Pre-isolation, isolation and regeneration protoplasts from leaf mesophyll of in vivo Malus domestica ‘Anna’ cv.

Abstract

To isolate protoplast, a pre-treatment was completed with the order to reduce and identify the phenolic contents round the year to encourage the isolation of protoplasts. Protoplasts from in vivo mesophyll leaves of apple cultivar “Anna” was isolated from 15 days old leaves by plasmolying in medium containing 90 g L-1 mannitol for half hour, then 130 g L-1 mannitol for half hour. Then using enzymatic mixture involving (1.5% cellulase + 0.5% pectianase + 1.5% Macrozyme) Prior to isolation. Anyhow, diverse factors, for example, Osmotic pressure, incubation period, sieve pore size, centrifugation period and hormonal balance was estimated using the techniques for isolation. The quantity of cells was computed as the quantity of cells per square on haemocytometer. A considerable higher yield of protoplast formation was noted in the CPW medium using a pore size of 25 µm with using incubation period for 20 hours. Moreover, the best protoplast regeneration with using of protoplast density of 2.0 x 105 in MS medium supplemented by 1.0 mg L-1NAA and 0.3 mg L-1BAP. We believed that our protocol might encourage the plant recovery using in apple somatic hybridization programs.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz)

“…Similar results were reported in carrot where somatic embryogenesis was strongly inhibited in high cell density cultures due to conditioning factors into culture medium from cell during culture [33]. Our results also compare with the finding of [39] who reported that increasing the leaf mesophyll protoplast plating density in Malus domestica 'Anna' from 0.5×10 5 p/mL to 2.0×10 5 p/mL resulted in enhanced protoplast development. Beyond the range, the development was adversely affected.…”

A More Accessible, Time-Saving, and Efficient Method for In Vitro Plant Regeneration from Potato Protoplasts

“…On the other hand, various pretreatments, such as 24 h of incubation, dark pretreatment, and preincubation with Ca 2+ have been shown to increase protoplast isolation [ 28 , 30 , 31 ]. In some plant species, pretreatment using antioxidants is needed to reduce phenolic compound accumulation and thus improve protoplast isolation [ 32 ]. In the case of in vitro-grown plants like the materials we used, it is thought that pretreatment may have little effect because there are few phenolic compounds present.…”