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

Abstract

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Fig 1. Cassava node propagation.

A. cassava nodes with auxiliary buds cultured on MS medium. B. Node germinating on MS medium after 2 weeks culture.

Fig 2

Protoplast solid media culture: A. Protoplast gradient centrifugation. Yellow arrow indicates the dead protoplast cells, blue arrow indicates the sucrose solution, green arrow indicate a cushion of living protoplast cells and orange arrow indicates the washing medium. B. Protoplast cells under ×100 after Evans-blue staining. Black arrows indicate non-viable cells. C. Protoplast growing on solid medium. D. Cell clumps/colonies on solid medium.

Fig 3. Protoplast suspension culture.

A. Freshly isolated protoplast. B. Dividing protoplast three days after culture initiation. C. Microcalli developing two weeks after culture initiation. D. Minicalli 4 weeks after culture initiation. E. Minicalli developing on solid medium. F Callus obtained from minicalli after 28 days of minicalli culture. G. Compact callus. H. Callus with somatic embryos after 28 days of culture. Black arrows indicate the developing somatic embryos. I. Callus with torpedo shaped somatic embryos.

Fig 4. Embryo maturation, shoot and root induction.

A. cotyledonary embryos after 14 days of culture in maturation media. B. Greening cotyledonary embryos after 3 days of culture in desiccation medium. C. Greening cotyledonary embryos with defined shoot and root apices. D. Wholly greening leafy embryogenic calli. E. Rooting plantlet with stunted shoot elongation. F. Rooting plantlet with elongating shoot. G. Shooting embryogenic calli in 0.4 mg/l BAP. H. Rooted plantlet after one month of culture in MM medium. I. Hardening plantlet in peat moss. J. plantlet growing in soil in the green house.