Production of embryogenic callus from leaf explants of Camellia sinensis (L.)

Abstract

An attempt was made to produce embryogenic callus from leaf explants of tea (Camellia sinensis L). The sterilized field-grown leaf explants were cultured on MS media with various combinations of benzyl aminopurine (BAP) and naphthalene acetic acid (NAA) to obtain embryogenic callus. The results showed that the rates of callus initiation (72.2%) and formation (56.9%) were relatively high in the medium containing 2.0 mg/L BAP and 3.0 mg/L NAA (referred as callus medium). To study the development of embryogenic callus morphologically and histologically, in vitro leaf segments were cultured on callus medium (0.8% agar) and then transferred at the 8th week to the same medium but solidified with 0.7% agar. Results revealed that leaf segments became slightly swollen at the cut end, subsequently whitish - yellow, friable cell masses were produced with bursting of swollen tissues. The percentage of callus initiation was 93.8% in the 6th week of incubation. 65.8% of explants formed caHus covering about half the surface of leaf segment in the 8th week. Cytological examination showed the development of collenchyma cells which were packed within the swollen tissues, and after bursting, parenchyma cells formed gradually as a result of cell division. These parenchyma cens de-differentiated into meristematic and embryogenic cells. A few somatic embryos (3.3%) developed from primary calli (16 weeks after incubation of leaf explants) in the preliminary study. The average number of single embryogenic cells was significantly more in liquid medium than in solid. The protocol developed in this study, will be used to obtain embryogenic callus from leaf explants and also genetic transformation.