ANTIFUNGAL AND AUXIN-LIKE ACTIVITY OF CARBENDAZIM IN MICROPROPAGATION AND ESSENTIAL OIL COMPOSITION OF EUCALYPTUS CAMALDULENSIS DEHNH

Abstract

An investigation on Eucalyptus camaldulensis Dehnh. was carried out using samples from matured field grown trees in Malaysia. Surface sterilization and callus induction protocols have been described intended for in vitro plant regeneration using leaf and stem explants. A multi-option surface sterilization protocol has been established using three common sterilizing agents such as Mercuric Chloride (HgCl2), Sodium Hypochlorite (NaOCl) and ethanol which were tested by varying their combinations and their time of exposure. Combined treatments with two and three of sterilizing agents proved efficient in getting higher rates of clean, especially, when 70% alcohol was included. Using of 2.63% NaOCl for 10 min, 0.1% HgCl2 for 2.0 min and 70% ethanol for 1.0 min was the most effective sterilizing method for E. camaldulensis leaf explants and could obtain low contamination and damage rates, which can be applied to other plants with the same rigidity. Callus induction was successfully obtained on MS medium from leaf and stem explants using different auxins and cytokinins i.e. 6-Benzylaminopurine (BA), Kinetin (Kn), 2,4-Dichlorophenoxyacetic acid (2,4-D), Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) in the concentration range of 0.5 - 3.0 mg/l. IBA and 2, 4-D were shown to be the best hormones for callus induction in leaves and stems, respectively. Combination of BA and NAA showed a better callus induction response, than individual hormones. Embryogenic callus was produced in leaf explants using IBA and the globular and heart stages were observed. The effects of the potent fungicide Carbendazim (MBC) were evaluated in in vitro cultures of E. camaldulensis. It showed significant reduction in fungal contamination, especially at ≥25 mg/l and low phytotoxic effects. Callus induced by NAA and BA from leaf and stem explants were not highly sensitive to MBC at low and moderate concentrations (≤15 and <50 mg/l, respectively). At higher MBC concentrations, callus iv induction and organogenesis were decreased. The highest MBC concentrations (200 and 300 mg/l), inhibited callus induction in leaf and callus development in stem. In addition to its cytokinin-like effect, the compound showed auxin-like effect by enhancement of callus induction in stem explants. The remarkable callus induction increment was observed when 3 mg/l MBC was combined with 3 mg/l 2,4-D or 0.1 mg/l BA and 3.0 mg/l NAA. Stem callus derived from MBC grown cultures could be efficient, regenerated into complete plants and established in field conditions. Direct shoot formation using plant nodal segments was also not sensitive to the fungicide concentrations (≥50 mg/l). Increasing concentrations of MBC in media caused some ultrastructural and anatomical alterations. Thus, these findings may have significance towards the understanding of the roles of the fungicide in plants. The essential oils compositions of E. camaldulensis various organs were investigated. Fifteen (15) is major compounds were identified represented 98.8, 97.5 and 99.5% of the compounds in the leaves, stems and immature flowers, respectively. Monoterpenes hydrocarbons were predominant in the three oil samples. The flower oil had the highest oxygenated monoterpenes content (19.6%). The most abundant compound in the three essential oils was γ-terpinene, as a principal oil component (57.4–72.5%) followed by o-cymene (14.6–26.3%) and terpinen-4-ol (6.6–16.2%). The regenerated plants from callus induced in stem using 3.0 mg/l NAA and 0.1 mg/l BA and 3.0 mg/l MBC, rooted on MS half strength medium fortified with 1.0 mg/l IBA and successively established under the field environment. They showed the same essential oil major components as those of the field-grown plants. In addition, E. camaldulensis leaf oils demonstrated cytotoxic effects in four cell lines; WEHI-3, HT-29, HL-60 and RAW 264.7 with WEHI-3 was the most sensitive to the essential oils with IC50 16.1.