Isolamento, cultivo e fusão de protoplastos de Dendrobium nobile
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Data
2022-11-28
Autores
Blasques, Gabriel Mazetti
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Dendrobium nobile é uma orquídea nativa do continente asiático que possui grande
potencial ornamental, além de ser considerada uma das principais plantas da farmacopeia
chinesa. Essa posição é garantida devido à grande variedade de propriedades medicinais da
espécie, que resultam dos diversos compostos bioativos como alcalóides, compostos bibenzil
e flavonoides. Baseado nisso, estratégias biotecnológicas como a tecnologia de protoplastos
podem conduzir ao melhor aproveitamento e uso potencial da espécie. Este trabalho teve
como objetivo o estudo dos parâmetros para o isolamento, cultivo e fusão de protoplastos
de D. nobile. Protoplastos foram isolados com sucesso a partir de folhas jovens de plantas
adultas utilizando solução enzimática composta por 2 % de celulase, 2 % de pectinase, 0,4 M
de sorbitol e meio MS a meia força com pH 5.8. O isolamento dos protoplastos demonstrou
maior eficácia no tratamento enzimático de 4 h alcançando 1,45 × 104 protoplastos por mL
de solução enzimática. O cultivo dos protoplastos isolados foi possível em meio B5
suplementado com BAP 0,8 mg/L, ANA 0,5 mg/L, sacarose 20 g/L e manitol 5 g/L. Foram
observados tecidos macroscópicos formados a partir do 31º dia de cultura, porém, esses
tecidos morreram com o prosseguimento dos experimentos. Experimentos de fusão dos
protoplastos utilizando o método do polietilenoglicol (PEG) demonstraram sucesso na junção
de citoplasmas apesar de causarem danos à membrana plasmática das células.
Dendrobium nobile is an orchid native to the Asian continent that has great ornamental potential and is considered one of the main plants of the Chinese pharmacopoeia. The range of therapeutic qualities possessed by the species, which are brought about by a variety of bioactive chemicals like alkaloids, bibenzyl compounds, and flavonoids, is what keeps this position in place. This allows for the best and most prospective utilization of the species to be the focus of biotechnological tactics like protoplast technology. In this research, the conditions for D. nobile protoplast isolation, culture, and fusion were examined. Protoplasts were successfully isolated from young leaves of adult plants using an enzymatic solution made up of 2% cellulase, 2% pectinase, 0.4 M of sorbitol, MS medium at half strength, and pH 5.8, protoplasts of the species were isolated from young leaves of adult D. nobile plants. Protoplast isolation performed more effectively during the 4 hour enzymatic treatment, yielding 1.45 × 104 protoplasts per mL of enzyme solution. In B5 medium supplemented with BAP 0.8 mg/L, NAA 0.5 mg/L, sucrose 20 g/L, and mannitol 5 g/L, isolated protoplasts could be grown. On the 31st day of culture, macroscopic tissues were visible, however as the experiments went on, these tissues perished. In spite of harming the cell's plasma membrane, protoplast fusion tests employing the polyethylene glycol (PEG) technique have shown success in fusing cytoplasms.
Dendrobium nobile is an orchid native to the Asian continent that has great ornamental potential and is considered one of the main plants of the Chinese pharmacopoeia. The range of therapeutic qualities possessed by the species, which are brought about by a variety of bioactive chemicals like alkaloids, bibenzyl compounds, and flavonoids, is what keeps this position in place. This allows for the best and most prospective utilization of the species to be the focus of biotechnological tactics like protoplast technology. In this research, the conditions for D. nobile protoplast isolation, culture, and fusion were examined. Protoplasts were successfully isolated from young leaves of adult plants using an enzymatic solution made up of 2% cellulase, 2% pectinase, 0.4 M of sorbitol, MS medium at half strength, and pH 5.8, protoplasts of the species were isolated from young leaves of adult D. nobile plants. Protoplast isolation performed more effectively during the 4 hour enzymatic treatment, yielding 1.45 × 104 protoplasts per mL of enzyme solution. In B5 medium supplemented with BAP 0.8 mg/L, NAA 0.5 mg/L, sucrose 20 g/L, and mannitol 5 g/L, isolated protoplasts could be grown. On the 31st day of culture, macroscopic tissues were visible, however as the experiments went on, these tissues perished. In spite of harming the cell's plasma membrane, protoplast fusion tests employing the polyethylene glycol (PEG) technique have shown success in fusing cytoplasms.
Descrição
Palavras-chave
Dendrobium nobile, Orchidaceae, regeneração in vitro, hibridação somática, protoplasto, in vitro regeneration, somatic hybridization, protoplast