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Aquatic Plants Tissue Culture
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mohan4060
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PostPosted: Sun Aug 05, 2012 9:46 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Yes   Madan  Sir,
 I  know  actually  it  is  not  a  rocket  science,  with  some  proper  technical  understanding  and  some  dedication  we  can  nail  it.  Good  one  but  i  will  not  be  able  to  join  the  group,  Instead  I  will  talk  to  some  hobbyists  who  are  interested  from  my  locality  will  discuss  on  this  topic  and  will  try  ourselfs.......  
 
 
 Regards,
 MMS
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Madan
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PostPosted: Sun Aug 05, 2012 9:59 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 The  idea  is  to  share  knowledge.  Especially  failures,  and  what  went  wrong,  so  people  can  learn.
 Have  fun  !
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rasikanayak
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PostPosted: Sun Aug 05, 2012 10:22 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 I  was  the  first  to  reach  the  venue.  Yipee,  in  spite  of  the  scaling  down  of  Madan's  aquariums  from  17nos(Out  of  which  7  were  planted)  there  was  no  dearth  for  a  aquarium  hobbyist's  fancy.  Was  discussing  with  Madan  when  Dinesh,  Ravi(Ravabi),  Ashwin(Mandragoran,  Vimal(Imperator)  and  Prabhu  arrived.  The  pleasant  surprise  was  Rufus  with  his  family.
 
 The  activity  started  at  11.00pm  sharp,  after  a  refreshing  cup  of  Coffee  from  Gayathri  Madan(as  usual).  Madan  had  already  mixed  the  Nutrient  solution  as  well  as  the  other  necessary  Chemicals.
 
 
 The  gang  getting  ready,  Madan  briefing  the  plan.
 
 
 Everybody  dirtying  their  hands.  There  were  no  Tourists.
 
 
 Ashwin,  in  front  of  the  Sterilizing  trays.  What  a  waste  of  Ethanol....  he  seems  to  think!
 
 
 To  be  continued......
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PostPosted: Mon Aug 06, 2012 10:22 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Madan  has  improved  the  process  in  the  afternoon  as  quoted....
 
                                                 
Madan  wrote  (View  Post):                

 So  have  now  learnt  the  lesson.  Prepare  the  medium  with  agar  agar  and  let  it  cool  -  do  this  the  first  thing.
 By  the  time  you  sterilise,  rinse,  and  cut  the  ex-plants,  the  medium  is  ready  for  the  explant.
 
 Tried  this  modified  method  again  just  now  with  the  few  tissue  culture  bottles  remaining  with  me  from  the  morning  trials,  and  was  done  in  less  than  an  hour.                

 
 But  I  will  be  posting  the  Pics  in  the  exact  sequence  that  we  did  it  yesterday.
 
 PART-1
 
 
                                                 
Madan  wrote  (View  Post):                

 in  the  Culture  medium.
 
 Cryptocoryne  lucens
 Cryptocoryne  willisii
 Cryptocoryne  pontederifolia
 
 All  NEW  emersed  leaves  were  used  for  the  explants.
 I  have  an  emersed  culture  of  7  crypts  going,  we  used  three  this  time.
 
 Preparation  of  the  ex-plants:
 
 1.  The  emersed  leaves  were  rinsed  in  50%  Ethanol  for  1  minute  (  Mixed  100%  pure  ethanol  +  equal  volume  of  water)
 
 2.  Then  the  leaves  were  transferred  to  a  container  with  10%  bleach  solution  and  a  couple  of  drops  of  Dettol  liquid  soap.
 The  leaves  were  rinsed  well  for  5  minutes  in  the  bleach  solution.
 
 3.  The  leaves  from  the  bleach  solution  were  then  transferred  to  a  container  with  boiled  and  cooled  water  with  a  pinch  of  Borax  added  to  it.
 Borax  is  a  biocide.  The  leaves  were  rinsed  well  in  this  water  for  over  30  minutes.                

 
 Madan  preparing  the  specimens.  From  L-R:  Water  with  a  pinch  of  Borax,  10%  bleach  solution  and  50%  Ethanol  beaker.
 
 
 The  emersed  Cryptocoryne  in  the  Background.  Vimal  recording  the  proceedings  in  the  foreground.
 
 
 Timing  the  Sterilizing  process  in  10%  bleach  solution  in  Microseconds  Very Happy  
 
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PostPosted: Mon Aug 06, 2012 10:51 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 PART-2
 
 
                                                 
Madan  wrote  (View  Post):                

 Preparation  of  the  Media:
 
 1.  6-BAP  (  6-Benzylaminopurine,  a  Plant  Cytokinin  )  1  gram  of  powder  bought  from  ebay.com,  was  dissolved  in  200  ml  of  Dimethyl  sulfoxide
 and  stored  in  a  dark  container  in  the  fridge.
 
 2.  3-  IAA  (  3-  Indol  acetic  acid,  a  plant  Auxin)  1  gram  of  powder  bought  from  ebay.com,  was  dissolved  in  200  ml  Ethanol  and  stored  in  a  dark  container  in  the  fridge.
 
 3.  0.2  ml  of  each  of  the  above  solutions  was  added  to  1  liter  of  boiled  and  cooled  water  to  get  a  concentration  of  1mg/l  of  each  hormone.
 
 4.  1  teaspoon  of  commercially  available  NPK  20-20-20  fertiliser  was  added  to  2  liters  of  water  to  make  the  media  solution.
 4a.  2ml  of  PMDD  was  added  to  400  ml  water,  and  2  ml  of  from  this  was  added  to  the  media  solution.
 4b.  2  tablespoon  of  Sugar  was  then  added  to  the  media  solution.
 4c.  2ml  of  the  1mg/l  hormone  solution  was  then  added  to  the  media  solution.
 4d.  0.1  gram  of  Borax  was  mixed  in  100  ml  water  and  1  ml  of  this  was  added  to  the  media  solution  to  act  as  a  biocide.
 4e.  80  mg  of  Gentamycin  an  antibiotic  was  added  to  the  media  solution.
 4f.  The  Ph  of  the  media  solution  was  checked  and  found  to  be  7.0
 4g.  A  little  Vinegar  was  added  to  reduce  the  pH,  pH  checked  and  found  to  be  about  5.0
 4f.  Sodium  bicarbonate  powder  was  then  added  to  the  media  solution  and  pH  adjusted  to  5.8  (approx).                

 
 Madan  had  already  prepared  the  media  upto  4d.  Vimal  brought  the  Gentamycin  which  was  added.  To  proceed....
 
 pH  controlling  chemicals.Sodium  Hydrogen  Carbonate  and  Vinegar.
 
 
 Checking  the  pH  using  pH  test  strips.
 
 
 Watching  what  the  elders  are  up  to  from  a  vantage  point.  Katlyn  on  Dada's  shoulder.
 
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PostPosted: Mon Aug 06, 2012 11:43 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 PART-3
 
 
                                                 
Madan  wrote  (View  Post):                

 14g.  50  ml  of  the  media  solution  was  then  transferred  to  each  tissue  culture  bottle  and  3.5  gms  of  Agar  added  to  each  bottle.
 4f.  The  bottle  and  media  solution  +  agar  was  then  microwaved  and  cooled.                

 
 Dinesh  got  the  Microwave  oven  able  Tissue  culture  bottles.  Transferring  50ml  culture  media  into  the  bottles.
 
 
 The  neatly  arranged  Tissue  culture  bottles  with  the  media.
 
 
 The  Agar  Agar  bottle.  Thanks,  Ravi(Ravabi).  How  much  to  add?  Perfecting  the  right  concentration  of  Agar  agar  to  get  the  right  media  density  took  time.  Madan  had  to  microwave  a  Tissue  culture  bottle  repeatedly  with  different  concentrations  to  get  it  right,  and  whew!  did  this  consume  time?
 
 
 Calibrating  the  Spatula,  Ravi  and  Dinesh.  Final  concentration  was  about  3.5  Tea  spoons  of  Agar  Agar  to  50ml  media.
 
 
 Once  the  concentration  was  fixed  the  pace  of  the  work  picked  up  again.  
 
 
 Caution!!!!  Disturb  at  your  own  peril.  Men  at  work!
 
 Madan
 
 
 Dinesh.
 
 
 Vimal.
 
 
 Ravi.
 
 
 And  these  two  goofing  off,  for  a  nicotine  stick  break!
 
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PostPosted: Mon Aug 06, 2012 11:50 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 ROFL  
 Nayak..  is  at  it  again,  Can  make  the  most  boring  topics  to  hilarious  ones..
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PostPosted: Mon Aug 06, 2012 12:15 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 PART-4
 
 
                                                 
Madan  wrote  (View  Post):                

 4f.  The  bottle  and  media  solution  +  agar  was  then  microwaved  and  cooled.
 
 5.  The  edges  of  the  washed  leaves  were  sliced  off  as  bacteria,  fungi  and  all  kinds  of  nonsense  generally  stick  to  the  edges.
 5a.  The  clean  leaves  were  then  sliced  to  get  the  explants.
 5b.  The  ex-plants  were  then  transferred  to  the  tissue  culture  bottle  with  the  cooled  and  hardened  media.  Bottles  closed  and  labelled.
 
 The  microwaving  might  have  affected  the  Gentamycin,  what  the  hell,  we  have  to  begin  somewhere  !                

 
 Microwaving  the  media.  carefull!  Gayathri  Madan  is  accommodating,  but  is  your  better  half  so?  A  thought  to  ponder.
 
 
 Handle  the  hot  Tissue  culture  bottles  with  caution.  The  solution  hardens  as  it  cools.
 
 
 The  cleaned  and  Sterilized  specimens.  Even  the  table  on  which  it  is  kept  is  sprayed  with  70%  Ethanol.
 
 
 The  Man  of  the  hour.  Ravi  is  doing  his  PhD  in  IISC.  And  had  already  done  Tissue  culture  in  the  lab.  So  his  experience  counts.
 Cleaning  his  hand  with  Ethanol.
 
 
 Slicing  the  Specimens  to  get  the  Ex-plants.
 
 
 The  ex-plants.  Ready  to  be  transferred  into  the  cooled  media.
 
 
 The  last  and  the  most  important  part.  Labeling  the  Tissue  culture  bottles.  Forget  this,  and  you  will  not  know  which  plant  is  which.
 
 
 That's  all  from  me.  Thanks.  Madan,  Gayathri  and  the  rest  of  the  Folks  for  making  this  possible.
 
 Regards,
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PostPosted: Mon Aug 06, 2012 12:31 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 We  did  this  all  over  the  place,  in  order  to  accommodate  so  many  people,  when  you  try  it,
 send  the  wives  and  kids  to  a  movie,  doing  it  alone  in  the  kitchen  and  dining  table.
 Sterilise  everything  with  70%  Ethanol.
 
 That  will  reduce  contamination.  I  frankly  don't  expect  any  success  this  time,  but  we  got  the  process  right,
 and  documented  it  to  show  it  is  not  difficult.  We  were  working  all  over  the  place.  
 Cleanliness  is  most  important,  and  cannot  be  stressed  enough.
 
 Ashwin  will  keep  trying  in  the  next  few  months  from  home  and  the  US  and  update  this  thread  regularly.
 
 Waiting  for  Vimal's  pictures.  Wink
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PostPosted: Mon Aug 06, 2012 12:51 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Just  wanted  to  check,  do  you  guys  expect  any  problems  in  the  sterilization  process  of  the  explants  ?  I  am  asking  because  Ethanol  sterilizes  by  causing  dehydration.
 
 Also,  a  tip  for  you  guys  for  the  next  TC  batch.  Take  a  look  at  Tropicas  1-2-Grow  series  of  plants  to  get  an  idea  of  what  plants  are  currently  being  cultured  this  way
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PostPosted: Mon Aug 06, 2012 1:01 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Mortis,  Tropica  uses  Tissue  culture  for  all  plants.
 That's  not  the  problem,  it  is  finding  out  which  plants  hobbyists  have  cultured  at  home,  believe  me  many  do  it,
 and  to  find  the  right  combo  for  the  medium  for  each  plant,  I  think  people  call  this  protocol.
 
 To  Succeed  we  need  emersed  grown  plants  to  start  with.
 Emersed  plants  sprayed  with  a  mister  also  does  not  help  !  
 Emersed  stem  plants  are  worthwhile  next  time,  so  on  track  for  that.
 
 Yes.  I  do  expect  trouble  with  sterilisation,  too  many  people  involved  is  the  main  thing,
 we  did  take  a  lot  of  care,  but  one  never  knows.  A  week  will  tell  if  bacteria  or  fungus  has
 taken  a  hold.


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PostPosted: Mon Aug 06, 2012 1:03 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 [quote="Madan";p="302121"]Prabuddha  ji,
 
 We  used  Agar  Agar,  there  was  some  confusion  initially  thinking  both  meant  the  same,  but  some  research  set  us  right.
 Thanks  for  the  insight.[/quote]
 
 Thanks  sir.  The  most  important  part  of  success  plant  tissue  culture  is  the  surface  sterilization  part  and  usage  of  hormones.  Diluted  ethanol,  HgCl2  are  the  things  I  have  used,  and  sometimes  diluted  savlon  etc....but  the  time  is  the  key  factor  here,  so  that  the  microbes  are  killed  and  plant  parts  survive.For  Aquatic  plant  you  can  also  use  semi  liquid  or  liquid  media  and  a  little  shaking.  That  might  help.  For  that  agar  concentration  must  be  kept  low  or  removed.  but  a  little  shaking  surely  will  help  in  good  growth.
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PostPosted: Mon Aug 06, 2012 1:09 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Prabuddha,  Mercuric  chloride  and  all  it's  toxic  effects,  kept  us  away  from  it.
 We  substituted  lab  grade  Borax  instead.  
 
 I  followed  Michael  Kane's  process,  except  that  we  didn't  use  PPM  and  half  strength  MS  medium.
 The  Medium  was  home  brewed.  People  have  had  success  with  this  too.
 
 Now  the  problem  is  if  we  succeed,  the  next  step,  we  can't  sterilise  the  growing  buds,
 have  to  pray  everything  is  sterile.   Very Happy
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PRAVU
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PostPosted: Mon Aug 06, 2012 1:12 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Another  good  thing  is,  you  can  use  pressure  cooker  instead  of  using  microwave  oven.  Just  make  sure  that  your  vials  and  caps  can  sustain  pressure  cooker.  Keep  it  for  15-20  min  inside  with  full  pressure.  To  avoid  contamination  not  only  the  antibiotic  will  work,  thought  it  is  a  broad  spectrum  Ab,  but  certain  microbes  specially  fungus  or  certain  bacteria  can  grow  in  presence  of  this.  So,  do  one  thing,  keep  two  small  sized  burners  (can  use  spirit  lamps)  with  a  gap  of  1  ft  or  slightly  less,  and  inoculate  the  surface  sterilized  explants  into  the  medium  in  between  the  two  lamps.  This  is  a  good  technique  to  avoid  contamination.  Otherwise  you  can  use  Laminar  Air  Flow,  if  you  access  anywhere.  But  the  method   mentioned  by  mentioned  by  me  is  a  good  one.
 Thanks
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PostPosted: Mon Aug 06, 2012 1:17 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Yup,  will  try  the  pressure  cooker  the  next  time.  Mom  too  will  have  a  go  at  me  for  that  !
 We  went  a  little  modern  tech,  with  the  Microwave  for  convenience,  as  it  was  the  first  time  and  25  bottles  in  a  pressure  cooker  will
 take  a  lot  of  batches.
 The  spirit  lamp  tip  is  good,  guys  you  listening  ?
 
 I  got  a  series  of  mails  from  another  IAHian,  about  Tissue  Culture,  lots  of  info.  I'll  post  that  below  this.
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