The below mentioned article provides a note on anther culture.
Explants used for anther culture is very critical as the anther lobes bearing the PMC (pollen mother cell) of correct divisional stage signify for being divided to form the callus mass or the direct haploid embryo.
Young flower buds with immature anther lobes are surface sterilised and the stamens are taken out with the fine forceps, the filaments are removed and then one of them is crushed in acetocarmine stain and checked for proper stage i.e., just released from the tetrad condition, released microspores are uninucleate and densely cytoplasmic preparing for the male gametophytic development. If it is found in the proper stage then the anthers are inoculated on proper media.
Within few days or weeks the anther walls may turn brown, the microspore within it divides to form either callus or the embryos. Due to the inside pressure of developing microspores, the anther-wall burst open and following the same tissue culture technique the embryos or the plantlets are sub-cultured in proper media to get the whole plant in rooted condition to be transferred into soil (Fig. 21.2A-B).
Flowchart for Anther Culture:
(i) The young flower buds are collected and washed under running tap water to remove the dirt’s.
(ii) These are then surface sterilized by immersing in 70% ethanol or sodium hypochlorite soln.
(iii) Then washed in sterile water and transferred into a sterile petridish.
(iv) With the help of sharp scalpel and using forceps the buds are split open and anther lobes are taken out.
(v) One of the anther lobes of each bud is checked by crushing into acetocarmine stain under microscope for the proper stage of microspore development.
(vi) The filament portions are removed from the selected anther lobes.
(vii) The damaged anther lobes should be discarded and intact anther lobes are placed into proper media.
(viii) Incubated at 24°-28°C in dark for 3-8 weeks.
(ix) The haploid embryos or plantlets develop, come out by bursting the anther lobes.
(x) Individually these embryos or plantlets are removed and sub-cultured on suitable media to develop further and root development.