I would like to separate individual cells from my Orchid callus. What is the best way to do this, without damaging the cells?
Individual cells without damaging can only be obtained via suspension cultures. It is a quick and easy technique with little precautions to be taken provided if you have a callus* ( *condition applied)
Put the callus in liquid shake culture, to facilitate the aeration as well as dissociation of cell clumps in to cells...the growth is much faster than callus culture... but requires a regular sub-culture are 7-10 days. But before that refine the medium also...
Over and above what you must do (as said by John and Kumar), be sure that your callus is the "friable type of callus" as it is easier to do liquid shake cultures with this type.
agree with Jimmy, Sanjeev and Koetle.. Suspension culture with regular subculturing gives you individual intact cells
By subculturing the callus three times on the same medium. Following are transferred to a liquid medium in erlenmyers containing 30 mL and placed on a shaking with a speed that depends on its goals, eg 50-110 rpm in 6.25 to 12.5 umol.m / 2. s / 1 irradiance. Cells are grown 3 to 4 weeks and subcultured for the same period.
Would this also work for the callus of a banana sprout? (Musa acuminata)
@ Sara Mohy
Yes you can. You need to proceed further with regeneration. You will have to use different hormone concentration to achieve shoots. Multiple shoots once achieved needs regular subculture to obtain roots and further for individual plantlets. Hormones concentrations required for shoots and roots are different.
@ Gijsje van Raalte
Yes it would. You can kindly refer to Dai et al (2010) Plant regeneration from embryogenic cell suspensions and protoplasts of dessert banana cv. ‘Da Jiao’ (Musa paradisiacal ABB Linn.) via somatic embryogenesis. In Vitro Cellular & Developmental Biology - Plant 46(5):403-410
Article Plant regeneration from embryogenic cell suspensions and pro...
Can anyone help me with the separation of single cells from regenerating callus? Can we have a suspension culture for a calli which is already regenerating? Is there any enzymatic method to achieve single cell suspension for FACS analysis?