Practice question

Protoplasts are plant cells that completely lack cell wall, defined as protoplasm enclosed only by plasma membrane after removal of cellulose hemicellulose and pectin components. Isolation employs enzyme mixture cellulase hydrolyzing beta 1,4 glucan backbone of cellulose microfibrils and pectinase macerozyme dissolving middle lamella pectin crosslinking cells, often adding hemicellulase driselase; incubation performed in osmoticum mannitol sorbitol 0.4-0.6 molar to prevent lysis due to loss of wall counteracting turgor. After digestion spherical protoplasts released from leaf mesophyll or cell suspension appear intact with visible chloroplasts. They retain nucleus vacuole mitochondria capable of resynthesizing new cell wall within 24-72 hours mediated by cellulose synthase complexes delivering glucan chains, re-entering cell cycle forming microcallus. Absence of wall makes protoplasts ideal recipients for direct gene transfer via electroporation PEG mediated DNA uptake and for somatic hybridization because membranes of different species can be forced to coalesce unrestricted by wall barrier. Viability assessed by fluorescein diacetate FDA green fluorescence. Totipotency retained ensures whole plant regeneration from single protoplast under appropriate division promoting medium.