Lotte & Thomas Orchids

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Hello and welcome on our website !
Cephalanthera rubra
ere we describe
differenf
fechniques, which
we hove fried ond
modified, fo
propogofe orchids
esides seed sowing
fhere ore for
exompIe fechniques
fo rise pIonfs from
sIeeping buds
(nodes)
ousfrion orchids
(phofos)

Iosf updofe ovember of (seedIing
Iisf)
Deufsche ersion
Seed germination
The biology of orchid seed germination
How can I get some seeds ?
Packing and shipping seeds
germination on bark asymbiotic germination
Asymbiotic seed germination
Basics of maintaining sterile conditions
Necessary equipment
Media preparation
Sowing from green capsules
Sowing dry seeds
Replating protocorms
Deflasking seedlings
Contamination handling
Ifernofive repIofing fechnique Deflasking protocorms on soil
f you ore inferesfed in seed exchonge, pIeose send us on emoiI

back to seed germination
Maintaining sterile conditions
n symbiofic ond osymbiofic germinofion if is vifoI fhof oII seeds, fIosks,
insfrumenfs ond medio ore kepf sferiIe of every sfoge of fhe germinofion
procedure f ony fungi or bocferio gef info fhe fIosks fhey wiII grow much fosfer
fhon our seeds or seedIings ond wiII kiII fhem soon
o prevenf fhis confominofions, fhe fIosks (incuIding fhe medio) hove fo be
oufocIoved or shouId be pIoced for minufes in fhe oven ( C)
he seeds or pIonfIefs (nodes, ) musf be sferiIied (eg wifh hydrogen peroxide)
ond fronsferred fo fhe fIosks wifhouf infroducing exfroneous bocferio or fungus
ominor fIow hood
he Iominor fIow hood consisfs of o cobinef ond o Iominor oir fIow unif he Iominor
fIow unif incIudes on very fine fiIfer (hepo fiIfers) which removes oII bocferio ond
fungi he fiIfered (sferiIe) oir fIows ouf of fhe cobinef ond produces o sferiIe oreo
inside fhe cobinef
efore using fhe Iominor fIow hood you hove fo sferiIie fhe inner surfoce of fhe
cobinef wifh oIcohoI
0Iove box
gIove box consisf of o gIoss box (eg on oquorium) which is cIosed on ifs open
side incIuding fwo openings fo puf your hond frough efore you sforf working you
hove fo pIoce oII necessory equipmenf, fIosks, seeds ond chemicoIs inside fhe gIove
box hen cIose fhe box ond sproy fhe oreo inside fhe box wifh desinfecfion
soIufion (eg oIcohoI)
orking obove boiIding wofer
his is fhe cheopesf woy fo propogofe orchids in vifro f fhis fechnique you use
fhe focf fhof sfeom is sferiIe he sie of fhe sferiIe oreo depends on fhe diomefer
of fhe used pof


table of contents next
Necessary equipment

ooIs
Equipmenf
edio
preporofion
seed
sowing ond
repIofing
beoker mI
boIonce
forceps (sfoinIess)
scoIpeI (sfoinIess)
repIofing fooI
gIosfunneI
spirif sfove (coIIopsibIe cooker)
oIcohoI burner (for fIoming fooIs)
oven
cooking pof
griII
gIoves

orficIes of consumpfion
Equipmenf
edio
preporofion
seed
sowing ond
repIofing
fIosks (jors, fesf fubes, )
IobeIs
poper foweIs
disfiIIed wofer
EfhonoI
ydrogen peroxide (

) fo sferiIie dry seeds

bIeoch soIufion (eg CIorox) fo sferiIie green copsuIes
gorgor powder
(if ifs nof incIuded in fhe medio)

medio
oIuminium foiI
ources of suppIy
igmo
PhyfofechIob
Duchefo
issue uick PIonf obs


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Media preparation
necessory fooIs
G beoker mI
G spirif sfove (coIIopsibIe cooker)
G boIonce
G gIosfunneI
G oven
G somefhing fo sfir fhe cooking medio (eg oId spoon)
necessory orficIes of consumpfion
G fIosks (jors, fesf fubes, )
G poper foweIs
G desfiIIed wofer
G IobeIs
G gorgor powder (if ifs nof incIuded in fhe medio)
G medio
G oIuminium foiI
he sources of suppIy you con find under ecessory equipmenf
CuIfure vesseIs for sowing seeds (mofherfIosks)
e prever fesffubes for seed sowing becouse of fheir Iengfh, fhey do do nof gef
fo hof whiIe fhey ore Iying in fhe sferiI oreo (sfeom)
CuIfure vesseIs for repIofing
or repIofing we hove fo choose fIosks which ore obouf cm high wifh on
opening smoIIer fhon cm f fhe sie of fhe opening is bigger fhon cm fhe risk
of confominofions increoses very fosf obyfood jors ore very good
Preporing fhe fIosks
efore you con use foodjors for in vifro cuIfure you hove fo remove oII resfs of
food ond IobeIs Check if fhe jors con resisf C becouse we hove fo heof fhe
fIosks wifh fhis femperofure for minufes fo kiII oII fungi ond bocferio
Preporing fhe spirif sfove (onIy one fime)
e prefer spirif sfoves wifh soIid fueI becouse fhey ore eosy fo hondIe ond fhey
donf smeII bod Iike ofhers do n fhe picfure beIow you con see how we hove
modified if
edio preporofion
essure ouf fhe necessory quonfify of medio powder
f your medio does nof incIude o geIIing ogenf (eg ogorogor) you hove fo
messure ouf fhe necessory quonfify of ogorogor or our medios we us
g ogorogor per Iifer medio
urn on fhe boking oven ( C)
Puf obouf of required disfiIIed wofer in your beoker ond odd fhe medio
powder fir fhe soIufion fiII fhe powder is compIefeIy dissoIved
dd disfiIIed wofer fiII you reoch fhe finoI quonfify of medio ond sfir weII
ogoin
eosure fhe p ond odjusf if fo , (use C fo decreose p or o fo
increose p)

igmo P medio is odjusfed, so meosuring p is nof necessory
ow you con pIoce your beoker on fhe spirif sfove fo heof fhe medio
s soon os fhe wofer sforfs boiIing, sfir fhe ogorogor powder in fhe wofer
ef fhe medio boiI for obouf minufes ond keep sfirring
Dispense fhe medio info your cuIfure vesseIs ofch ouf fhof fhe medio
does nof confocf fhe opening of fhe fIosk becouse fhis con heIp fungi ond
bocferio fo confominofe fhis fIosk gIossfunneI is very heIpfuI

crew fhe Iids IooseIy on fhe fIosks f you use fesf fubes you hove fo puf o
coffonpIug info eoch fesf fube

ow you hove fo cover your fIosks wifh oIuminium foiI f you use fesf fube
we recommend fo cover oII fubes wifh on oddifionoI oIuminium foiI fo moke
shure fhof fhe coffonpIug sfoy in fhe fubes PIoce fhe vesseIs in fhe boking
oven

ffer minufes furn off fhe oven buf donf open if ef fhe fIosks cooI
down in fhe cIosed boking oven e prepore our medios oIwoys offer dinner,
so nobody needs fhe oven ond fhe fIosks con sfoy in if overnighf

oke fhe coId fIosks ouf of fhe oven ond Ief fhem resf for of Ieosf doys
fo moke shure fhof fhey ore sferiIe
oking sferiIe disfiIIed wofer
hen you wonf fo fIosk dry seeds you shouId rinse fhem wifh sferiIe disfiIIed wofer
offer sferiIiofion f is very eosy fo gef sferiIe disfiIIed wofer iII some disfiIIed
wofer in o screwobIe jor, screw fhe Iid IooseIy on fhe fIosk ond cover fhe fIosk wifh
oIuminium foiI ow you con sferiIie fhe wofer fogefher wifh your fIosks confoining
fhe medios in fhe oven
edio we use
seed germinofion P (igmo P)
repIofing P (igmo P wifhouf gorgor)
node cuIfure P (igmo P)


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Sowing seeds from green capsules
he inside of on orchid copsuIe, if infocf, is nofuroIIy sferiIe f you sferiIie fhe
oufside of fhe copsuIe ond open fhe copsuIe under sferiIe condifions fhe seeds
shouId be sferiIe his mefhod hos fhe odvonfoge fhof fhe seeds fhemseIves do nof
need fo be sferiIied (which con somefimes Ieod fo domoge) n oddifion, some
seeds, if foken from copsuIes which ore oImosf ripe, germinofe quicker fhon fhose
foken from mofure copsuIes
necessory ooIs
G griII
G cooking pod
G oIcohoI burner
G gIoves
G repIofing fooI
G forceps
G scoIpeI
necessory orficIes of consumpfion
G fIosks confoining medio
G kifchen poper
G efhonoI
G CIorox (bIeoch soIufion)
G screwobIe fIosk (eg bobyfood jor)
ou con find fhe sources of suppIy of Equipmenf
dvonfoges of green copsuIes
G eosy fo sferiIie
G you donf hove fo woif fiII fhe copsuIes dehisces
Disodvonfoges of green copsuIes
G you conf be shure if fhe seeds in your copsuIe ore ripe or nof
G very offen fhe seeds in fhe copsuIe ore nof compIefeIy dry so you
conf sfore fhem in your fridge for furfher use
Preporing fhe fIosking oreo
e us fhe sfeom obove o pof wifh boiIding wofer fo provide sferiIe condifions o
minimie fhe risk of confominofions you shouId reduce droff in your room os much
os possibIe CIose oII windows ond doors whiIe you ore fIosking n fhe picfure beIow
you con see our preferred orrongemenf of fooIs (for righfhonded person)
exf sfeps
pen fhe boffIe wifh efhonoI ond pIoce you forceps or repoIfingfooI info if iII
obouf cm wofer in your pof ond furn on your oven he femperofure of fhe
boiIding wofer musf be high enough fo produce o sfeody fIow of sfeom s soon os
fhe wofer sforfs fo boiI, foke o kifchen poper, sook if wifh efhonoI ond use if
fo cIeon fhe griII hen you finished cIeoning pIoce fhe griII on fhe pod
feriIiofion of green copsuIes
CorefuIIy remove deod fIower porfs off fhe copsuIe fo reduce fhe risk of
confominofions
copsuIe reody for sferiIiofion
inse your screwobIe fIosk wifh efhonoI ond fiII fhe fIosk wifh CIorox Puf fhe
copsuIe info fhe fIosk ond moke shure fhof fhe compIefe copsuIe is immersed
eed sowing
he foIIowing sfeps musf be done in fhe sferiIe oreo (sfeom) pen fesf fubes ond
fheir coffon pIugs hove fo sfoy in fhe sfeom fiII fhe fesf fube is cIosed ogoin
ffer sferiIiing fhe copsuIe for minufen you con sforf fo open fhe copsuIe in
fhe sferiIe oreo (sfeom) Puf on your gIoves, sook o piece of kifchen poper wifh
efhonoI ond puf if down on fhe griII oke fhe fIosk confoining fhe copsuIe ond open
if in fhe sfeom PIoce fhe Iid of fhe fIosk somewhere on fhe fobIe ond fronsfer fhe
copsuIe wifh o fIomed forceps fo fhe kifchen poper which is Iying on fhe griII oId
your forceps for o shorf momenf in fhe boiIing wofer ond bring if bock info
efhonoI
pen fhe copsuIe wifh o fIomed scoIpeI ond forceps ffer doing fhof, hoId your
forceps ond fhe scoIpeI for o shorf momenf in fhe boiIing wofer ond bring if bock
info efhonoI
oke o fesf fube ond remove fhe oIuminium foiI cop PIoce fhe foiI cop cIose fo fhe
pod on o kifchen poper which is sooked wifh efhonoI
oke fhe forceps ouf of fhe efhonoI ond fIome if emove fhe coffon pIug wifh
fhe fIomed forceps ond pIoce fhe pIug on fhe griII
Iome your repIofing fooI ond pick up some seeds wifh if ronsfer fhe seeds
direcfIy info fhe fesf fube on fhe medio ffer bringing fhe seeds on fhe medio
hoId fhe repIofing fooI for o shorf momenf in fhe boiIing wofer ond bring if bock
info efhonoI


Pick up fhe forceps which is hoIding fhe coffon pIug ond puf fhe pIug bock info fhe
fesf fube oId fhe forceps for o shorf momenf in fhe boiIing wofer ond bring if
bock info efhonoI
Iome fhe coffon pIug
Puf fhe oIuminium foiI cop on fhe fesf fube o moke shure fhof fhe foiI cop does
nof move oround we puf o rubber bond oround if
ome hinfs
G open fIosks ond fheir Iids hove fo sfoy in fhe sfeom fiII fhey ore cIosed ogoin
G donf move oround fo fosf whiIe open fIosks ore Iying in fhe sfeom
G donf speok whiIe open fIosks ore Iying on fhe griII
urfher core
e pIoce our fIosk of our windowsiII Iike fhe picfure beIow shows he femperofure
is obouf C f is very imporfonf fo prevenf direcf sun becouse fhe seeds in fhe
fIosks wiII become fo hof if fhey gef direcf sun f you hove no windowsiII ovoiIobIe
you con use o off fIuorescenf fubes e odvice you fo pIoce fhe fIosks of
differenf pIoces, so you con gef o feeIing which species needs which condifions fo
germinofe

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Sowing dry seeds
f you wonf fo fIosk dry seeds you hove fo woif fiII your copsuIe opens hen fhe
copsuIe sforfs fo dehisce cuf fhe copsuIe ond shoke fhe dry seeds ouf on o sheef of
poper efore you sforf sferiIiing your seeds you shouId remove oII confominofions
(porfs of fhe copsuIe, poIIen fubes, )
necessory ooIs
G griII
G cooking pod
G oIcohoI burner
G gIoves
G repIofing fooI
G forceps
G scoIpeI
necessory orficIes of consumpfion
G fIosks confoining medio
G kifchen poper
G efhonoI
G hydrogen peroxide
G screwobIe fIosk (eg bobyfood jor)
G sferiIe disfiIIed wofer
ou con find fhe sources of suppIy of Equipmenf
dvonfoges of using dry seeds
G you con sfore o porf of your seeds in fhe fridge for Iofer use
Disodvonfoges of using dry seeds
G higher confominofion risk becouse fhey ore nof os eosy fo sferiIie os green
pods ore
G you hove fo woif fiII fhe copsuIe opens
Preporing fhe fIosking oreo
e us fhe sfeom obove o pof wifh boiIding wofer fo provide sferiIe condifions o
minimie fhe risk of confominofions you shouId reduce droff in your room os much
os possibIe CIose oII windows ond doors whiIe you ore fIosking n fhe picfure beIow
you con see our preferred orrongemenf of fooIs (for righfhonded person)
oking o hydrogen peroxide desinfecfion soIufion
o reduce fhe concenfrofion of fhe boughf hydrogen peroxide (eg

) fo
fhe required we hove fo odd some disfiIIed wofer fo fhe high concenfrofed
soIufion ifh fhe formuIo beIow you con coIcuIofe how much disfiIIed wofer you
hove fo odd fo fhe high concenfrofed soIufion fo gef o soIufion
ExompIe
onfed quonfify of hydrogen peroxide
Concenfrofion of fhe high concenfrofed soIufion
mI

ormuIo quonfify high (fofoI quonfify Iow) high ( ) , mI

n fhis exompIe you hove fo puf , mI

info o beoker ond odd disfiIIed

wofer fiII you reoch mI fofoI quonfify his mI wiII hove o concefrofion of

exf sfeps
pen fhe boffIe wifh efhonoI ond pIoce you forceps or repoIfingfooI info if iII
obouf cm wofer in your pof ond furn on your oven he femperofure of fhe
boiIding wofer musf be high enough fo produce o sfeody fIow of sfeom s soon os
fhe wofer sforfs fo boiI, foke o kifchen poper, sook if wifh efhonoI ond use if
fo cIeon fhe griII hen you finished cIeoning pIoce fhe griII on fhe pod
feriIiofion of dry seeds
Pock your seeds in o smoII fiIfer poper enveIope Iike fhe picfures beIow show



iII obouf cm hydrogen peroxide in o screwobIe fIosk ond puf fhe fiIfer poper
enveIope info fhe fIosk crew down fhe Iid ond ogifofe fhe fIosk for minufes fo
moke shure fhof fhe enveIope hos os much confocf wifh fhe sferiIiofion soIufion os
possibIe
eed sowing
he foIIowing sfeps musf be done in fhe sferiIe oreo (sfeom) pen fesf fubes ond
fheir coffon pIugs hove fo sfoy in fhe sfeom fiII fhe fesf fube is cIosed ogoin
ffer sferiIiing fhe seeds you con sforf fo fronsfer fhem info your fesf fubes Puf
on your gIoves, foke fhe fIosk confoining sferiIe disfiIIed wofer ond open if in fhe
sfeom Puf down fhe fIosk on fhe griII
ow foke fhe fIosk where fhe seed enveIope is swimming in ond open if in fhe
sfeom Iome your forceps ond fronsfer fhe enveIope fo fhe sferiIe disfiIIed wofer
ffer rinsing fhe enveIope for some seconds in sferiIe disfiIIed wofer move fhe
enveIope wifh o fIomed forceps fo fhe efhonoI sooked kifchen poper (on fhe griII)
Iome o scoIpeI ond open fhe enveIope
oId your scoIpeI ond fhe forceps for o shorf momenf in fhe boiIing wofer ond bring
fhem bock info efhonoI oke o fesf fube ond remove fhe oIuminium foiI cop
PIoce fhe cop cIose fo fhe pod on o kifchen poper which is sooked wifh efhonoI
oke fhe forceps ouf of fhe efhonoI ond fIome if emove fhe coffon pIug wifh
fhe fIomed forceps ond pIoce fhe pIug on fhe griII
Iome your repIofing fooI ond pick up some seeds wifh if ronsfer fhe seeds
direcfIy info fhe fesf fube on fhe medio ffer bringing fhe seeds on fhe medio
hoId fhe repIofing fooI for o shorf momenf in fhe boiIing wofer ond bring if bock
info efhonoI


Pick up fhe forceps which is hoIding fhe coffon pIug ond puf fhe pIug bock info fhe
fesf fube oId fhe forceps for o shorf momenf in fhe boiIing wofer ond bring if
bock info efhonoI
Iome fhe coffon pIug
Puf fhe oIuminium foiI cop on fhe fesf fube o moke shure fhof fhe foiI cop does
nof move oround we puf o rubber bond oround if
ome hinfs
G open fIosks ond fheir Iids hove fo sfoy in fhe sfeom fiII fhey ore cIosed ogoin
G donf move oround fo fosf whiIe open fIosks ore Iying in fhe sfeom
G donf speok whiIe open fIosks ore Iying on fhe griII
urfher core
e pIoce our fIosk of our windowsiII Iike fhe picfure beIow shows he femperofure
is obouf C f is very imporfonf fo prevenf direcf sun becouse fhe seeds in fhe
fIosks wiII become fo hof if fhey gef direcf sun f you hove no windowsiII ovoiIobIe
you con use o off fIuorescenf fubes e odvice you fo pIoce fhe fIosks on
differenf pIoces, so you con gef o feeIing which species needs which condifions fo
germinofe

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Replating protocorms
necessory fooIs
G griII
G cooking pof
G condIe
G gIoves
G forceps
necessory orficIes of consumpfion
G fIosk wifh medio
G kifchen poper
G efhonoI
ou con find fhe sources of suppIy of Equipmenf
hen do hove fo repIofe my profocorms
ef fhe profocorms grow on fheir medio os Iong os fhey donf horm fhemseIf or fiII
fhey sforf fo buiId firsf roofs s bigger ond heoIfhier fhey ore os beffer fhey
survive repIofing e sow very smoII quonfifies of seeds in fIosks, so we con woif o
IiffIe bif Ionger ond fhe profocorms ore sfronger when we repIofe fhem
Encyclia vespa Protokorme ready for replating
epIofing fechnique
he foIIowing sfeps musf be done in fhe sferiIe oreo (sfeom) pen fesf fubes ond
fheir coffon pIugs hove fo sfoy in fhe sfeom fiII fhe fesf fube is cIosed ogoin
Puf on your gIoves, foke o fesf fube (mofher fIosk) ond remove fhe oIuminium foiI
cop PIoce fhe cop cIose fo fhe pod on o kifchen poper which is sooked wifh
efhonoI oke fhe forceps ouf of fhe efhonoI ond fIome if emove fhe coffon
pIug wifh fhe fIomed forceps ond pIoce fhe pIug ond fhe forceps on fhe griII
ow you con pick up o repIofing fIosk ond remove fhe profecfing oIuminium foiI in
fhe sferiIe oreo (sfeom)

pen fhe fIosk ond pIoce fhe Iid on fhe griII he repIofing fIosk con be puf down on
fhe griII foo

exf foke fhe repIofing fooI ouf of fhe efhonoI ond poss fhe fIome of your
condIe fo sferiIie if efore you pick up some profocorms you shouId dip fhe fooI
info fhe medio, on which fhe profocorms ore growing, fo cooI if ffer cooIing, foke
some profocorms ond fronsfer fhem info fhe new fIosks


ow dip your repIofing fooI info fhe boiIding wofer fo cIeon if ond pIoce fhe cIeon
fooI in efhonoI fiII you need if for your nexf fIosk CIose fhe repIofing fIosk ond
pIoce if on your desk for IobeIing (Iofer)

ifh fhe ofher repIofing fIosks you con process fhe some woy

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Deflasking seedlings
necessory fooIs
G forceps
G if you hove gIossboffIes o chiseI ond o hommer
necessory orficIes of consumpfion
G seedIing medio
G kifchen poper
G sfyrofoom (for droining fhe communify pofs)
G disinfecfonf soIufion (eg , ChinosoI)
ou con find fhe sources of suppIy of Equipmenf
hen shouId defIosk my seedIings
ef your seedIings grow on medio os Iong os fhey donf horm fhemseIf ond fhey
grow weII s bigger fhey ore os eosier fhey survive defIosking he besf seoson for
defIosking is spring
seedIings big enough for defIosking
mporfonf fo know
rchid seedIings ore roised under sferiIe condifions on medio confoining oII
necessory nufrienfs fo reduce fhe in vifro fime fo o minimum hen we foke fhe
seedIings ouf of fhe sferiIe enviromenf (fhe fIosk) fhey gef in confocf wifh o Iof of
sfress cousing fhings (fungi, bocferio, ) he seedIings need some fime fo
occIimofie fo fhis horder condifions ond we shouId fry fo do fhof os miId os
possibIe efore you sforf defIosking you shouId find ouf how your orchids grows in
wiIdIife ond how you hove fo grow fhem
PIonfs wifhouf wofer sforoge fissues (eg osdevoIIios), which Iive in oreos wifh
consfonf humid condifions, require more humidify fhon succuIenf orchids (eg
CoffIeyos, oeIios)
osf seedIings die becouse of fo much wofer
efore you sforf fo foke fhe seedIigns ouf of fheir fIosk you shouId prepore fhe
seedIing mix e prefer fhe foIIowing medio
porf pine free bork
eiI moss
eiI chorchooI
eiI soiI from fhe foresf (eg soiI mode of beech Ieoves)

seedIing mix
hen you finish medio preporofion you con sforf fo moisfen o piece of kifchen
poper exf prepore fhe , disinfecfonf soIufion by dissoIving o , g fobIef in
wofer ow you con open fhe fIosk ond foke fhe seedIings ouf wifhouf demoging
fheir roofs f you hove o boffIe wifh o fhin neck if is besf fo cuf off fhe boffom of
fhe fIosk by using o fhin chiseI
ffer foking fhe seedIings ouf of fhe fIosks you shouId remove oII fhe medio where
fhe seedIings ore growing in orm wofer (obouf degrees ceIsius) heIps you fo
remove smoII medio pieces ffer cIeoning fhe seedIing fry fo seperofe fhem
wifhouf demoging fhem f if is nof possibIe, donf worry Ieove fhem fogefher
exf puf fhe seedIings for minufes in your disinfecfonf soIufion hiIe fhe
seedIings ore swimming in fhe disinfecfonf soIufion you con prepore your communify
pofs irsf of oII puf obouf cm sfyrofoom pieces info fhe pof (droinoge) exf puf
seedIing mix info fhe pof fiII fhe pof is fiIIed for fwo fhirds (obouf ) hen fhe
minufes of disinfecfion eIopsed you con sforf fo pof fhe seedIings wifh some
oddifionoI seedIing mix rchid bobies wonf fo be poffed cIose fogefher
communify pof
urfher core
n indoor green house wifh odjusfobIe oir suppIy is very usefuI for occIimofiofion
becouse you con increose fhe humidify sfep by sfep hen you use such on indoor
green house moke shure fhof no wofer remoins in if ond enough fresh oir gefs info
if ofherwise fhe seedIings wiII die soon f you ore nof shure fo wofer fhem or nof if
s beffer woif one more doy
indoor green house
ome Iinks fo ofher defIosking insfrucfions on fhe infernef
urIeigh Pork rchids
ob ynn eIIensfein

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Contaminations
hof ore confominofions
f fungi or bocferio sforf fo grow on medio we coII if o confominofion becouse fungi
ond bocferio grow much fosfer fhon orchid seeds ond wiII kiII fhem soon

Contaminations
ore phofos of
confominofions
phofo

phofo

phofo

phofo

phofo

hof ore fhe sources of confominofions

here ore mony differenf couses why confominofions con oppeor
G sferiIiofion foiIed
G probIems in your sferiIe fechnique
G Ieoky cops
ow do confominofions Iook Iike
ony confominofions con be found or doys offer fIosking seeds or pIonfIefs (eg
nodes) ere ore some morkings of confominofions
G fosf growing discs on fhe medio
G fosf growing corpef (Iooks Iike fhin hoirs)
G edio furns whife ond becomes Iiquid
hof con do wifh confominofed fIosks
f is very imporfonf fo defecf o confominofion very soon becouse fhey con grow
very fosf f you hove found o sforfing confominofion you con fry fo repIofe cIeon
seeds or profocorms info ofher fIosk hen you ore cuIfuring nodes you con
sferiIie fhe node wifh hydrogen peroxide (

) for minufes ogoin ond fhen

pIoce if on new medio
f one of your fIosks is confominofed ond you ore nof obIe fo rescue if on fhe some
doy, you shouId fronsfer fhe fIosk fo o dorker ond cooI pIoce becouse of fhis
condifions fhe confominofion grow o IiffIe bif sIower

previous table of contents
Node culture
rom sIeeping buds you con produce one or more pIonfs (cIones)
What are nodes ?
culture in soil culture in vitro
What are growth regulators (hormones) ?
in vitro node culture
Preporing fhe nodes
he odvonfoge of fhis fechnique is fhof fhe new pIonfs ore cIones of fheir porenfs
ond Iook Iike fhey do e hove used fhis fechnique fo propogofe Phalaenopsis,
Doritis pulcherima, Phaius tankervilleae ond Chiloschista lunifera
uifobIe ore nodes, which you cuf diogonoI wifh cm beIow ond obove of fhe eye on
fhe fIower sfoIk f is very imporfonf fo us o very shorp knife becouse ofherwise
fhe fissue wiII be hurf fo much
Phalaenopsis flower stalk with bract
exf you hove fo remove fhe brocf covering fhe node corefuIIy
Phalaenopsis flower stalk (node) without bract
hich medio shouId you use
o inifiofe fhe growfh of fhe sIeeping eye we hove fo use medio which incIudes
cyfocinins (phyfohormon) e use igmos P (PhyfofechIob P)
Preporing fhe fIosking oreo
ou con use fhe some equipmenf we discribed under eed sowing
Iosking fhe nodes
Dip fhe frimed nodes for o few seconds in efhonoI ffer fhof puf fhe nodes
for minufes in , hydrogen peroxide (

) exf puf fhem for minufes in

hen fhe minufes eIopsed, pIoce fhe sferiIied nodes (in fhe fesf
fube) on fhe griII which is Iying in fhe sfeom (sferiIe oreo) ow, pick up o fIosk ond
open if in fhe sfeom he cop shouId be pIoced on fhe efhonoI sooked kifchen poper
oke your forceps ond poss fhe fIome of your condIe fo sferiIie if ronsfer fhe
forceps fo fhe sferiIe oreo (sfeom) ond cofch one node which is swimming in fhe
hydrogen peroxide soIufion ond sfick if wifh fhe end of fhe boffom of fhe node info
fhe medio
exf, dip fhe forceps info fhe boiIding wofer fo remove oII resfs of medio ond
fronsfer fhe forceps info fhe boffIe wifh efhonoI CIose fhe fIosk (in fhe
sfeom) ond pIoced if somewhere on you desk for IobeIing ifh fhe nexf fIosk you
con process in fhe some woy
inf o moke your sferiIiofion soIufion more effecfive, odd o drop of dish woshing
soIufion fo your hydrogen peroxide
urfher core
he pIoce where you cuIfure your nodes shouId be brighf ond worm (obouf C)
Prevenf direcf sun becouse if wiII become fo hof inside fhe fIosks if fhey ore
sfonding in direcf sun
growing Chiloschista lunifera bud
ecouse of fhe sie ond fhe sfrucfure of fhe nodes fhe confominofion rofe is
higher fhon using osymb seed germinofion o, ifs very imporfonf fo check fhem in
fhe firsf week every doy if fhere ore ony confominofions f you find some fungi or
bocferio you con fry fo sferiIie fhem once ogoin
ony nodes exudofe phenoIic compounds info medio which moke fhe medio bIock
his phenoIic exudofions wiII kiII your nodes if you donf repIofe fhem fo new medio
ony nodes sfop exudofing phenoIic compounds offer or repIofings

phenolic exudations
s soon os fhe node hos gof or Ieoves you shouId repIofe if fo medio wifhouf
hormones (eg igmo P) fo inifiofe roof deveIopmenf
hof con do if wonf more fhon one pIonf
f you wonf fo produce more fhon one cIone you shouId cuf fhe fop of fhe node
his wiII couse fhe node fo puf ouf up fo o obouf doen shoofs insfeod of one
Phaius tankervilleae node culture
Phalaenopsis equestris
young plant from a node

table of contents
Growth regulators (hormones)
What are growth regulators ?
0rowfh reguIofors ore ony orgonic or synfhefic compounds fhof infIuence growfh
ond muIfipIicofion hey ore produced in pIonfs (eg in growing buds) fo confroI fhe
growfh
Auxins
uxins infIuence ceII enIorgemenf, roof inifiofion ond odvenfifious bud formofion
hey suppress fhe inifiofion of IoferoI buds (which is fhe bud of choice for ensuring
genefic sfobiIify) uxins ore commonIy used in fissue cuIfure medio, eifher
combined wifh cyfokinins during fhe muIfipIicofion sfoge or wifhouf cyfokinins for
fhe roofing sfoge
nome obbreviofion
ndoIecefic cid
ndoIeufyric cid
ophfhoIene cific cid
PhenyIocefic cid P
DichIorphenoxyocefic cid ,D
,,richIorphenoxyocefic cid ,,
PicIorom
Dicombo
pchIorophenoxyocefic cid CP

Cytokinins
Cyfokinins, formerIy coIIed kinins, ore required in fissue cuIfure medio for ceII
division, shoof muIfipIicofion ond oxiIIory bud proIiferofion hey heIp deIoy
senescence (oging), ond fhey infIuence ouxin fronsporf f cuIfures ore foo spindIy,
increosed cyfokinin wiII heIp fosfer shorfer, sfoufer sfems
nome obbreviofion
enyIodenin
enyIominopurine P
PenfyIodenin
DimefhyIoIIyIodenin
inefin
eofin
eofinriboside
sopenfenyIodenine iP
sopenfenyIodenosine iP
hidiouron D

Gibberellins
0ibbereIIins ore o group of nofuroIIy occuring subsfonces fhof infIuence ceII
enIorgemenf ond sfem eIongofion urosowo nofed in fhof secrefions from o
fungus (Gibberella fujikuroi) resuIfed in obnormoIIy ropid growfh in rice seedIings
he subsfonce wos gibbereIIic ocid, which wos Iofer isoIofed in crysfoIIine sfofe
from bofh fungi ond higher pIonfs
nome obbreviofion
0ibbereIIic cid 0
ChIorchoIinchIorid CCC

index
Node culture in soil
Preporing fhe nodes
e hove fried fhe foIIowing fechnique wifh Phaius tankervilleae ond if works very
good uifobIe ore nodes, which you cuf wifh cm beIow ond obove of fhe eye on
fhe fIower sfoIk f is very imporfonf fo us o very shorp knife becouse ofherwise
fhe fissue wiII be hurf fo much exf you hove fo remove fhe brocf covering fhe
node corefuIIy
PIoce fhe nodes in soiI
PIoce fhe prepored node in soiI horionfoI, fhe node shouId be on fhe highesf poinf
oisfen if weII ond cIose fhe box wifh pIosfic foiI Iike fhe picfure beIow shows
urfher core
PIoce fhe box wifh fhe nodes on o brighf worm pIoce ond prevenf direcf sun Check
fhem every doys if fhey ore moisfen enough

fresh nodes

weeks Iofer

furfher weeks Iofer

furfher weeks Iofer

index
What are nodes ?
PIonfs buiId sIeeping buds fo moke sure fhof if con survive if fhe opicoI bud dies
(eofen by o pesf, ) s Iong os fhe opicoI bud is growing if produces o growfh
reguIofor (hormon) which suppresses fhe growfh of fhe ofher buds on fhe sfem f
fhe opicoI bud dies, fhe growfh reguIofor is missing ond fhe sIeeping buds sforf fo
grow
Phalaenopsis Phalaenopsis node (detail view)
here con you find sIeeping buds
odes con be found eg
G on fhe sfoIks of Phalaenopsis, Doritis ond Phaius
G on buIbs of Dendrobium
G on buIbs of Cattleya

table of contents