Berta E. Llorente

LEAF ANATOMY OF CYNARA SCOLYMUS L. IN SUCCESSIVE MICROPROPAGATION STAGES

SummaryLeaf structure along the successive stages of Early French artichoke Cynara scolymus L. micropropagation was characterized using light and transmission electron microscopy. The mesophyll presents disorganized spongy and palisade parenchyma with large intercellular spaces and a few small chloroplasts in the leaves of plants cultured in vitro. In addition, both epidermal surfaces of such leaves invariably show a cell wall of the same thickness with a very thin cuticle and open stomata. In the root differentiation stage in vitro, structural changes take place in the leaves that are favorable for survival in the acclimatization stage: conspicuous cuticle, greater cell wall thickness, functional stomata, better mesophyll organization, developed vascular bundles, and the presence of sclerenchymatous tissue are observed. These features found in later in vitro stages are maintained in the following ex vitro stages, some becoming more evident. Our results demonstrate that the structural changes required to ensure appropriate acclimatization of micropropagated artichoke plants begin at the root differentiation stage, which can reduce in vivo acclimatization time and achieve greater survival of transferred plants.

Anatomy of normal and hyperhydric leaves and shoots of in vitro grown Simmondsia chinesis (link) schn

SummaryThe anatomy of normal and hyperhydric in vitro shoots and leaves from micropropagated simmondsia chinensis (Link.) Schn. (jojoba) was compared with that of seedlings (control plants). In vitro normal plantlets displayed good development and survived during the acclimatization stage. In vitro hyperhydric plantlets presented numerous anatomical defects, such as hypertrophy of the mesophyll and of the stem cortex, malformed non-functional stomata, epidermal discontinuity, and xylem hypolignification; they did not survice acclimatization. The study of the anatomical features of in vitro jojoba shoots and leaves allowed determination of the structural condition of the plantlets and prediction of which plantlet would survive the critical acclimatization stage.

Structural differences between hyperhydric and normal in vitro shoots of Handroanthus impetiginosus (Mart. ex DC) Mattos (Bignoniaceae)

The anatomy of normal and hyperhydric shoots (leaves and stems) of in vitro Handroanthus impetiginosus was compared using light microscopy, scanning electron microscopy, and transmission electron microscopy. In contrast to normal shoots, hyperhydric shoots presented numerous anatomical abnormalities at the proliferation stage. Disorganized cortex, epidermal holes, epidermal discontinuity, collapsed cells, and other structural characteristics were observed in hyperhydric shoots. So, by using anatomical analysis of in vitro H. impetiginosus shoots at the proliferation stage, we can predict which plants will survive the rhizogenesis and acclimatization stages.

In Vitro Propagation of Jojoba

Jojoba (Simmondsia chinensis (Link) Schn.) is a nontraditional crop in arid and semi-arid areas. Vegetative propagation can be achieved by layering, grafting, or rooting semi-hardwood cuttings, but the highest number of possible propagules is limited by the size of the plants and time of the year. Micropropagation is highly recommended strategy for obtaining jojoba elite clones. For culture initiation, single-node explants are cultivated on Murashige and Skoog medium (MS) supplemented with Gamborgs vitamins (B5), 11.1 μM BA (N(6)-benzyl-adenine), 0.5 μM IBA (indole-3-butyric acid), and 1.4 μM GA(3) (gibberellic acid). Internodal and apical cuttings proliferate on MS medium containing B5 vitamins and 4.4 μM BA. Rooting is achieved on MS medium (half strength mineral salt) amended with B5 vitamins and 14.7 μM IBA during 7 days and transferred to develop in auxin-free rooting medium. Plantlets are acclimatized using a graduated humidity regime on soil: peat: perlite (5:1:1) substrate. This micropagation protocol produces large numbers of uniform plants from selected genotypes of jojoba.

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.

Stimulation of root development with cyclodextrins on jojoba shoots in Vitro

SummaryWe studied the effect of α- and β-cyclodextrins on in vitro rooting of jojoba (Simmondsia chinensis (Link) Schn.) shoots. When 0.03–0.5 mM of cyclodextrins were added to modified Murashige and Skoog culture medium containing 0.015 mM of indolebutyric acid as 1-wk pulse treatment, rooting percentage increased by approximately 100% with respect to controls. Addition of cyclodextrins induced earlier rooting. An increase in rooting following treatment with cyclodextrins was also observed in the absence of indolebutyric acid, indicating that both compounds promote rooting per se.

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.