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Aquatic Plants Tissue Culture
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Madan
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PostPosted: Wed Jul 25, 2012 5:11 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 I  have  a  method  for  sterilising  the  ex  plants.  10  minute  rinse  under  running  water.  Then  dipped  in  70%  ethanol  for  1  minute,  then  25%  bleach  +  few  drops  of  liquid  detergent  for  10  mins.
 Followed  by  rinsing  in  boiled  and  cooled  water.
 
 I  am  looking  at  a  biocide  for  the  storage  itself.  Plant  Preservative  Mixture  is  one,  but  not  available.
 I  guess  adding  a  few  drops  of  an  antibiotic  injection  like  amoxyillin  to  the  medium  might  help.
 I  have  Formalin  tabs  which  you  can  use  for  sterilising  the  external  holding  container.
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Cherry
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PostPosted: Wed Jul 25, 2012 6:02 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 This  Is  another  good  read  I  found  for  surface  sterilization,  almost  covering  all  the  points  suggested  by  Madan  JI:
 
 Surface-sterilizing  Plant  Material  
 1.  Preparation  of  Stock  Plants  
 Prior  good  care  of  stock  plants  may  lessen  the  amount  of  contamination  that  is  present  on  
 explants.  Plants  grown  in  the  field  are  typically  more  “dirty”  than  those  grown  in  a  greenhouse  or  
 growth  chamber,  particularly  in  humid  areas  like  Florida.  Overhead  watering  increases  
 contamination  of  initial  explants.  Likewise,  splashing  soil  on  the  plant  during  watering  will  
 increase  initial  contamination.  Treatment  of  stock  plants  with  fungicides  and/or  bacteriocides  is  
 sometimes  helpful.  It  is  sometimes  possible  to  harvest  shoots  and  force  buds  from  them  in  clean  
 conditions.  The  forced  shoots  may  then  be  free  of  contaminants  when  surface-sterilized  in  a  
 normal  manner.  Seeds  may  be  sterilized  and  germinated  in  vitro  to  provide  clean  material.  
 Covering  growing  shoots  for  several  days  or  weeks  prior  to  harvesting  tissue  for  culture  may  
 supply  cleaner  material.  Explants  or  material  from  which  material  will  be  cut  can  be  washed  in  
 soapy  water  and  then  placed  under  running  water  for  1  to  2  hours.  
 
 2.  Sodium  Hypochlorite   
 Sodium  hypochlorite,  usually  purchased  as  laundry  bleach,  is  the  most  frequent  choice  for  
 surface  sterilization.  It  is  readily  available  and  can  be  diluted  to  proper  concentrations.  
 Commercial  laundry  bleach  is  5.25%  sodium  hypochlorite.  It  is  usually  diluted  to  10%  -  20%  of  
 the  original  concentration,  resulting  in  a  final  concentration  of  0.5  -  1.0%  sodium  hypchlorite.  
 Plant  material  is  usually  immersed  in  this  solution  for  10  -  20  minutes.  A  balance  between  
 concentration  and  time  must  be  determined  empirically  for  each  type  of  explant,  because  of  
 phytotoxicity.  
 
 3.  Ethanol  (or  Isopropyl  Alcohol)  
 Ethanol  is  a  powerful  sterilizing  agent  but  also  extremely  phytotoxic.  Therefore,  plant  material  is  
 typically  exposed  to  it  for  only  seconds  or  minutes.  The  more  tender  the  tissue,  the  more  it  will  
 be  damaged  by  alcohol.  Tissues  such  as  dormant  buds,  seeds,  or  unopened  flower  buds  can  be  
 treated  for  longer  periods  of  time  since  the  tissue  that  will  be  explanted  or  that  will  develop  is  
 actually  within  the  structure  that  is  being  surface-sterilized.  Generally  70%  ethanol  is  used  prior  
 to  treatment  with  other  compounds.  
 
 4.  Calcium  Hypochlorite  
 Calcium  hypochlorite  is  used  more  in  Europe  than  in  the  U.S.  It  is  obtained  as  a  powder  and  
 must  be  dissolved  in  water.  The  concentration  that  is  generally  used  is  3.25  %.  The  solution  must  
 be  filtered  prior  to  use  since  not  all  of  the  compound  goes  into  solution.  Calcium  hypochlorite  
 may  be  less  injurious  to  plant  tissues  than  sodium  hypochlorite.  
 5.  Mercuric  Chloride  
 Mercuric  chloride  is  used  only  as  a  last  resort  in  the  U.S.  It  is  extremely  toxic  to  both  plants  and  humans  and  must  be  disposed  of  with  care.  Since  mercury  is  so  phytotoxic,  it  is  critical  that  
 many  rinses  be  used  to  remove  all  traces  of  the  mineral  from  the  plant  material.  
   
 6.  Hydrogen  Peroxide  
 The  concentration  of  hydrogen  peroxide  used  for  surface  sterilization  of  plant  material  is  30%,  
 ten  times  stronger  than  that  obtained  in  a  pharmacy.  Some  researchers  have  found  that  hydrogen  
 peroxide  is  useful  for  surface-sterilizing  material  while  in  the  field.   
 
 7.  Enhancing  Effectiveness  of  Sterilization  Procedure  
 •  Surfactant  (e.g.Tween  20)  is  frequently  added  to  the  sodium  hypochlorite.
 •  A  mild  vacuum  may  be  used  during  the  procedure.
 •  The  solutions  that  the  explants  are  in  are  often  shaken  or  continuously  stirred.
 
 8.  Rinsing  
 After  plant  material  is  sterilized  with  one  of  the  above  compounds,  it  must  be  rinsed  thoroughly  
 with  sterile  water.  Typically  three  to  four  separate  rinses  are  done.  
 
 Using  Sterile  water  is  a  big  precautionary  measure  that  must  be  ensured  while  doing  the  process.
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aregias
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PostPosted: Wed Jul 25, 2012 6:05 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 awesome  guys  are  on  IAH,  your  dedication  is  infectious   Thumbs Up  :D
 
 when  do  we  start  cloning  projects?  Cheering
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Cherry
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PostPosted: Wed Jul 25, 2012 6:10 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 For  those  who  have  no  idea  how  to  culture,  a  very  basic  read  on  how  to  do  culturing  without  a  laboratory  and  high  tech  euipments:
 
 Sterile  Culture  Techniques  
    Successful  control  of  contamination  depends  largely  upon  the  operator’s  techniques  in  aseptic  culture.   You  should  
 always  be  aware  of  potential  sources  of  contamination  such  as  dust,  hair,  hands,  and  clothes.   Obviously,  your  hands  
 should  be  washed,  sleeves  rolled  up,  long  hair  tied  back,  etc.   Washing  your  hands  with  95%  ethyl  alcohol  is  an  easy  
 precautionary  measure  that  can  be  taken.   Talking  or  sneezing  while  culture  material  is  exposed  also  can  lead  to  
 contamination.   When  transferring  plant  parts  from  one  container  to  another,  do  not  touch  the  inside  edges  of  either  
 vessel.   By  observing  where  contamination  arises  in  a  culture  vessel,  you  may  be  able  to  determine  the  source  of  
 contamination.
   
 In  plant  tissue  culture,  small  pieces  of  plant  tissue  are  placed  on  or  in  a  medium  rich  in  nutrients  and  sugar.   If  bacteria  
 or  fungi  come  in  contact  with  the  plant  tissue  or  the  medium,  the  culture  becomes  contaminated.   The  contaminants  
 (bacteria  and  fungi)  will  use  nutrients  from  the  medium  and  the  plant,  which  quickly  destroys  the  plant  tissue.   Our  aim  
 is  to  surface  sterilize  the  plant  tissue  and  put  it  on  a  sterile  growth  medium  without  any  bacteria  or  fungi  getting  on  the  
 plant  or  medium.   This  is  not  easy  because  bacteria  and  fungal  spores  are  in  the  air,  on  us,  in  us,  and  under  us!  
 When  you  see  sunlight  shining  in  a  window  you  can,  from  certain  angles,  see  dust  particles  in  the  air.   There  are  
 hundreds  of  bacteria  attached  to  each  dust  particle.   A  horizontal  laminar  flow  unit  is  designed  to  remove  the  particles  
 from  the  air.   Room  air  is  pulled  into  the  top  of  the  unit  and  pushed  through  a  HEPA  (High  Energy  Particle  Air)  filter  
 with  a  uniform  velocity  of  90  ft/min  across  the  work  surface.   The  air  is  filtered  by  the  HEPA  filter  so  nothing  larger  
 than  0.3  um  (micrometer)  can  pass  through.   This  renders  the  air  sterile.  The  flow  of  air  from  the  unit  discourages  any  
 fungal  spores  of  bacteria  from  entering.   All  items  going  inside  the  unit  should  be  sterile  or  sprayed  with  ethanol  or  
 isopropyl  alcohol.   They  will  remain  sterile  unless  you  contaminate  them.  
 A  transfer  cabinet  provides  an  enclosed  environment  that  is  not  sterile  but  can  be  sterilized.   A  cardboard  box  lined  
 with  aluminum  foil  or  plastic,  or  a  well-cleaned  aquarium,  provides  a  shield  to  reduce  contamination.   A  box  that  is  20-
 24  inches  wide,  20-24  inches  high,  and  12-16  inches  deep  provides  a  good  work  area.   Working  inside  any  of  these  
 does  not  guarantee  success.     
 
 The  following  precautions  are  necessary  for  all  work  areas.  
 1.  The  room  should  be  swept  and  if  possible,  mopped.  
 2.  Each  work  surface  should  be  washed  with  a  10%  Clorox,  or  lysol  or  other  disinfectant  solution.  
 3.  Doors  and  windows  should  be  closed.  
 4.  Air  conditioners  and  fans  should  be  turned  off.  
 5.  If  possible,  each  student  must  have  a  work  space  that  can  be  properly  treated  against  contamination.   For  
 example,  the  box  or  aquarium  mentioned  earlier,  or  a  piece  of  poster  paper  lying  on  the  table  to  indicate  the  
 student’s  sterile  workspace.  
 6.  Have  spray  bottles  filled  with  70%  ethanol  or  isopropanol  (never  methanol)  placed  so  each  student  has  access  
 to  one  bottle.   Spray  everything  going  into  the  sterile  area.  7.  Have  a  central  supply  area  so  all  necessary  items  can  be  picked  up  and  taken  to  the  workspace  as  needed.   
 Items  can  be  returned  to  the  central  supply  area  after  being  used.  
 8.  Sterile  instruments  will  be  needed  for  each  person.   One  way  to  accomplish  this  is  to  have  a  ½-pint  jar  of  70  
 %  ethanol  for  scalpels  and  short  forceps.   When  tissue  has  to  be  positioned  in  a  vessel,  long  10-inch  forceps  
 are  needed.   The  long  forceps  need  to  be  placed  deep  enough  in  alcohol  so  that  any  part  of  the  forceps  that  
 might  come  into  contact  with  the  vessel  is  sterilized.   A  100-ml  graduated  cylinder  can  be  used  to  hold  the  
 alcohol  and  long  forceps.   A  ½-pint  jar  of  sterile  water  is  needed  for  dipping  the  instruments  to  remove  the  
 residual  alcohol  that  might  dry  out  plant  tissues.  
 9.  A  sterile  work  surface  is  needed  on  which  to  place  the  sterile  tissue  to  trim  it.   The  easiest  thing  to  use  is  a  
 sterile  petri  dish.   If  you  have  glass  ones,  you  can  autoclave  and  reuse  them.   Presterilized  plastic  dishes  are  
 used  and  discarded.   Spray  the  bag  of  dishes  with  70  %  alcohol  before  you  open  it  and  place  the  desired  
 number  of  unopened  dishes  at  each  station.   Each  dish  has  two  sterile  surfaces-the  inside  top  and  inside  
 bottom.  
 10.  Long  hair  should  be  tied  back  or  covered.   Hands  should  be  washed,  not  scrubbed  (scrubbing  dries  hands  and  
 creates  flakes  of  skin  that  have  bacteria)  and  sprayed  with  70  %  ethyl  or  isopropyl  alcohol  or  coated  with  
 isopropyl  alcohol  gel.   Gloves  and  masks  provide  extra  protection.   Do  not  talk  while  performing  sterile  
 operations.   Do  not  lean  over  your  work.   Keep  your  back  against  the  backrest  of  your  chair.   Try  to  work  
 with  your  arms  straight:  this  position  may  feel  awkward,  but  it  will  reduce  contamination.   Do  not  pass  
 nonsterile  items  over  sterile  areas  or  items.   Reach  around  rather  than  over.   Make  your  movements  smooth  
 and  graceful  so  that  you  do  not  disturb  the  air  more  than  is  necessary.   Work  quickly  though,  the  longer  it  
 takes  to  manipulate  the  tissues  the  greater  the  chance  of  contamination.   Have  only  the  necessary  items  in  
 sterile  work  area.   Remove  items  that  are  no  longer  needed  as  quickly  as  possible.   Act  out  each  step  before  
 beginning  so  that  you  understand  what  you  are  about  to  do.  
 
 Store  cultures  in  a  well-lit  area  (not  in  direct  sunlight),  and  do  not  allow  the  temperature  to  exceed  80  degree  F  where  the  
 cultures  are  stored.   If  you  are  placing  cultures  under  lights,  use  only  fluorescent  light.   The  preferred  schedule  is  16  
 hours  of  light  and  8  hours  of  dark.   Check  the  temperature  prior  to  placing  the  cultures  under  the  lights  because  
 temperature  will  build  even  under  fluorescent  lights.  
 
 Check  cultures  every  3-5  days  for  contamination.  Slimy  areas  mean  bacterial  contamination  while  fuzzy  areas  are  due  
 to  fungal  contamination.   Do  not  open  containers  that  are  contaminated.   The  contaminants  could  be  disease  causing  or  
 pathogenic.   The  only  safe  way  to  dispose  of  these  is  to  autoclave  (or  pressure-cook)  them  for  15  minutes  at  15  psi.   
 Contaminated  plastic  dishes  can  be  placed  inside  a  large  can  or  autoclavable  bag  to  be  sterilized  before  discarding.
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saravana
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PostPosted: Wed Jul 25, 2012 7:24 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Interesting  thread.  Keep  it  going.  Thumb Up  
 Got  to  learn  new  thing  today.  Thanks  Madan  sir  for  starting  this  and  Thanks  Cherry  sir  for  explaining  the  basics.
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uv_umesh
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PostPosted: Wed Jul 25, 2012 8:07 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

                                                   
saravana  wrote  (View  Post):                
Interesting  thread.  Keep  it  going.  Thumb Up  
 Thanks  Cherry  sir  for  explaining  the  basics.                

 
 Not  sir  saravana  -  cherry  is  'Ms'
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Madan
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PostPosted: Wed Jul 25, 2012 9:51 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 So  who  are  all  coming  ?
 We  need  to  make  a  list  of  what  everyone  needs  to  bring  !
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Ravabi
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PostPosted: Wed Jul 25, 2012 11:21 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Hi  Madan
 
 I  have  a  single  runner  of  Hydrocotyle  vulgaris,  given  to  me  by  Asif   (Zipper  boy).  We  can  try  with  it  as  it  seems  to  be  emersed,  not  sure  though.  The  leaves  were  waxy  and  exposed  on  water  surface.
 Will  check  with  VASA  and  get  back  to  you  about  DMS,  Agar  and  some  glass  containers.  
 I  will  also  pick  up  scalpel,  Gloves  and  forceps  from  them  if  necessary(for  cutting  and  replanting).  Let  me  know  about  it.
 
 ravi
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Madan
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PostPosted: Thu Jul 26, 2012 12:04 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 I'll  be  seeing  my  chemist  tomorrow  about  the  DMS  and  agarose  and  some  containers,  I  don't  think  the
 fellow  has  containers,  and  scalpels,  check  with  Vasa  too  and  update,  we'll  need  both  the  guys  it  looks  like,
 or  will  have  to  head  elsewhere  for  a  scalpel.
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vidhyasagar99
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PostPosted: Thu Jul 26, 2012 8:15 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 hi  madan  even  i  might  want  to  have  a  dash  at  it,where  to  come?
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Madan
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PostPosted: Thu Jul 26, 2012 10:59 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 It'll  be  at  my  place  as  I  have  all  the  stuff  to  make  what  is  required  and  a  few  emersed  plants.
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Scorpio
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PostPosted: Thu Jul 26, 2012 11:16 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Madan  Ji,  last  year  I  have  already  worked  on  Tissue  Culture  of  Aquatic  plants.  But  in  fourth  week,  due  to  fungus,  I  have  lost  all  the  work.  I  have  all  the  essentials  required  for  tissue  culture  of  plants.  This  year  I  will  start  again  the  process  in  the  end  of  September.
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nikhilsood1
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PostPosted: Thu Jul 26, 2012 1:05 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Hi  Madan,
 
 As  stated  earlier,  I  want  to  give  this  a  try  too.  Let  me  know  what  needs  to  be  picked  up  and  I  will  try  and  get  it.
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Cherry
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PostPosted: Thu Jul 26, 2012 1:45 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Madan  Ji,
 
 Kindly  let  me  know  what  all  resources  you  are  left  to  arrange  for  the  project.
 Abhishek  and  I  can  try  arranging  some  if  we  can.
 
 I  assume  it  will  be  after  ASK  meet  only.Since  we  don't  know  your  place,so  might  be  we  can  coordinate  from  there.
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Madan
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PostPosted: Thu Jul 26, 2012 2:44 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Shanai,  I'll  do  that,  I  am  going  shopping  for  a  few  items  today,
 I'll  get  what  I  can  find,  and  the  rest  I'll  post  what  each  one  has  to  bring  along.
 I'll  try  to  limit  it  to  the  minimum  possible.
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