodal !e%ment! o* "idelia
chinensis @ere collected *rom Botani!al +arden> FRI. Nodal
!e%ment! @ith !in%le axillary #ud! @ere u!ed a! an explant material
*or !hoot induction , lar%e !ized leaBe! @ith proper mid ri# @a! ta,en
*or callu! induction *rom leaBe! and node! *or callu! induction *rom
node!
EAlant SteriliCation* 5he mo!t important and rather di**icult a!pect
o* the in Bitro techniCue! i! the reCuirement to carry out Bariou!
operation! under a!eptic condition!1 Bacteria and *un%i are the mo!t
common contaminant! o#!erBed in culture!1 5he!e micro#e! are
omnipre!ent in our enBironment and on comin% into contact @ith the
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 50
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
culture media, *ine optimal condition! and %ro@ much *a!ter than the
cultured ti!!ue1 &on!eCuently, the ti!!ue i! ,illed due to
contamination1 5he contaminant! may al!o %iBe out meta#olic @a!te!
@hich are toxic to plant ti!!ue!1 In Bie@ o* thi!, the maintenance o* a
completely a!eptic enBironment in!ide the culture Bial @here ti!!ue
%ro@! i! a#!olutely e!!ential1
Pro!ed(re*
Nodal explant! @ere cut and @iped @ith ?<O o* alcohol1
2xplant! @ere @a!hed in runnin% tap @ater to remoBe the du!t
particle! adherin% to it! !ur*ace1
2xplant! @ere @a!hed in liCuid deter%ent la#,lin )7.0< drop! E
0<<ml+ in a Bial #y %entle a%itatin% condition! *or 0<.07 min!
2xplant! @ere a%ain @a!hed @ith di!tilled @ater to remoBe exce!!
o* deter%ent1
2xplant! @ere then treated #y *un%icide1 0O o* BaBi!tin and 0O o*
Blitox treatment! @ere %iBen !eCuentially *or 0<.07 min!
2xplant! @ere a%ain @a!hed until the *un%icide %et! remoBed1
5he explant! !o @a!hed @ere !ur*ace !terilized @ith <10O o* $%&l
2
in ;aminar Air *lo@ *or 0. 7 min! dependin% on the explant! to #e
u!ed1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 51
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
4. INOCULATION FINCUBATION

PROCEDURE
:
Sur*ace o* the laminar air *lo@ @a! @iped @ith ?<O alcohol1
All the autoclaBed material! )media, *orcep!, !calpel!, empty *la!,,
cotton, di!tilled @ater etc1+ and !pirit and alcohol #ottle!, !pirit lamp,
etc @ere ,ept in!ide the laminar air *lo@1
6/ li%ht @a! !@itched on *or 07 minute!1
Be*ore !@itchin% FF the 6/ li%ht , Blo@er i! !@itched N and then
$%&l
2
@a! di!!olBed in 0<< ml di!tilled @ater
A%ain 6/ li%ht @a! !@itched N and then Blo@er @a! !@itched FF
*or next 07 minute!1
No@ Blo@er @a! !@itched on *or 0< minute!1
Final !terilization treatment o* mercuric chloride @a! %iBen to the
explant! )*or 7 minute! to the node! and *or 2 minute! to the leaBe!+1
5reated explant! @ere @a!hed thrice @ith di!tilled @ater *or 2 minute!
each1
Forcep! and !calpel @a! heated upto red hot1
4ith the help o* *orcep! and !calpel explant! @ere inoculated in the
media near the !pirit lamp1
Inoculated explant! @ere tran!*erred into incu#ation cham#er *or
%ro@th1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 52
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
RESULTS*
Table 7* RESULTS FOR CALLUS INDUCTION &ob)er?ed a#ter 4=5 /ee.)
o# ino!(lation'
SL no 3>5=D
&$+Fl'
BAP&$+Fl' EEPLANT RESULTS
01 <17 <10 ;eaBe! In 0 explant
3. 0 <10 ;eaBe! In 7 explant!
31 2 <10 Node! In 7 explant!
41 3 <10 ;eaBe! In 0 explant
6. 5 ;.2 Node) In 8 eAlant)
>1 <17 <17 Node! In 7 2xplant!
PEa!, eAeri$ent !on)i)t) o# ten reli!ate).

5he re!earch @or, i! oriented on direct a! @ell a! indirect or%ano%ene!i! on
"am#or% media1 For indirect ora%no%ene!i! callu! *ormation i! done *ir!t
and then *rom callu! the @hole plant i! deBeloped 1For callu! *ormation
leaBe! a! @ell a! nodal explant! @ere ta,en and inoculated in "am#or%
media !upplemented @ith Bariou! hormonal treatment!1 "ro@th o* callu! @a!
increa!ed !i%ni*icantly and callu! coBered the entire !ur*ace o* the explant!
in 4.> @ee,! 1&allu! i! an unor%anized ma!! o* plant cell! and it! *ormation
i! controlled #y %ro@th re%ulatin% !u#!tance! pre!ent in medium1 $ormone!,
e!pecially auxin! @ere a#!olutely nece!!ary *or callu! induction1 Amon% the
di**erent concentration! o* 2, 4.D and BAP tried hi%he!t re!pon!e o*
callu!in% o#!erBed @ith 2, 4.D )4m%El+ and BAP )<10 m%El+ and minimum
re!pon!e o* callu!in% @a! o#!erBed @ith 2, 4.D )<17 m%El, 3m%El+ and BAP
)<10m%El+1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 53
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 54
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
Table ? * RESULTS FOR SHOOT INDUCTION &Readin+ a#ter 4; da") o#
ino!(lation'
Sl. No. TREAT-ENT NO. OF
SHOOTS F
EEPLANT
-ean
S,oot
Len+t
,
BAP&$+Fl' NAA&$+Fl'
0 01< <127 0 212
2 21< <127 2 21>
3 31< <127 3 014
4 41< <127 2 214
7 2.; ;.6; 2 2.9
> 3.; ;.6; 5 4.4
? 31< <17<
8 41< <17<
HEa!, treat$ent !on)i)t) o# ten reli!ate).
In direct or%ano%ene!i!, the plant i! deBeloped *rom the part o* a mother
plant1 &allu! in not %ro@n in thi! type o* or%ano%ene!i! 1For !hoot induction
axilliary #ud! @ere ta,en nd inoculated in "am#or% media !upplemented
@ith Bariou! hormonal concentration1 Bud #rea, and !hoot induction @a!
o#!erBed in 4.> @ee,! 1It @a! o#!erBed that cyto,inin @a! needed *or !hoot
induction1 Amon% the di**erent concentration! and o* BAP and NAA tried
hi%he!t re!pon!e o* !hoot induction @a! o#!erBed @ith BAP )21<m%El+ and
NAA )<17< m%El+1 'inimum re!pon!e o* callu!in% @a! o#!erBed @ith BAP
)01< m%El+ and NAA )<17<m%El+1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 55
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
Table 9* SHOOT -ULTIPLICATION IN GA-BORG -EDIA
Sl. No. Treat$ent Con!entraton -(ltili!ation
rate
-ean ),oot
len+t,&!$'
0 &ontrol . . . .
3 BAPIIBA ;.6 ;.6 ;.5 ;.2
3 BAPQIBA 01< <17 <1: <12
4 BAP QIBA 21< <17 017 <14
6 BAP IIBA 3.6 ;.6 4.; 2.;
> BAP QIBA 31< <17 217 <1?
? BAP QIBA 317 <17 210 <18
8 BAPQIBA 41< <17 01: <1:
For multiplication micro !hoot! containin% one !hoot @a! tran!*erred *rom
!hoot induction media to multiplication media o* Bariou! concentration o*
BAP )cyto,inin+1 'aximum multiplication rate and maximum !hoot len%th
@a! o#!erBed in "am#or% media !upplemented @ith BAP )217m%El+ *ollo@ed
#y IBA )<17m%El+ @hile the minimum multiplication rate and minimum !hoot
len%th @a! o#!erBed @ith BAP )<17m%El + and IBA) <17m%El+1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 56
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
ROOT INDUCTION IN GA-BORG -EDIA
For rootin% , micro!hoot! @ere tran!*erred *rom !u#culturin% media to
rootin% media i1e1, V "am#or% !upplemented @ith Bariou! auxin )IBA+
concentration!1 Since rootin% it!el* ta,e! 3.4 @ee,! )reported+, re!ult!
re%ardin% root induction are a@aited1
IN VITRO HARDENING D ACCLI-ATIBATION
Plantlet! @ith @ell deBeloped root! @ere remoBed *rom the culture
tu#e! and a*ter @a!hin% the root! %ently under runnin% tap @ater, the
plantlet! @ere placed into #ottle! *illed @ith !terilized Bermiculite in culture
room at 27W2
<
& *or t@o @ee,! #e*ore they @ere tran!*erred to poly#a%!1
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 57
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
PHOTOPLATE ;3* IN VITRO SHOOT INDUCTION AND -ULTIPLICATION
IN WIDELIA CHINENSIS
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 58
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
P!"T"PLATE. 0# IN VITRO $ALL%& 'ND%$T'"N 'N WIDELIA CHINENSIS
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 59
IN VITRO MICROPROPOGATION OF WEDELIA CHINENSIS WITH CALLUS INDUCTION AND
SHOOT INDUCTION
D'&$%&&'"N&
Prs#$ s$u%& #com"asss '#%'rc$ orga#og#s's o# MS m%'a a#% %'rc$ orga#og#s's o# (am)org m%'a. *or %'rc$ orga#og#s's a+'llar& )u%s
,rom -al$-& mo$-r "la#$ .r $a/# .-'l ,or '#%'rc$ orag#og#s's la0s as .ll as #o%al +"la#$s .r $a/# a#% '#ocula$% '# MS m%'a su""lm#$%
.'$- 0ar'ous -ormo#al $ra$m#$s. (ro.$- o, callus '#cras% s'g#','ca#$l& a#% co0r% $- #$'r sur,ac o, $- +"la#$s '# 416 ./s .Callus 's a#
u#orga#'2% mass o, "la#$ clls a#% '$s ,orma$'o# 's co#$roll% )& gro.$- rgula$'#g su)s$a#cs "rs#$ '# m%'um. Hormo#s3 s"c'all& au+'#s .r
a)solu$l& #cssar& ,or callus '#%uc$'o#. !mo#g $- %',,r#$ co#c#$ra$'o#s o, 23 414 a#% 5!P $r'% -'g-s$ rs"o#s o, callus'#g o)sr0% .'$- 23 414
(4mg6l) a#% 5!P (0.1 mg6l). M'#'mum rs"o#s o, callus'#g .as o)sr0% .'$- 23 414 (0.5 mg6l3 3mg6l) a#% 5!P (0.1mg6l).
5!P .as ,ou#% $o ,a0or s-oo$ lo#ga$'o#. !lo#g .'$- 5!P3 N!! a#% 75! .r also a%%% ,or s-oo$ '#%uc$'o# a#% s-oo$ mul$'"l'ca$'o# rs"c$'0l&.
!$ $- co#c#$ra$'o# o, 2.0mg68 o, 5!P alo#g .'$- 0.50mg6l o, N!! s-o.% )s$ rsul$s ,or s-oo$ '#%uc$'o# a#% 2.5mg68 o, 5!P alo#g .'$- 0.5mg68 o,
75! s-o.% )s$ mul$'"l'ca$'o# rsul$ amo#g all. 9- ac$'0'$& o, 5!P com"ar% $o o$-r c&$o/'#'#s 's also r"or$% '# ma#& suc- "la#$s '..3
sylvestre (Komala0all' et al.3 2000)3 Holostemma annulare (Su%-a et al.3 2000)3 Hyptis suaveolens (:o-# 5r'$$o
et al.3 2001))3 Zehneria scabra (!#a#% a#% :&ac-a#%ra# 2004)3 Smilax zeylanica (Hassa# a#% ;o& 2004) a#%
2009).
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 60
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9- "rc#$ag o, s-oo$s ,orm'#g roo$s a#% %a&s $o roo$'#g s'g#','ca#$l& 0ar'% .'$- %',,r#$ co#c#$ra$'o#s a#% com)'#a$'o# o, 75! < 5!P. 7# "rs#$ s$u%&
= s$r#g$- (am)org m%'a .as su""lm#$% .'$- 0ar'ous -ormo#al $ra$m#$s )u$ rsul$s ar a.a'$%.7# co#clus'o#3 $- "ro$ocol s$a#%ar%'2% $-roug- $-'s
s$u%& %mo#s$ra$s a ra"'% a#% ,,c$'0 m$-o% o, in vitro s-oo$s mul$'"l'ca$'o# a#% "la#$ rg#ra$'o# o,
&%((A)* AND $"N$L%&'"N#
9- "rs#$ .or/ .as u#%r$a/# $o g$ a .ll %,'#% "ro$ocol ,or in vitro "ro"aga$'o# o,
'#%uc$'o#.
7# $- "rs#$ s$u%& #o%al +"la#$s .r $a/# ,or in vitro "ro"aga$'o# .No%al sgm#$s a#% la0s .r '#ocula$% '#$o MS m%'a .'$- %',,r#$ -ormo#al
co#c#$ra$'o#s u#%r s"c','c co#%'$'o#s3 Ma+'mum callus '#%uc$'o# .as o)sr0% '# MS m%'a su""lm#$% .'$- 23414 (4mg6l) a#% 5!P (0.1
mg6l).Ma+'mum s-oo$ '#%uc$'o# .as o)sr0% '# (am)org m%'a su""lm#$% .'$- 5!P (2.0mg6l) a#% N!! (0.50mg6l).Ma+'mum mul$'"l'ca$'o# ra$ .as
o)sr0% '# (am)org m%'a su""lm#$% .'$- 5!P (2.5mg6l) a#% 75! (0.5mg6l)
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+L", $!A)T
Mo$-r "la#$.
>+"la#$ slc$'o#
?as-'#g o, +"la#$s .'$- $a" .a$r
?as-'#g o, +"la#$s .'$- la)/'l#
*u#g'c'%s $ra$m#$s ( 5a0's$'# < 5l'$o+)
S$r'l'2a$'o# o, +"la#$s .'$- HgCl
2
7#ocula$'o# o, +"la#$s '# MS a#% (am)org m%'a
Callus a#% 5u% '#%uc$'o# ,rom +"la#$
Mul$'"l'ca$'o#s o, s-oo$s
Su)cul$ur'#g
;oo$'#g
Har%#'#g a#% !ccl'ma$'sa$'o#
)E+E)EN$E&#
1. 5-a%raS/3 !/-$r 93 Hossa'# MM3 (2009) . In vitro m'cro"ro"aga$'o# o, Plumbago indica
P9C. 5'o$c-#ol.3 19(2)@1691175.
2. 5-a$$ac-ar& 7#%'a# "-arm 3(1951).7 12AMa'$'3 5ull.)o$.Sur0. 7#%'a3 (1968).10311A?'llama# < 8'3 7lo&%'a 3(1970). 33(3!)3 Su""l.3 81.
ADITI SINGH, A.P.S UNIVERSITY, REWA (MADHYA PRADESH) Page 62
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3. 5's.as! 3 ;o& M 3 M'a- M!5 3 5-a%ra SK 3(2007) . In vitro "ro"aga$'o# o, !)rus"rca$or'ous 83 1a rar m%'c'#al "la#$ o, C-'$$ago#g -'ll $rac$s
P9C. 5'o$c-#ol.3 17(1)@ 59164.
4. C-a#%ra3 Buar$.:. Cru% 4rug ;s.3(1969) 3 9 3 1461AKar#'c/3 !c$a"-&$o$-r.3 !#rs$.3 (1970)3 173 181A .'$- 7N47!1 ;a. ma$r'als37773 127
5. C-m. !)s$r 3 (1969) 370317567A(o0'#%ac-ar' .3 :.C -m. Soc.319563629A (o0'#%ac-ar' .3')'%3(1957). 545A(o0'#%ac-ar' 39$ra-%%ro#3(1961)3153 129
6. ;a-ma#1>7 3 ?a/'l H1>73 4'#1H73 Sa'%1>8 3 K-l',a H.4 (2008)3 Journal of pplied Sciences !esearch
7. Hasa# !KMS3 ;o& SK3 (2005). M'cro"ro"aga$'o# o, (lor'osa su"r) 8. 9-roug- -'g- ,rCu#c& s-oo$ "rol',ra$'o#.P9C.15(1)@67174.
8. :a0a-ar. M3 ;a0'"aul S3 :&asla#.M.3 (2008). In vitro rg#ra$'o# o, 5'$+#gu#%o 81 a mul$'"ur"os .oo%& aroma$'oc m%'c'#al s-ru)s. P9C .
5'o$c-318(1)@ 37142
9. K'r$ a#% 5asu 3 77 3 13641653 C-a#%ra3(1968). 8oc.c'$A Pal .37#%'a#:.ma%3 ;s.3 3563445A5-a/u#' 3(1971).3 7#%'a# :. >+". 5o'l.39391
10. Mrr'll s&# . ? .cal#%ulacalss.3 #o# r'c- 4.>.P3 D7 (4)3 301A *7 . 5r 7#% .3 777 3 306 A K'r$<5asu 3 P7.531
11. Mu$as'm Mo-am% K-ala,alla3 >'sa7)ra-m>lgllal' 3 Mag%a Mo-am% !-m% 3(2007). frican "rop Science "onference Proceedings
12. Sar'$-a 3 Na'%u 3 (2007). Pla#$ Sc'#c3 847185
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13. S'0a#sa#7 3:o#g 5; 3(2009). M'cro"ro"aga$'o# o, Plumbago zeycalani 8'##. 4"ar$m#$ o, Hor$'cul$ur3 4'0's'o# o, a""l'% sc'#c (5K 21
Program3 :'#Eu3 Kora 6601701.
14. 9'la/3 :a'3 !%-'/ar'3 Soum&a/a#$'3 40asaga&am3 9-omas3 (2004). !#$'o+'%a#$ "ro"r$'s o, P.2&la#'ca3 Ma#& Pu)l'ca$'o# (!ug 2004)3 Dol. 93
P.g.2191227.
15. Fas'rHasa#3 S'%%'Cu3 (uls-a#!ra3 Mo-omma%*a'2a3 Mo-omma%!,2al3 (2011). Pro$c$'0 rol o, Plum)ag'# aga'#s$ $-&'#&ls$ra%'ol3 Ma#&
Pu)l'ca$'o# 3(2011).
16. K.D Pa$l3 4.H Pa$l3 Su-'la Pa$l3 (1997198). >,,c$ o, s% ra$ a#% cro" %ura$'o# o# roo$ &'l%3M%'c'#al < !roma$'c Pl$. ProEc$3 (uEra$ agr'cul$ur
u#'0rs'$&3!#a#%13881103 7#%'a.
17. ;.P. Ma-ar's-' 3(2006). ,,c$ o, *.sola#' 7 ;.)a$a$'cola o# s%l'#g mor$al'$& o, !s-.aga#%-a3:our#al o, M%'c'#al < !roma$'c Pla#$ Sc'#cs3
(2006). 0ol.28 No.314 (N.*.8.7.C)
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