Plant Tissue Culture Practical Note Book

Plant Tissue culture
(Practical Note Book)

By;
Nasir Hussain
(M.Phil Roll NO#05)
Subitte! "o;
#r. $sh Muhaa!
#r. %&bal Muhaa!
Plant 'enoics (Biotechnolo)y
National *ni+ersity o, $)ricultural Sciences
National $)ricultural Research centre %slaaba!
1
-./erient No0 01
%n +itro see!lin) )ro2th o, tobacco
Ob3ecti+es0
In vitro explants source for onward experiments (in vitro multiplication, in vitro rooting,
callus induction and regeneration and hardening in green house)
Me!iu0
MS (plain) (Murashige and Skoog, 1!") medium was used as #asal medium in present
investigation$ %omposition of MS media is given in ta#le$
4o/osition o, e!iu0
1. Solution A (KNO3, NH3NO3, CaCl2.2H2O),
2. Solution B (MgSO4.7H2O),
3. Solution C (KH2PO4),
4. Solution D (MnSO4.4H2O2, H3BO3, ZnSO4.4H2O, KI, Na2MoO4.2H2O,
C4SO4.5H2O, CoCl2.6H2O),
5. Solution E (FeSO4.7H2O, Na2 E!".2H2O)
6. Solution F (#$%a&$n'), Sucrose, Agar an( distilled water.
"able0 4o/osition o, Murashi)e an! Skoo) (1567) Me!iu
Me!ia 8olue (1000l)
S. No 4onstituents 9orula 4onc. in stock
solutions ):l
8olue o, stock:l
o, e!iu (l)
Macronutrients
1 Magnesium sulphate MgS&'$()"& ($'
*+
" %alcium chloride %a%l"$")"& ,$,
- Potassium nitrate ./&- -,
' 0mmonium nitrate /)'/&- --
* Potassium di
h1drogen phosphate
.)"P&' -$'
Micronutrients
! Manganese sulphate MnS&'$ )"& '$' *
( 2inc sulphate 2nS&'$)"& 1$("
"
, %opper Sulphate %uS&'$")"& +$+1
%o#alt chloride %o%l"$!)"& +$++*
1+ Potassium iodide .I 1$!(
11 3oric acid )-3&- 1$"'
1" Sodium mol1#date /a"Mo&'$")"& +$+*
%ron Source
1- 4errous sulphate 4eS&'$()"& *$*!
*
1' Sodium 56T0 /a"56T0$")"& ($'!
Or)anic Su//leents (8itains)
1* M1o7inositol "+
*
1! /icotinic acid +$1
1( P1ridoxine7)%l +$1
1, Thiamine7)%l +$1
1 8l1cine +$'
4arbon Source
"+ -+ g 9l of Sucrose was used in MS medium$
PR-P$R$"%ON O9 1 ;%"-R MS M-#%*M0
&ne liter medium was prepared according to the following protocol$
*+ ml of macronutrients, * ml of micronutrients, * ml of vitamins and *ml of iron source
from their stock solutions were added #1: "+ml of solution 0 and solution 3, 1+ml of
solution % and solution 6 and vitamin solution, *ml of solution 5 and -+g of sucrose -;
(w9v) was dissolved in 1++ ml of distilled water contained in a "7litre flask and then
poured in graduated c1linder$ <olume was raised up to one liter with distilled water$
/H a!3ustent0
To ad=ust p) of the media, p) meter was cali#rated$ 4irst the lens of p) metre was
washed with distilled water and dr1 with clean washing paper$ Then #uffer ' was used to
ad=ust the p) to ', error was ad=usted from slope #utton$ >ash the lens with distilled
water and dr1 with clean washing paper$ %hecked with #uffer ( and ad=ust the p) to (,
error was removed from #uffer #utton$ 0gain washed the lens with distilled water,dr1 it
and check with #uffer ' for conformation$ Then p) of media was ad=usted to *$,+$The
-
error was removed #1 using /a&) (+$1 /) or )%l (+$1 /) in case of high or low p) of
media$
Soli!i,yin) $)ent0
0gar ,$+g (w9v) was added and medium was #oiled with continuous stirring for
thorough mixing of agar into medium$
Pourin)0
The medium was dispensed into culture vessels i$e$ *+ ml test tu#es$
0pproximatel1 1+71* ml of medium in test tu#es was poured$
Plu))in)0
%ulture vessels were plugged with test tu#e caps and wrapped with aluminum
foil$
$utocla+in) ,or Sterili<ation0
Medium was autoclaved at 1* I#9in
"
pressure for "+ minutes at 1"1
o
%$0fter
sterili?ation the medium was kept for "'7', hours in growth room #efore inoculation to
check whether it was satisfactoril1 sterili?ed$
-=P;$N"S PR-P$R$"%ON0
The to#acco seed explant were sterili?ed #1 soaking in sodium h1po chlorate for 1+mins
and then rinsed with distal water$ Si?e of explant is ver1 important factor as if the si?e of
explant was smaller than re@uired, it produced small amount of callus and if the si?e of
explant was larger than re@uired then more microorganisms were likel1 to enter the
culturing medium$
#$"- O9 %N%"%$"%ON0 15
"H
O4"> 700?
%NO4*;$"%ON PRO4-#*R-0
1$ 5xplants were transferred under Aaminar 4low %a#inet with )5P0 ()igh
5fficienc1 Particulate 0ir 4ilters)$4ollowing procedure was used$
"$ Aaminar 4low %a#inet was turned on and all floors and walls were swa##ed down
with (+ ; eth1l alcohol carefull1$
-$ 0utoclaved test tu#es with culturing media, Petri7plates, distilled water: forceps,
cutters, mask, cap, gloves and match #ox were place inside %a#inet$ 4lask
'
containing +$1; )g%l" wiped with (+ ; eth1l alcohol and 3enson #urner was
also placed in Aaminar 4low %a#inet$
'$ Transfer room was treated with B< ra1s (Peak emission of "*-(0+) for 1*
minutes #efore use$
*$ B< lamp was turned of and hands were sterili?ed with spirit$
!$ 4or surface sterili?ation, explant were dipped in +$1; )g %l" (w 9 v) solution$
0fter one minute the1 were rinsed with autoclaved distilled water for three times
to remove an1 traces of Mercuric chloride$
($ 4orceps and cutter were dipped in pure eth1l alcohol then incinerated under flame
of 3enson #urner to ensure an1 likel1 contamination$ Then cooled in distilled
water and dried with filter paper$
,$ 5xplant were chopped to prepare for inoculation i$e$ stem, leaf and shoot tips as
given a#ove, then transferred to test tu#es containing media in them near flame$
$ 0fter each inoculation in a test tu#e the manipulated tools were dipped in (+;
eth1l alcohol then incinerated under flame, cooled and reused$
%N4*B$"%ON O9 4*;"*R-S0
%ulturing test tu#es were then placed in growth cham#er for incu#ation$ Standard
conditions were prevailed in culture room$ Through out the temperature of growth room
was "*C1+% for present research$ Photoperiod was 1!9, hours light9dark c1cle$ Aight
intensit1 was 1+++ lux and relative humidit1 was (* C*;$ 5xperiments conducted #1
Murashige (1(') with 0sparagus, 8er#era, Saxifraga, and #romeliads indicated an
optimum light intensit1 of 1,+++ lux during culture initiation and shoot proliferation
$ Obser+ations0
0fter one week, the test tu#es were examined thoroughl1 for the germination and
if an1 contamination and following o#servations were noted$
6ate of o#servationD "!
th
&ct "++,
*
%n +itro see!lin) )ro2th o, tobacco
Test Tu#e /o /o of cultures (Eeplicates) Eesponded %ontaminated T1pe of response
1 * ' + Seedling growth
" " " + Seedling growth
- - - + Seedling growth
' - - + Seedling growth
* " " + Seedling growth
! - " + Seedling growth
( - " + Seedling growth
, - - + Seedling growth
" 1 + Seedling growth
1+ ' " + Seedling growth
11 - - + Seedling growth
1" " 1 + Seedling growth
1- - " + Seedling growth
1' ' " + Seedling growth
1* - " + Seedling growth
1! ' ' + Seedling growth
1( - " + Seedling growth
1, - " + Seedling growth
1 * - + Seedling growth
"+ * * + Seedling growth
"1 - - + Seedling growth
"" " " + Seedling growth
"- - " + Seedling growth
"' - " + Seedling growth
7@ A6 55 0
!
-./erient No0 07
4allus in!uction
Ob3ecti+es0
&ptimi?ation of medium and explants for callus induction of to#acco plant
Me!iu0 MS F "$'$6 (1ml9A)$
4o/osition o, e!iu0
). Solution B (MgSO4.7H2O),
*. Solution C (KH2PO4),
1+. Solution D (MnSO4.4H2O2, H3BO3, ZnSO4.4H2O, KI, Na2MoO4.2H2O,
12. Solution F (#$%a&$n'), Sucrose, Agar an( distilled water.
Pre/aration0
'++ml of distilled water were taken in a #eaker$ "+ml of solution 0 and solution
3, 1+ml of solution % and solution 6 and vitamin solution, *ml of solution 5 and -+g of
sucrose were added to the #eaker$ Then 1ml of "$'$6 solution was added and the #eaker
contents were mixed on an electrical stirrer$ 0fterwards the p) of the mixture was
ad=usted at *$,$ Then !g of agar were added in the a#ove mixture and the mixture was
heated for a#out *mins in an oven$ The mixture was then diluted with distilled water up
to 1+++ml and was again heated for a#out !mins in oven$ 0fter that 1*7"+ml of li@uid
media was poured into * glass =ars under sterili?ed conditions and the mouths of =ars were
covered with plastic sheets #1 means of ru##er #ands$ The glass =ars were then wrapped
with paper and were then su#=ected to the autoclave at 1"1G% for a#out "+mins in order to
maintain sterili?ed conditions in glass =ars$

(
#ate o, initiation0 +3 e, 2++).
Materials0
Sterili?ed To#acco seeds (sterili?ed #1 soaking in sodium h1po chlorate for
1+mins), Safet1 ca#inet (Aaminar 4low Bnit), (+; ethanol, Spatula, Sterili?ed glass =ars
containing MS F "$'$6 media, 0 control =ar containing plane MS media, Metal racks,
%otton plugs, Plastic covers, Eu##er #ands, Markers, 5thanol spra1 #ottle, 3en?ene
#urner and Petri plates$
Ste -./lant0
Stem explants were also excised from plant 1oung stems #1 cutting the stem
transversel1 into pieces of +$* cm $These were also cut longitudinall1 to produce
maximum cuts on the surface that would explant touch the surface of the medium$
Metho!olo)y0
The work was carried out in the inoculation room$ 3efore starting, the hands and
safet1 ca#inet were sterili?ed with (+; ethanol to avoid contamination$ In safet1 ca#inet
under the presence of #en?ene #urner, first the to#acco seedlings grown in the previous
experiments were pulled out of the test tu#es with help of sterili?ed spatula and were
placed in a Petri plate$ The rooting potion of each seedling was then cut with the help of a
#lade$ These seedlings without the rooting portions are our explants$ - to ' of these
explants were inoculated in each of the glass =ars containing the MS F "$'$6 media and
the control =ar containing the plane MS media$ The inoculation tools, i$e$ spatula and
#lade were sterili?ed with ethanol after each inoculation to minimi?e the chance of
contamination$ 0ll the work was done ver1 carefull1 and attentivel1$ The inoculated glass
=ars were then la#eled with marker and were placed in the growth room under optimum
conditions for growth$
,
Obser+ations0
0fter one week, the glass =ars were examined thoroughl1 for the germination and
6ate of o#servationD 1(
th
6ec "++,
'lass 3ars No No o, cultures (Re/licates) Res/on!e! 4ontainate! "y/e o, res/onse
%ontrol * * + Seedlings
1 ' ' + %allus
" ' ' + %allus
- * * + %allus
' * * + %allus
* - - + %allus
6 76 76 0
-./erient No0 0B
%n 8itro Multi/lication o, "obacco

Ob3ecti+es0 Mass scale plant production
Me!iu0 MSF30P (1mg9l)
M$"-R%$;S $N# M-"HO#S
PRO4*R-M-N" O9 M$"-R%$;S0
The present research work was carried out in Plant Tissue %ulture la#orator1 in
Plant 3iotechnolog1 program at /0E%, Islama#ad$ (/>4P) Pakistan$ 0 #rief account of
the materials and methods used and the procedures adopted is given #elow$
';$SS C$R-0
In tissue culture techni@ues, the glass >are used include 5rlenme1er flasks(1+++
ml, *++ ml, "*+ ml, test tu#es (*+ ml ), #eakers (1+++ ml, *++ ml, "*+ ml, 1++ ml ),
pipettes ( "* ml,1+ ml, etc), micropipettes (+$171$+ ml) 8raduated c1linder (1+++ ml, *++
ml ) and Petri7plates$ /ew glassware ma1 release chemicals that are toxic to the cultured
tissues$ 0dditional information can #e o#tained from Street (1(-) and 3iondi and
Thorpe (1,1)$ 0ll the glassware used in the stud1 was made up of P1rex and
3orosilicate$
-D*%PM-N"0
The e@uipment used in tissue culture techni@ues include 5lectrical #alance (847
-++), 3urner, p) meter (Henwa1 --+*), 0utoclave (.P7-+A, 0AP Tok1o, Hapan), Aaminar
4low Transfer %a#inet (5S%&), Magnetic )eating Stirrer (8uohua electric appliance
%o$AT6) and >ater 6istillation Plant$
S"-R%;%E$"%ON "-4HN%D*-S0
0ll t1pe of glass ware like 5rlenme1er flasks, pipettes, Petri7plates, #eakers and
test tu#es were washed with commercial detergent (lemon Max li@uid)$0fter scru##ing
with a #rush, the glassware is rinses repeatedl1 with tap water, and then given two or
three rinses in distilled water until all traces of dirt were removed$ >ashed test tu#es and
1+
flasks were plugged after pouring 8rowth 0gar Media in it to avoid entrance of an1
traces of contamination$ %ontaminated cultures and discarded culture were first
autoclaved to kill all t1pes of micro#es and fungal spores as well as to li@uef1 the agar
that made it eas1 to wash$
Sterili<ation o, %noculation $rea0
Transfer room was cleaned with a commercial detergent on monthl1 #asis and
spra1ed with meth1lated spirit #efore start of work$ 3efore using Aaminar 4low Transfer
%a#inet, all floors and walls were swa##ed down with (+ ; eth1l alcohol carefull1$
Transfer room was treated with B< ra1s (Peak emission of "*-(0+) for 1* minutes
#efore each use$ 0septic transfer of tissues was carried out in a Aaminar 4low Transfer
ca#inet fitted with a )5P0 filter$
S"O4F SO;*"%ON PR-P$R$"%ON0
6ifferent stock solutions prepared for M$S media include macronutrient stock,
micronutrient stock, vitamin stock, iron stock and hormonal stock(30P, ",'76) in
concentration of 1mg91ml$30P stock(+$1g91++ml) was stored in free?er at 7"+c and ",'7
6(+$1g91++ml) was stored in refrigerator at 'c$
4o/osition o, e!iu0
14. Solution B (MgSO4.7H2O),
15. Solution C (KH2PO4),
16. Solution D (MnSO4.4H2O2, H3BO3, ZnSO4.4H2O, KI, Na2MoO4.2H2O,
1). Solution F (#$%a&$n'), Sucrose, Agar an( distilled water.
11
#$"- O9 %N%"%$"%ON0B1:.17:700?
%NO4*;$"%ON PRO4-#*R-0
1+$ 5xplants were transferred under Aaminar 4low %a#inet with )5P0 ()igh
5fficienc1 Particulate 0ir 4ilters)$4ollowing procedure was used$
11$ Aaminar 4low %a#inet was turned on and all floors and walls were swa##ed down
with (+ ; eth1l alcohol carefull1$
1"$ 0utoclaved test tu#es with culturing media, Petri7plates, distilled water: forceps,
cutters, mask, cap, gloves and match #ox were place inside %a#inet$ 4lask
containing +$1; )g%l" wiped with (+ ; eth1l alcohol and 3enson #urner was
also placed in Aaminar 4low %a#inet$
1-$ Transfer room was treated with B< ra1s (Peak emission of "*-(0+) for 1*
minutes #efore use$
1'$ B< lamp was turned of and hands were sterili?ed with spirit$
1*$ 4or surface sterili?ation, explant were dipped in +$1; )g %l" (w 9 v) solution$
0fter one minute the1 were rinsed with autoclaved distilled water for three times
to remove an1 traces of Mercuric chloride$
1!$ 4orceps and cutter were dipped in pure eth1l alcohol then incinerated under flame
of 3enson #urner to ensure an1 likel1 contamination$ Then cooled in distilled
water and dried with filter paper$
1($ 5xplant were chopped to prepare for inoculation i$e$ stem, leaf and shoot tips as
given a#ove, then transferred to test tu#es containing media in them near flame$
1,$ 0fter each inoculation in a test tu#e the manipulated tools were dipped in (+;
eth1l alcohol then incinerated under flame, cooled and reused$
%N4*B$"%ON O9 4*;"*R-S0
%ulturing test tu#es were then placed in growth cham#er for incu#ation$ Standard
conditions were prevailed in culture room$ Through out the temperature of growth room
was "*C1+% for present research$ Photoperiod was 1!9, hours light9dark c1cle$ Aight
intensit1 was 1+++ lux and relative humidit1 was (* C*;$ 5xperiments conducted #1
1"
Murashige (1(') with 0sparagus, 8er#era, Saxifraga, and #romeliads indicated an
optimum light intensit1 of 1,+++ lux during culture initiation and shoot proliferation
Obser+ations0
0fter one week, the test tu#es were examined thoroughl1 for the germination and
#ate o, obser+ation0 1'th Han: "++
S9/o of Hars /o$ of culture
(replicates)
Eespond %ontaminated T1pe of
Eesponse
1 ' ' + 1es
" ' ' + 1es
- ' ' + 1es
' ' ' + 1es
* ' ' + 1es
!(control) ' ' + >eak Seedlings
1-
-./erient No0 0@
4$;;*S R-'-N-R$"%ON

Ob3ecti+e0
To produce disease free plants

Me!iu0 MS F +$1ul9ltr /00, +$*ul9ltr 30P
#ate o, initiation0 -171"7"++,
Materials an! Metho!s0
Pre/aration o, e!iu0
4irst of all MS medium was prepared #1 using six stock solutions 0, 3 ,%, 6, 5 and a
stock solution containing vitamins$ 0 #eaker was taken and was filled with distilled water
up to a#out !++ ml$ Then the following compositions of solutions was added
Stock 0 "+ml9ltr
Stock 3 "+ml9ltr
Stock % 1+ml9ltr
Stock 6 1+ml9ltr
Stock 5 *ml9ltr
0nd 1+ml9ltr of stock solutions containing vitamins were added to #eaker$ The vitamins
solution contain the following ingredients,
1) /icotinic acid +$*ug9ltr
") P1ridoxine +$*mg9ltr
-) Th1mine )%A +$1mg9ltr
') M1oenositol +$1mg9ltr
0nd +$1ul of /00 and +$*ul of 30P were taken with the help of a micropipette and
added to the #eaker$-+ g sugar (sucrose) was weighed on an electric #alance and was
added to the #eaker$ Then the sugar was dissolved with a stirrer and P) was measured
with the P) meter$ P) was ad=usted at *$(+ #1 adding few drops of /0&)$ Then ! g
agar was added to the #eaker and the volume of the solution was made 1+++ ml #1
adding the re@uired volume of distilled water$ Then the #eaker containing the solution
was kept in microwave oven for !7, minutes and #oiled to dissolve the agar$ 0fter the
dissolution of agar, #eaker was taken out of the oven and solution was stirred$ Then the
solution was immediatel1 added to the glass #ottles$ 0#out "*+ ml of the solution was
added to each glass #ottle$ These glass #ottles were then su#=ected to steam sterili?ation
in an autoclave for 1*7"+ minutes at 1* l#s and 1"1%$
1'
%noculation o, the e./lants ,or ulti/lication0
)ands were sterili?ed with the (+; alcohol to ensure aseptic conditions$ %allus that were
growing in the glass tu#es were taken out$ The calli were excised with the #lade and were
carefull1 cultured in the glass #ottles containing the medium for callus regeneration$'7*
explants were cultured per #ottle$ 0ll this inoculation work was done under aseptic
conditions provided #1 the laminar air flow ca#inet$
#$"$ R-4R#-#
#ate o, obser+ation; 1BG01G700?
No o,
obser+ation
No o, cultures Res/on!e! 4ontainate! "y/e o,
res/onse

1

-

-

+
%allus #ecome
regenerated to
plants

" - " +
%alli #ecome
#rown and one
regenerated
- - - +
%allus #ecome
regenerated

' - " +
%allus #ecome
regenerated

* - - +
%alli #ecome
regenerated to
green plants
1*
-./erient No0 05
ROO" %N%"%$"%ON O9 %N8%"RO M*;"%P;%4$"-# SHOO"S
Ob3ecti+e0
To produce roots of in7vitro multiplicated shoots from single shoots
Me!iu0 plain MS media was used
#ate o, initiation0 "17+17"++,
4irst of all plain MS medium was prepared #1 using six stock solutions 0, 3 ,%, 6, 5
and a stock solution containing vitamins$ 0 #eaker was taken and was filled with distilled
water up to a#out !++ ml$ Then the following compositions of solutions was added
Stock 0 "+ml9ltr
Stock 3 "+ml9ltr
Stock % 1+ml9ltr
Stock 6 1+ml9ltr
Stock 5 *ml9ltr
0nd 1+ml9ltr of stock solutions containing vitamins were added to #eaker$ The
vitamins solution contain the following ingredients,
-+ g sugar (sucrose) was weighed on an electric #alance and was added to the
#eaker$ Then the sugar was dissolved with a stirrer and P) was measured with the P)
meter$ P) was ad=usted at *$,+ #1 adding few drops of /0&)$ Then ! g agar was added
to the #eaker and the volume of the solution was made 1+++ ml #1 adding the re@uired
volume of distilled water$ Then the #eaker containing the solution was kept in microwave
oven for !7, minutes and #oiled to dissolve the agar$ 0fter the dissolution of agar, #eaker
was taken out of the oven and solution was stirred$ Then the solution was immediatel1
added to the glass #ottles$ 0#out "*+ ml of the solution was added to each glass #ottle$
These glass #ottles were then su#=ected to steam sterili?ation in an autoclave for 1*7"+
minutes at 1* l#s and 1"1%$
1!
)ands were sterili?ed with the (+; alcohol to ensure aseptic conditions$
Multiplicated shoots growing in glass flasks were taken out$ The larger shoots were
excised with the #lade into smaller pieces and were carefull1 cultured in the glass #ottles
containing the medium for root initiation$"7- explants were cultured per #ottle$ 0ll this
inoculation work was done under aseptic conditions provided #1 the laminar air flow
ca#inet$
#$"$ R-4R#-#
#ate o, obser+ation; 7AG01G700?
No o,
obser+ation
res/onse
1 " " + Eooting started
to all plants
" " " + Eooting started
- " " + Eooting started
' " " + Eooting started
* " /& I5S /ill
1(
-./erient No0 06
4-;; 4*;"*R- %N ;%D*%# M-#%$

Ob3ecti+e0
To maintain media for cell suspension culture so those to o#tain disease free
plants from single cells$

Me!iu0 MS F *mg9 ltr ",' 6 F1mg9 ltr 30P
#ate o, initiation0 "(71"7"++,
4irst of all MS medium was prepared #1 using six stock solutions 0, 3 ,%, 6, 5
Stock 0 "+ml9ltr
Stock 3 "+ml9ltr
Stock % 1+ml9ltr
Stock 6 1+ml9ltr
Stock 5 *ml9ltr
0nd *mg9ltr ",'6and 1 mg9ltr 30P were taken with the help of a micropipette and added
to the #eaker$-+ g sugar (sucrose) was weighed on an electric #alance and was added to
the #eaker$ Then the sugar was dissolved with a stirrer and P) was measured with the P)
meter$ P) was ad=usted at *$(+ #1 adding few drops of /0&)$ )ere no agar was added
and the volume of the solution was made 1+++ ml #1 adding the re@uired volume of
distilled water$ Then the #eaker containing the solution was kept in microwave oven for
!7, minutes and #oiled to dissolve the agar$ 0fter the dissolution of agar, #eaker was
taken out of the oven and solution was stirred$ Then the solution was immediatel1 added
to the glass #ottles$ 0#out "*+ ml of the solution was added to each glass #ottle$ These
glass #ottles were then su#=ected to steam sterili?ation in an autoclave for 1*7"+ minutes
at 1* l#s and 1"1%$
&nl1 *+ Ml Media >as Taken In 5ach 4lask
1,
Maintenance o, e!ia0
)ands were sterili?ed with the (+; alcohol to ensure aseptic conditions$ callus that were
growing in the glass tu#es were taken out$ The calli were excised into smaller pieces with
the #lade and were carefull1 cultured in the glass #ottles containing the medium$"7-
explants were cultured per #ottle$ 0ll this inoculation work was done under aseptic
conditions provided #1 the laminar air flow ca#inet$ Then media #ottles were kept on
shaker to #reak clumps of calli into tin1 pieces so this media should #e read1 for
suspension culture$
#$"$ R-4R#-#
#ate o, obser+ation; 7AG01G700?
No o,
obser+ation
res/onse
1 - - +
%alli clumps
#roken to
smaller pieces
and remain
alive$
" - " +
%alli clumps
#roken down
#ut calli
#ecome dead
1
-./erient No0 0A
%NG8%"RO ROO"%N' M-#%$
Ob3ecti+e0
To produce roots of in7vitro multiplicated shoots from single shoots
Me!iu0 MSFvitFI00 1mg9ltr
#ate o, initiation0 +'7+"7"++,
4irst of all plain MS medium was prepared #1 using six stock solutions 0, 3 ,%, 6, 5
Stock 0 "+ml9ltr
Stock 3 "+ml9ltr
Stock % 1+ml9ltr
Stock 6 1+ml9ltr
Stock 5 *ml9ltr
0nd 1mg9ltr of I00 added to solution for root induction-+ g sugar (sucrose) was weighed
on an electric #alance and was added to the #eaker$ Then the sugar was dissolved with a
stirrer and P) was measured with the P) meter$ P) was ad=usted at *$,+ #1 adding few
drops of /0&)$ Then ! g agar was added to the #eaker and the volume of the solution
was made 1+++ ml #1 adding the re@uired volume of distilled water$ Then the #eaker
containing the solution was kept in microwave oven for !7, minutes and #oiled to
dissolve the agar$ 0fter the dissolution of agar, #eaker was taken out of the oven and
solution was stirred$ Then the solution was immediatel1 added to the glass #ottles$ 0#out
"*+ ml of the solution was added to each glass #ottle$ These glass #ottles were then
su#=ected to steam sterili?ation in an autoclave for 1*7"+ minutes at 1* l#s and 1"1%$
"+
)ands were sterili?ed with the (+; alcohol to ensure aseptic conditions$ Multiplicated
shoots growing in glass flasks were taken out$ The larger shoots were excised with the
#lade into smaller pieces and were carefull1 cultured in the glass #ottles containing the
medium for root initiation$"7- explants were cultured per #ottle$ 0ll this inoculation work
was done under aseptic conditions provided #1 the laminar air flow ca#inet$
#$"$ R-4R#-#
#ate o, obser+ation; 7@G07G700?
No o,
obser+ation
res/onse
1 - - + Eooting started
to all plants
" - - + Eooting started
- - - + Eooting started
' - - + Eooting started
* - - + Eooting started
"1
-./erient No0 0?
$44;%M%"%E$"%ON
Ob3ecti+e0
To acclimati?e the cultured plants in growth room so that the1 ma1 a#le to grow
in field9green house
Me!iu0 peat moss cla1
#ate o, initiation0 "'7+"7"++,
4irst of all peat moss soil containing plent1 of organic compounds was taken into
plastic #ags$
"rans/lantation
The plants growing in growth room in =ars were taken out and roots were washed
to clean the growing media on them$ Then plantation was done into plastic #ags and some
water was poured into #ags and were kept in growth room where proper s1stem of
aeration and light was maintained$ This was aimed to harden and acclimati?e the plants
son that the1 ma1 a#le to grow in green house and su#se@uentl1 to field$
JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ
$

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