PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

More documents

Recommendations

Info

FSB1 – 2004 Food Science and Biotechnology in Developing Countries The aim of this study was to establish the callus and cell suspension culture of B. hemisphaerica in order to investigate the production of protease(s) under in vitro conditions. And investigate the effects of the auxins and cytokinins in the medium on the initiation and growth of callus culture. Abbreviations: 2,4-dichlorophenoxyacetic acid (2,4-D), naphtalenacetic acid (NAA), Indolacetic acid (IAA), 4-amino-3,5,6-Trichloropiridin-2-carboxilic acid (Picloram) and Bencilaminopurine (BAP). MATERIALS AND METHODS Plant germination Seeds were obtained from a specimen of B. hemisphaerica maintained at Centro de Desarrollo de Productos Bióticos, IPN Experimental Field. Surface-sterilized seeds were placed on Knop medium with 0.5% (w/v) of agar. Cultures were maintained at 25 °C in darkness for three weeks and then under photoperiod of 16 h light, 8 h darkness for four weeks. Callus initiation The calli were induced with stem explants from B. hemisphaerica plants obtained under aseptic conditions in Murashige and Skoog (MS) ¼ basal medium with 20 g l -1 sucrose and 6 g l -1 agar. The medium also were supplemented with four auxins (2, 4-D, NAA, IAA and Picloram) and two cytokinins (Kinetin and BAP) in different proportions giving twelve combinations (MS1 to MS12). And two auxins concentrations (1.0 y 0.5 mg/L) were used. The pH was adjusted to 5.7 before autoclaving at 121 °C for 20 min. The cultures were carried out at 30 + 3 °C and under photoperiod as described above. After that the calli were induced, they were transferred to fresh medium each two weeks. The proteolytic activity was determined in the medium where the callus was growing. Suspension culture Cell suspension cultures were established by suspending 3-5 g fresh weight of calli into 50 ml of MS medium containing 0.5 mg l -1 Picloram, 0.5 mg l -1 BAP and 4 % (w/v) of sucrose in 125 ml Erlenmeyer flasks. Medium culture was subcultured every week. The cell suspension cultures were carried out at 27 + 2 °C and under 13 hours of light and 11 hours of darkness conditions and shaken at 150 rpm. The proteolytic activity was determined every week in the culture medium. Protease assay The proteolytic activity was measured both in callus and cell culture medium by Kunitz method, modified by Ortega and del Castillo (8) according with the protocol described by Badillo et al., (2), using 1% casein in sodium phosphate buffer 0.05 M pH 7.6 as substrate at 37°C.
RESULTS AND DISCUSSION FSB1 – 2004 Food Science and Biotechnology in Developing Countries Plants grown in aseptic conditions were obtained after to 50 days with germination of 80 %. Callus induction were obtained with several combination of BAP or Kinetin and Picloram or 2,4-D concentration in agar medium. With 1.0 mg l -1 of auxins callus induction was observed in the mediums MS 4, MS 5, MS 8 and MS 12 after to 60 days. Response was better with 0.5 mg l -1 of auxins because callus induction was observed in the mediums MS 1, MS 5, MS 8, MS 9 and MS 12 after to 50 days (Figure 1 and Table 1). In some cases organogenesis was obtained. The organs were formed with low concentration of both ANA or AIA and BAP, especially with 1.0 mg/L of ANA and without cytokinins roots were induced (Table 1). Table 1. The effect of auxins concentration on callus induction MEDIUM COMPOSITIÓN RESULTS (MS) AUXINS CYTOKININS (mg l -1 ) AUXIN 1.0 mg l -1 AUXIN 0.5 mg l -1 MS 1 2,4-D - Oxidation Callus MS 2 ANA - Roots Oxidation MS 3 AIA - Oxidation Oxidation MS 4 Picloram - Organogenesis Oxidation MS 5 2,4-D 0.25 Kinetin Callus Callus MS 6 ANA 0.25 Kinetin Organogenesis Oxidation MS 7 AIA 0.25 Kinetin Organogenesis Organogenesis MS 8 Picloram 0.25 Kinetin Callus Callus MS 9 2,4-D 0.5 BAP Oxidation Callus MS 10 ANA 0.5 BAP Organogenesis Organogenesis MS 11 AIA 0.5 BAP Oxidation Oxidation MS 12 Picloram 0.5 BAP Callus Callus In this study we get green to brown callus with hard to soft texture, callus approx. 0.5 -2 cm thick after 60 days of culture (Figure 1). A B C D Figure 1. Callus induction: (A) callus inducted in MS 8 medium with 0.5 mg l -1 Picloram. (B) callus inducted in MS 9 medium with 0.5 mg l -1 2,4-D. (C) callus inducted in MS 12 medium with 0.5 mg l -1 Picloram. (D) callus inducted in MS 12 medium with 0.5 mg l -1 Picloram.
Page 1: FSB1 - 2004 Food Science and Biotec
Page 5 and 6: UT/L 25000 20000 15000 10000 5000 F
Page 8 and 9: FSB1 - 2004 Food Science and Biotec
Page 30 and 31: Food Science and Biotechnology for
Page 40: FSB1 - 2004 Food Science and Biotec

PROTEASES FROM CELL CULTURE OF Bromelia hemisphaerica ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?