Abstract
The technique of hybridization using plant protoplasts is widely used in plant breeding programs. The purpose of our study is to further characterize the process of protoplast isolation from the ornamental species Etlingera elatior (Jack) R. M. Smith. Protoplasts were isolated from different tissues: in vitro leaves, in vitro pseudostem, and leaves from plants cultivated hydroponically. We tested six enzymatic combinations, four incubation time periods, the rotary system (40 rpm) or steady in the dark, and three concentrations of mannitol (0.5, 0.6 and 0.7 M). The diameter and viability of obtained protoplasts were evaluated. The best source of explants used for protoplast isolation was the in vitro leaves, which yielded 22x105 protoplasts g-1 of fresh matter. The optimal incubation period was 15 hours. The in vitro leaves presented a greater viability (96%) and larger protoplasts (36.7 µm diameter). Greater yields were obtained using a rotatory system with protoplasts incubated in the dark. The best enzymatic combination was 3% Cellulase “Onozuca” R-10 + 2% Meicelase + 1% Driselase + 1% Dextran + 5 mM MES, followed by the addition of 0.6 M mannitol.
Highlights
IntroductionSomatic hybridization by protoplast fusion is a promising technique for breeding ornamental species and requires reliable in vitro protocols
The enzyme combinations B and E after 15 hours produced the best results for protoplast isolation and were significantly different according to the SNK test (5%) compared to other enzyme combinations
Incubation periods longer than 15 hours resulted in a decrease in yield of isolated protoplasts caused by increased membrane instability and nonselectivity of the enzymatic solution
Summary
Somatic hybridization by protoplast fusion is a promising technique for breeding ornamental species and requires reliable in vitro protocols. Somatic hybridization offers the following possible genomic manipulations: (1) overcoming sexual incompatibility; (2) producing amphyploids; (3) transferring part of one species genome to another (cybrids); (4) transferring cytoplasmatic DNA to produce male-sterile plants; and (5) producing plants resistant to environmental stresses, pests and diseases (WU et al, 2009). Somatic hybrids can be obtained when protoplasts of different species are fused. This study further characterizes the conditions used for isolating protoplasts from E. elatior because different tissues (in vitro leaves, in vitro pseudostems and leaves from a hydroponic system), different combinations of enzymes, different incubation periods, using a rotating or stationary system in light or dark, and mannitol concentrations can affect the diameter and viability of isolated protoplasts
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