THE USE OF CONTINUOUS, TEMPORARY IMMERSION BIOREACTOR SYSTEM AND SEMISOLID CULTURE MEDIUM FOR THE PRODUCTION Of <i>Eucalyptus camaldulensis</i> CLONES

Abstract

The plant micro-propagation in bioreactor systems is regarded as one way to reduce cost by automation and production scheduling. This research was carried out in order to obtain an efficient procedure for clone production of Eucalyptus camaldulensis on different types of bioreactor including continuous and temporary immersion bioreactor. To do so, the apical meristems (1 mm) and the apical meristems with adjacent tissue (2,5 mm) were used as initial explants. These tissues were cultured, for 60 days, in semisolid culture medium supplemented with 1 mg L -1 indole acetic acid (IAA) and 0.32 mg L -1 benzylaminopurine (BA). After 60 days, the meristems with adjacent tissue were transferred to a continuous immersion bioreactor and maintained in dark or light conditions. In order to verify the effect of the explant source on bioreactor multiplication, the explants subcultured from meristems multiplied in semisolid culture medium and the meristems multiplied in continuous immersion bioreactor were tested and maintained in dark conditions. After establishing this parameters, the multiplication experiments were carried out in continuous and temporary immersion and the multiplied explants were then rooted in MS medium supplemented with 0, 2, 4, 8 and 20 mg L -1 indole butyric acid (IBA) and kept in the dark or under controlled lighting conditions. After that, the rooting the plants were acclimatized in mist chamber. The meristem with adjacent tissue favored a greater number of buds/explants. The continuous immersion bioreactor in the dark provided higher shoots number and multiplication rate. The rooting was better on culture medium without auxin and kept in the dark for 15 days or the culture medium supplemented with auxin and maintained under light with 100% plantlet rooting. The Eucalyptus camaldulensis acclimatization was efficient, with high survival rate (76%). It was possible to establish the procedure for bioreactor micro-propagation of Eucalyptus camaldulensis large-scale clones.

Use of bioreactor systems in the propagation of forest trees

“…Shoots of E. camaldulensis with their bases continuously exposed to liquid medium were cultured in glass flasks with forced ventilation or in small flasks with natural ventilation . In both cases, the multiplication coefficients were higher than obtained in semi‐solid medium, although hyperhydricity was detected . This disorder also affected the propagation of apple shoots in a 5 L balloon with a net (Figure D), indicating that other parameters beside forced ventilation influence shoot quality .…”

“…CIS with natural or forced ventilation have been tested for solving these problems. Shoots of E. camaldulensis with their bases continuously exposed to liquid medium were cultured in glass flasks with forced ventilation or in small flasks with natural ventilation . In both cases, the multiplication coefficients were higher than obtained in semi‐solid medium, although hyperhydricity was detected .…”

Tissue Culture of Corymbia and Eucalyptus

“…Temporary immersion has increased proliferation more than 2-fold and increased nursery survival from 5% to 67% for shoots of E. grandis × E. nitens [168], and increased proliferation more than 5-fold for shoots of E. grandis × E. urophylla [171]. Temporary immersion and continuous immersion systems have both provided high rooting (100%) and nursery survival (76%) with E. camaldulensis shoots [161]. Another technique that supports the acclimatisation capacity of eucalypt shoots is photoautotrophic culture, in which shoots are maintained under conditions of high CO 2 concentration, but low sugar concentration, to promote photosynthetic carbon fixation and transpiration [173][174][175][176][177].…”

“…Eucalypt shoots sometimes produce roots spontaneously in hormone-free medium or potting mix [45,46,64,88,92,96,104,113,119,143,144,150,161]. However, adventitious rooting on eucalypt shoots is usually induced with an auxin rooting hormone, typically IBA at a concentration between 0.49 and 49 µM (Table A1).…”

“…One of the innovations in tissue culture that has been expected to improve ex-flasking capacity of eucalypt shoots has been the use of temporary immersion systems that provide repeated cycles of shoot wetting and drying [95,161,[168][169][170][171]. These systems have the potential to increase nutrient and hormone uptake by repeatedly refreshing the medium in contact with the shoot surface during the wetting cycles while also conditioning the shoot for ex vitro conditions during the drying cycles (e.g., by promoting cuticle formation [170][171][172]).…”