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Aquatic Plants Tissue Culture
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Madan
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PostPosted: Mon Aug 06, 2012 1:21 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Hi  Madan,
 
 Its  good  to  know  that  you  are  starting  with  tissue  culture  of  aquatic  plants.  I  am  sure  its  a  wonderful  initiative  you  have  undertaken  and  I  am  sure  you  will  succeed.  
 I  wish  I  could  be  in  Bangalore  to  join  you.
 
 A  little  more  about  me:   I  am  a  Biotechnologist  by  profession  and  have  hands-on  experience  in  Tissue  Culture  (both  plant  and  animal)  
 and  my  Masters  thesis  involved  extensive  Microbiology.  Besides  my  curriculum  (  I  hold  a  B.tech  and  M.tech  in  Biotech),  
 I  have  also  been  trained  at  Institute  of  Himalayan  Bioresource  Technology  (CSIR)  in  tissue  culture.  I  have  been  a  not-so-regular  member  of  this  forum  for  
 the  last  few  years  -  my  research  leaves  me  little  time  for  posting  on  forums,  I  hope  you  can  understand,  though  I  do  follow  the  forum  regularly  and  read  as  many  posts  as  I  can.
 As  far  as  the  hobby  is  concerned,  I  have  kept  different  types  of  fish,  snails,  inverts,  and  FW  critters  of  all  sorts  since  childhood.  
 Started  with  plants  and  moss  in  a  7G  Nano  two  years  back.  Time  has  been  the  only  limiting  factor  for  my  devotion  to  the  hobby.
 
 Anyways,  I  wish  you  all  the  best  in  your  endeavor.   The  motive  for  writing  this  message  to  you  is  -  if  you  need  any  kind  of  help  please  feel  free  to  contact  me.  
 Besides  my  experience,  I  have  access  to  labware,  chemical  suppliers  and  lot  of  technical  literature.  
 Had  I  been  there  in  Bangalore,  I  would  have  loved  to  join  you.  But  the  least  I  can  do  for  you,  sitting  in  Delhi  is,   supply  you  with   as  much  info,  tips,  
 troubleshooting  or  any  kind  of  information  you  need.  
 The  only  reason  I  did  not  post  this  in  public  forum  is  because  I  may  not  be  able  to  keep  too  regular  with  this  post  and  sending  replies  to  everyone.  
 With  all  your  experience  and  devotion  to  this  hobby  and  IAH,  you  the  right  person  to  pass  on  all  the  info  to  others.
 I  am  not  so  regular  with  the  forum  so  kindly  contact  me  via  mail  -  djmankotia  [at]  gmail  [dot]  com.  I  will  try  to  respond  as  early  as  I  can.
 
 Best  of  luck!
 
 Regards,
 Dhananjay  Singh
 
 *************************************************************
 
 From  Dhananjay  Singh  Mankhotia,
 
 Hello  Mr.  Madan,
 
 Good  to  hear  from  you.  At  the  outset,  I  would  recommend  that  you  get  in  touch  with  a  lab  supplies  store/dealer  because  
 they  would  have  all  the  equipment/reagents/chemicals  you  would  need  for  the  tissue  
 culture.  For  reagents  and  chemicals,  I  can  recommend  some  manufacturers  of  Lab  grade  materials  -  Qualigens  
 (Owned  by  Thermo-Fisher  Scientific),  SRL,  Mumbai,  HIMEDIA,  etc.
 
 
 To  maintain  sterility,  there  are  two  main  aspects  during  Tissue  culture:
 
 1.  All  work  is  done  under  aseptic  conditions.  In  laboratory  we  use  an  aseptic  chamber  which  has  a  flow  of  laminar  air  to  avoid  
 outside  air  to  come  in  contact  with  the  culture  material.  The  insides  of  this  chamber  are  wiped  with  spirit/70%  ethanol  in  water,  
 Followed  by  UV  light  treatment  of  the  entire  chamber.  All  equipment  are  sterilized  by  autoclaving  -  instead  you  can  dip  the  steel-ware  like  forceps,  
 Needles,  etc   in  spirit  and  lightly  run  over  the  flame  of  a  lamp  to  remove  contaminants.  To  mimic  a  sterile  chamber,  you  can  light  3-4  spirit  lamps
 (you  can  DIY  the  lamps  or  procure  them  from  lab  supply  store  but  make  sure  the  fuel  is  efficient,  leaves  no/less  carbon  residue)  and  try  and  work  within  this  area.  
 Due  to  flame,  the  area  will  be  devoid  of  contaminants  and  will  give  you  a  temporary  small  sterile  zone.
 
 
 2.  The  next  aspect  in  tissue  culture  is  surface  sterilization  of  the  explant  (starting  plant  material).  In  earlier  days  HgCl2  was  used  
 but  now  is  more  or  less  replaced  by  other,  more  environmentally  acceptable  agents  like  Hypo  or  7%  chlorox  solution.  
 After  treatment  with  surface  sterilizer,  washing  is  done  in  sufficient  amounts  of  sterile  water  (you  can  use  thoroughly  boiled  water  here).  
 The  explant  is  then  blotted  onto  a  piece  of  sterile  paper  to  remove  excess  water.  Following  this,  the  explant  is  placed  in  the  culture  media  with  a  sterile  forcep.  
 
 The  culture  media  most  commonly  used  in  laboratory  -  MS  Media  -  Murashige  and  Skoog  media  HAS  NO  ANTIBIOTIC/BIOCIDAL  agent   added  to  it.  
 You  can  check  the  composition  online  or  contact  me  for  details.  
 It  just  has  minimal  salts  (both  Macro  and  micro,  and  Fe-stock  -  similar  to  the  fert  regime  we  follow  for  dosing  in  aquariums,  plus  a  gelling  agent  (agar-agar),  and  also  some  phytohormones).  
 I  repeat,  the  plant  tissue  culture  is  based  on  use  of  sterile  explant  in  the  beginning  itself  and  not  on  the  addition  of  any  biocidal  agents  -  at  least  not  for  routine  purposes.  
 Such  biocidals  are  non-selective  and  may  affect  the  growth  of  the  explant  as  well  as  composition  of  the  media.  
 There  are  other  specific  media  available  that  contain  antibiotics  for  selection  but  these  are  not  used  for  routine  purposes  IMO.  
 They  are  used  for  genetically  engineered  plant  material  where  the  antibiotic  resistance  has  been  incorporated  into  the  plant  material  through  genetic  manipulation.  
 This  not  the  usual  approach  especially  where  the  explant  has  been  taken  from  Nature.
 
 
 Now  a  question  may  arise  in  your  mind  that  what  if  contamination  does  enter  the  growth  chamber  (in  your  case,  the  bottle).  Actually,  no  matter  how  much  precaution  you  take,  
 some  contamination  always  gets  in  (Even  if  some  biocide/antibiotic  has  been  added  because  even  these  chemicals  have  a  restricted  shelf  life  at  room  temperature).  
 Hence  the  tissue  culture  approach  is  based  on  repeated  sub-culturing  -  that  is  every  few  days,  the  explant  is  changed  to  a  fresh  media.  
 Another  reason  why  this  is  necessary  is  because  the  media  also  loses  its  nutritive  power  after  some  days.  Tissue  culture  is  always  performed  in  bulk.
 To  get  one  callus  (I  shall  explain  below),  one  starts  with  around  50-100  bottles/cultures.  Many  are  lost  during  sub-culturing  and  due  to  contamination.
 However,  out  of  these  100  or  so  cultures,  even  if  one  survives  and  forms  a  callus,  we  can  get  several  thousand  identical  plantlets  from  a  single  callus.  
 That  is  why  tissue  culture  is  such  a  versatile  technique.
 
 Callus  is  a  mass  of  unorganized  dividing  cells  -  from  one  callus,  you  can  get  several  explants.  
 From  here,  The  formation  of  root/shoot  is  a  process  called  "differentiation"  and  it  is  governed  by  what  
 phytohormone  you  add  in  the  media.  This  is  where  the  sub-culturing  step  also  comes  in  handy.  
 In  labs,  we  first  grow  the  explant  in  rooting  media  (rich  in  auxin)  and  later  for  aerial  shoot  development,  the  
 explant  is  shifted  to  a  shooting  media  (same  media  but  containing  cytokinin  now).  
 
 Now,  THis  is  another  issue  you  guys  need  to  divert  your  attention  to  -  we  do  the  above  steps  because  once  the  roots  
 develop,  enhanced  nutrient  uptake  starts  thanks  to  the  roots.  But  will  this  strategy  work  with  aquatic  plants,  that  you  need  to  figure  out.  
 Also,  what  would  be  the  strategy  for  mosses,  liverworts  and  other  
 lower  plants  where  no  roots  are  present??
 
 
 I  think  before  starting  you  guys  need  to  read  a  lot  and  do  lots  of  pondering  to  do.  If  you  are  willing  I  can  suggest  some  basic  literature  and  protocols.  
 Owing  to  lack  of  equipment/reagents/infrastructure  of  functional  lab,  you  guys  need  to  improvise  (  as  we  say  here  in  North  India  -  "Jugaad")  
 but  its  not  a  difficult  task  we  just  need  to  get-together  and  ponder.  Even  with  all  the  things  available  in  lab,  we  
 researchers,  especially  in  India,  have  to  use  jugaad  for  some  thing  or  the  other  -  its  all  a  part  of  a  researcher's  life  -  to  improvise!  
 So  am  here  with  you  for  any  troubleshooting.
 
 
 I  am  writing  this  from  my  lab  itself  in  a  jiffy,  I  hope  all  of  the  above  makes  sense.  Else  give  me  some  time,  and  I  will  try  to  come  up  with  something  more  basic  and  comprehensible,  
 easy  for  everyone  to  understand.  If  there  is  anything  in  the  above  text  that  you  may  not  understand  or  need  clarifications  (which  I  am  sure  you  would  -  a  lot!)  please  feel  free  to  contact  me!
 
 
 Regards,
 DJ
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Ravabi
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PostPosted: Mon Aug 06, 2012 1:38 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Prabuddha,  Do  remember  that  we  are  doing  it  as  hobbyist  and  with  minimum  budgets.
 
 Getting  autoclavable,  branded   bottles  and  caps  are  expensive.  Laminar  Air  Flow  Chamber  and  bunsen  burners  are  also  out.  Instead  we  are  planning  to  flame  only  the  bottle  mouths  for  a  minute  are  so  using  cooking  LPG  (Nayak  said  he  will  try  it).
 FYI,  Agarose  can  undoubtedly  be  used  for  plant  cell/tissue  culture.  Only  problem  is  it  is  way  too  expensive  as  it  is  an  ultra  purified  version  of  agar-agar  and  is  modified  for  sensing  even  small  amounts  of  impurities  .
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PRAVU
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PostPosted: Mon Aug 06, 2012 1:51 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Yes  that  I  know,  because  of  the  cost  factor  and  it  is  more  purified  than  agar  agar,  we  use  it  in  different  purpose,  not  in  tissue  culture,  I  have  mentioned  it.  We  never  use  agarose  in  cultures  in  labs.  On  case  of  agarose,  its  mess  size  is  important  along  with  gelling  capacity.  And  for  Agar  Agar,  the  only  thing  needed  is  the  anchorage,  for  the  tissue.  But  in  liquid  media  also  PTC  can  be  done,  in  this  way  this  will  be  more  cheaper.  But  for  other  things,  I  haven't  told  to  use  laminar  air  flow,  I  said  if  you  have  access  anywhere,  you  can  use,  but  a  cheaper  method  is  to  use  pressure  cooker.  And  I  dont  think,  the  container  which  can  sustain  pressure  cooker  is  expensive.  You  can  do  one  thing,  just  use  glass  flasks  with  narrow  mouth  (easily  available),  and  tightly  cap  it  with  non-absorbant  cotton  and  then  wrap  it  with  4  folds  of  news  paper  with  a  rubber  band  (all  are  cheap),  put  the  media,  cap  and  wrap  it  and  then  your  are  ready  to  go  with  pressure  cooker.  Is  this  not  a  cheaper  method,  this  way  you  can  prevent  contamination,  which  is  very  problematic  in  PTC.  I  have  personal  experiences.  Anyways,  good  luck.  
 Sorry  if  I  have  mentioned  anything  wrong  which  don't  satisfy  the  goal  of  the  venture.  
 Thanks.
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PRAVU
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PostPosted: Mon Aug 06, 2012 1:57 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Actually,  microwave  oven  is  not  going  to  do  good  for  the  sterilization  as  it  only  heat  the  media,  for  sterilzation,  you  need  atleast  15  lb/  inch2  pressure  with  121  C  temp,  which  is  done  in  autoclave.  But  pressure  cooker  is  also  a  good  option  as  that  will  also  provide  kind  of  same  parameters.  And  for  the  spirit  lamp,  you  just  heat  the  neck  of  the  vials  as  I  have  mentioned  before  inoculating,  and  close  the  neck  with  non-absorbant  cotton  as  I  mentioned  tightly.  (So  tight,  so  that  it  sounds  while  removing).  I  regularly  do  all  these  in  my  lab.
 Thanks.
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Madan
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PostPosted: Mon Aug 06, 2012 2:03 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Prabuddha,  Will  look  at  the  cotton  wool  option  too.  Everybody  associated  with  a  lab  advocates  it.
 I  went  by  -  This  Link
 
   one  more
 
 Specifically  about  Aquatic  Plants
 
 Your  analysis  of  what's  there  in  the  links  will  help  refine  what  we  are  trying.  
 Please  comment.
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PRAVU
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PostPosted: Mon Aug 06, 2012 2:12 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Give  me  some  time  sir  ji,  I  will  go  through  these  and  will  revert  back  soon.  Apparently,  the  ideas  they  gave  are  pretty  good.  I  will  try  to  put  some  diagramatic  figures  of  my  own,  that  may  help.
 Thanks.
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Madan
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PostPosted: Mon Aug 06, 2012 2:13 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Thank  you,  will  wait  for  your  reply.
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PostPosted: Mon Aug 06, 2012 6:27 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 As  mentioned  earlier,  had  a  very  wonderful,  fun  filled,  learning  experience.  We  are  well  aware  we  might  fail.  And  Madan  and  Ravi  did  warn  us  that  even  if  we  succeed  in  one  Tissue  culture  bottle  from  this  batch,  we  would  be  very  lucky.   Very  lucky  indeed.
 
 We  are  not  worried.  As  Madan  put  it,"We  have  got  the  process  right".  With  all  the  inputs  we  are  getting  from  Prabuddha,  Dhananjay  Singh  and  other  knowledgeable  members  we  will  definitely  improve,  improvise  and  do  a  much  better  job.
 
             
Link

 
 Regards,
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menonabhishek
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PostPosted: Mon Aug 06, 2012 8:48 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 i  missed  it  Sad  It  looked  like  fun.
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random2
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PostPosted: Mon Aug 06, 2012 10:18 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

                                                   
Madan  wrote  (View  Post):                
The  idea  is  to  share  knowledge.  Especially  failures,  and  what  went  wrong,  so  people  can  learn.
 Have  fun  !                

 
 This  is  why  I  love  this  forum.
 
 I  can  say  that  whatever  success  I  have  had  is  because  of  experience  shared  by  people  here.  I  can  say  that  I  have  learnt  from  others  mistakes.
 
 Nayak  &  Vimal,
 
 Great  effort  in  getting  things  documented.
 
 As  said,  its  just  the  beginning.  Once  you  guys  get  this  right,  this  is  going  to  be  very  useful  in  propagating  native  and  exotic  hard  to  find  flora.
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vidhyasagar99
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PostPosted: Mon Aug 06, 2012 11:36 pm Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 u  guys  rock,  its  so  exciting  to  see  ,  but  great  information,thank  u  all  ,  i  missed  it  a  lot,  my  work   schedule  is  screwing  me  up,best  of  luck  :thumb:
 
 but  will  try  to  give  u  guys  emmeresed  anubias  flowers
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PRAVU
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PostPosted: Tue Aug 07, 2012 2:18 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 The  first  link  is  a  very  good  one,  you  can  follow  that.  IMO,  use  pressure  cooker,  other  things  are  ok.  
 Now  check  for  any  contamination  in  your  current  vials,  if  its  not  working,  then  go  for  the  first  link.  very  well  demonstrated.  
 Only  green  house  is  not  needed  I  think.  MS  media  is  very  widely  used  medium.  a  conical  flask  is  a  good  choice  with  cotton  plug,  
 but  the  mouth  of  the  bottle  should  be  such  wide,  so  that  you  can  put  your  explants.  The  knife,  forceps  etc  can  be  sterilized  by  flaming  in  the  lamp  before  use,  
 but  better  if  wrapped  in  aluminium  foil  and  kept  in  pressure  cooker  for  15-20  min.  Over  all  the  information  in  the  link  are  very  good.  
 Preparation  of  media  and  pH  checking  is  readily  available  in  the  net.  Just  make  sure  you  heat  the  neck  in  the  flame  before  putting  the  plant  parts.
   It  should  also  be  done  in  front  of  the  spirit  lamp  to  avoid  contamination.  After  putting  the  plant  parts,  flame  the  neck  again  and  put  cotton  plug  tightly.  
 These  are  all  the  small  things  you  should  remember  to  avoid  contamination,  but  all  the  things  have  to  be  standerdized.
 you  will  be  able  to  figure  out  the  problems,  of  your  own.  And  better  to  keep  the  cultures  at  25  C.  All  the  chemicals  you  will  get  in  Himedia  laboratories.  
 At  first  you  should  develop  roots,  then  shoots.  for  doing  that,  you  have  to  use  different  hormones  (available  in  the  link),  if  you  are  doing  the  culture  from  the  seeds.  
 If  you  are  using  cut  leves,  you  have  to  go  for  callus  culture.  For  that  too  you  have  to  use  hormones.
 After  you  get  the  callus,  cut  the  upper  surface  in  front  of  the  lamp,  surface  sterilize  if  needed,  
 otherwise  directly  put  it  in  fresh  medium  for  root  development,  after  root  go  for  fresh  media  and  put  hormone  for  shoot  development.....
 Whatever  you  do,  remember  tht  after  a  certain  time  period,  you  have  to  put  the  developed  plant  part  in  fresh  media  ascetically.  
 And  you  can  try  for  a  semi  liquid  medium  and  little  bit  shaking,  its  a  kind  of  experiments,  it  might  help  in  rapid  growth.
 Any  problem,  please  tell  me,  I  will  try  to  help.  And  follow  the  links,  they  are  good  as  I  have  already  said,  with  some  modifications,
 thanks.
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PRAVU
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PostPosted: Tue Aug 07, 2012 2:39 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 http://www.google.co.in/imgres?hl=en&sa=G&biw=1280&bih=681&tbm=isch&tbnid=yMUOC-
 6PZ2dOJM:&imgrefurl=http://users.aber.ac.uk/lum/culture.htm&docid=ckdigPolEmb5hM&imgurl=http://users.aber.ac.uk/lum/somzyg.jpg&w=1275&h=922&ei=PikgUMy4LobkrAfa6IGACQ&zoom=1
 
 http://www.google.co.in/imgres?hl=en&sa=G&biw=1280&bih=681&tbm=isch&tbnid=8Ykqobb_oxvPzM:&imgrefurl=http://mrzacbio.blogspot.com/&docid=memsgX9Uqs_h8M&
 imgurl=http://2.bp.blogspot.com/_vgPvhHSRhhQ/TAoOQAjhOZI/AAAAAAAAAA8/GbL40o34x2E/s1600/plant%252Btissue%252Bculture.jpg&w=1051&h=401
 &ei=PikgUMy4LobkrAfa6IGACQ&zoom=1&iact=hc&vpx=370&vpy=15&dur=479&hovh=138&hovw=364&tx=179&ty=61&sig=115386956136270192477&
 page=1&tbnh=81&tbnw=213&start=0&ndsp=16&ved=1t:429,r:7,s:0,i:95
 
 You  can  also  use  slant  tubes,  I  mean  bigger  test  tubes.  And  instead  of  using  the  filter  in  the  first  link  of  mine,  sterilize  r\everything  as  I  have  mentioned  and  use  cotton  plug  in  place  of  the  filter  on  top  of  conical  flask.
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Madan
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PostPosted: Tue Aug 07, 2012 9:05 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Pics  From  Vimal  are  here  
 
 http://indianaquariumhobbyist.com/gallery2/v/User_Galleries/imperator/TissueCulture/
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PostPosted: Tue Aug 07, 2012 9:08 am Post subject: Re: Aquatic Plants Tissue Culture Reply with quote

 Flame  Sterilisation
 
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