Plant regeneration of mulberry (Morus indica) from mesophyll-derived protoplasts

Abstract

A protocol is presented for regenerating plants from protoplasts of tropical mulberry. Leaves from seedling node cultures maintained in vitro were used as donor tissue. Optimal cell wall digestion was achieved with a combination of cellulase (2%) and macerozyme (1%). The plant growth regulator (PGR) combination zeatin (2.3 lM) and 2,4-dichlorophenoxyacetic acid (2,4-D D ) (2.3 lM) resulted in the highest number (29%) of cell divisions. First cell divisions were observed at day 4 after plating. Only zeatin (2.3 lM) and 2-methoxy-3,6-dichlorobenzoic acid (dicamba) (13.5 lM) supplemented medium supported subsequent divisions in protoplast cultures. Microcolonies reached a cell number of approximately 50, after 40 to 42 days of culture. The cells of these colonies continued dividing, leading to formation of microcalli. Whole plants were obtained after culture of microcalli on Murashige and Skoog (MS) medium containing thidiazuron (TDZ) (4.5 lM) and indole-3-acetic acid (IAA) (17.1 lM). The regenerated shoots were rooted on MS medium supplemented with 4.9 lM indole butyric acid (IBA). With a low survival rate during acclimation, regenerated plants were established in the greenhouse.

Thidiazuron: A multi-dimensional plant growth regulator

“…Few reports endorse the combination of auxin and cytokinin in medium promoted/stimulated protoplast division and development. So far, there is no report on combination of TDZ with different auxins like NAA or 2, 4 D at altered concentrations to stimulate cell wall synthesis around protoplast to initiate cell division and to regenerate plant from protoplast-derived callus (Hassanein and Dorion, 2006;Umate et al, 2005;Chupeau et al, 1993). It is reported that TDZ is the best choice than other phytohormones for protoplast proliferation (Murthy et al, 1998;Xiao et al, 2007).…”

Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss.

“…Thus, the best treatment for isolation of Lilium protoplasts was 4% 2006). Technology and regeneration procedures play an increasingly significant role in plant improvement through somatic hybridization and protoplast transformation (Umate et al, 2005). Protoplasts have served as recipient hosts for DNA transformation and are required in somatic hybridization by protoplasts fusion (Stephen,l995).…”

Mulberry improvements via plastid transformation and tissue culture engineering

“…The protoplast cultures grown in media supplemented with 13.5 µM dicamba were capable of further cell divisions and colony formation. We have shown a more specific role of the auxin dicamba in inducing cell divisions in mulberry that is different from other auxins like NAA and 2,4-D. 5 The protoplast derived colonies the in vitro tissue culture and micropropagation studies for morus spp., a pivotal sericulture plant, are well established. the rapid and reproducible in vitro response to plant growth regulator treatments has emerged as an essential complement of transformation studies for this plant species.…”

“…4 A protocol for regenerating plants from mulberry protoplasts was established for the genotype S36. 5 The first cell divisions were observed at day 4 with cell division frequency (CDF) ranging from 1 to 29% as on day 6. With the density of approximately 1.0 x 10 5 protoplasts, the combination of zeatin and 2,4-D induced highest percentage of cell divisions (29%) followed with zeatin and NAA (10%).…”