Ezequiel E. Larraburu

In Vitro Propagation of Pink Lapacho: Response Surface Methodology and Factorial Analysis for Optimisation of Medium Components

Handroanthus impetiginosus, pink lapacho, is a timber, ornamental, and medicinal tree. Experiments on the in vitro propagation of H. impetiginosus were conducted using nodal segments cultivated in both Murashige and Skoog salts with Gamborg vitamins (MSG) and Woody Plant Medium (WPM) with different concentrations of 6-benzylaminopurine (BA) and indole butyric acid (IBA). Morphogenic responses were differentially affected by salt compositions and their interactions with plant growth regulators in each micropropagation stage. According to response surface analysis, the optimum multiplication rate with 1  μM IBA ranged from 16.7 to 21.3  μM BA in WPM, and the inhibitors of endogenous auxins could increase multiplication rates. A pulse with 50  μM IBA in MSG produced 83% rooting with 3.2 roots per shoots and higher fresh and dry weights of shoots and roots. In the acclimatisation stage, 50% of plants survived after 1 year. This methodology optimised the culture media for the in vitro propagation of the H. impetiginosus clonal pool and could be applied to related species, several of which are categorised as vulnerable on the International Union for the Conservation of Nature Red List.

In vitro propagation of fraser photinia using Azospirillum-mediated root development.

Fraser photinia (Photinia × fraseri Dress.) is a woody plant of high ornamental value. The traditional propagation system for photinia is by rooting apical cuttings using highly concentrated auxin treatments. However, photinia micropropagation is an effective alternative to traditional in vivo propagation which is affected by the seasonal supply of cuttings, the long time required to obtain new plants, and the difficulties in rooting some clones.A protocol for in vitro propagation of fraser photinia using the plant growth-promoting ability of some rhizobacteria is described here. Bacterial inoculation is a new tool in micropropagation protocols that improves plant development in in vitro culture. Shoots culture on a medium containing MS macro- and microelements, Gamborgs vitamins (BM), N (6)-benzyladenine (BA, 11.1 μM), and gibberellic acid (1.3 μM) produce well-established explants. Proliferation on BM medium supplemented with 4.4 μM BA results in four times the number of shoots per initial shoot that develops monthly. Consequently, there is a continuous supply of plant material since shoot production is independent of season. Azospirillum brasilense inoculation, after 49.2 μM indole-3-butyric acid pulse treatment, stimulates early rooting of photinia shoots and produces significant increase in root fresh and dry weights, root surface area, and shoot fresh and dry weights in comparison with controls. Furthermore, inoculated in vitro photinia plants show anatomical and morphological changes that might lead to better adaptation in ex vitro conditions after transplanting, compared with the control plants.

Biofertilization with Azospirillum brasilense improves in vitro culture of Handroanthus ochraceus, a forestry, ornamental and medicinal plant

Biofertilization with plant growth-promoting rhizobacteria is a potential alternative to plant productivity. Here, in vitro propagation of Handroanthus ochraceus (yellow lapacho), a forest crop with high economic and environmental value, was developed using the Azospirillum brasilense strains Cd and Az39 during rhizogenesis. Epicotiles of in vitro plantlets were multiplied in Woody Plant Medium (WPM). For rooting, elongated shoots were transferred to auxin-free Murashige-Skoog medium with Gamborgs vitamins and WPM, both at half salt concentration (½MSG and ½WPM), and inoculated with Cd or Az39 at the base of each shoot. Anatomical studies were performed using leaves cleared and stained with safranin for optical microscopy and leaves and roots metalized with gold-palladium for scanning electron microscopy (SEM). In ½WPM auxin-free medium, A. brasilense Cd inoculation produced 55% of rooting, increased root fresh and dry weight (45% and 77%, respectively), and led to lower stomata size and density with similar proportion of open and closed stomata. Both strains selectively increased the size or density of glandular trichomes in ½MSG. Moreover, bacteria were detected on the root surface by SEM. In conclusion, the difference in H. ochraceus response to A. brasilense inoculation depends on the strain and the plant culture media. Cd strain enhanced rooting in auxin-free ½WPM and produced plantlets with features similar to those expected in ex vitro plants. This work presents an innovative in vitro approach using beneficial plant-microorganism interaction as an ecologically compatible strategy in plant biotechnology.