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- Author or Editor: Eugenio Perez-Molphe Balch x
An efficient system for in vitro regeneration by organogenesis starting from internodal stem segments from seedlings of Mexican lime (Citrus aurantifolia Christm. Swing.) and mandarin (C. reticulata Blanco cv. Monica) was developed. The best results were obtained when the wounded edges of internodal stem segments cut longitudinally were placed downward on the surface of the culture medium. The optimal culture medium from both species was Murashige and Skoog with vitamins from B5 medium, 5% sucrose, 33.3 μm BA and 5.4 μm NAA. The best response was obtained when the segments were incubated at 25 ± 2 °C for 21 d in darkness, followed by 29 d on a 16/8-h light/dark cycle (fluorescent light, 54 μmol·m-2·s-1). The best regeneration system tested allowed the attainment of adventitious shoots from 96% and 88% of the explants in Mexican lime and mandarin, respectively. In Mexican lime an average of 7.8 well-differentiated shoots per explant was obtained, and in mandarin the yield was 5.1. Rooting of 70% of the shoots was achieved in culture medium with NAA (2.7–5.4 μm) or IBA (2.5–4.9 μm). Of the rooted plants, 85% adapted well to soil conditions. Chemical names used: 6-benzylaminopurine (BA), α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA).
In vitro propagation systems were developed for Carnegiea gigantea (Engelm.) Britt & Rose, Pachycereus pringlei (Berger) Britt & Rose and Stenocereus thurberi (Engelm.) Buxb, three North American species of columnar cacti. In vitro germinated seedlings were used as a source of explants. Multiple shoot formation from areoles was achieved for three types of explants (apical, lateral, and transverse) cultured on Murashige and Skoog (MS) basal media supplemented with 3% sucrose, 10 g·L-1 agar and various treatments with growth regulators. The highest shoot production efficiency for C. gigantea was obtained on transverse explants cultured on a medium with 2 mg·μmL-1 (8.87 μm) BA, where 5.3 shoots per explant were obtained. In P. pringlei and S. thurberi the best response was obtained using transverse explants on medium with 1 mg·L-1 (4.44 μm) BA (3.8 and 4.3 shoots per explant, respectively). Rooting of the in vitro generated shoots was achieved most efficiently on MS basal media with 3% sucrose, 10 g·L-1 agar and 1 mg·L-1 (4.9 μm) indole-3-butyric acid. Rooting frequencies were 92%, 88%, and 96% for C. gigantea, P. pringlei and S. thurberi, respectively, and the frequency of survival of the plants once transferred to soil was 86% on average. Chemical name used: benzyladenine (BA).
The present work is the first report in vitro on root induction of Agave salmiana Otto, using Agrobacterium rhizogenes. Several concentrations of bacteria and acetosyringone were used, and different inoculation sites were tested, such as leaves, shaft, and root. Incubation time in darkness was 6 days. The transformed adventitious roots appeared 25 days after inoculation. The best treatment was when the shaft was inoculated with: 1 × 108 bacteria/mL and 100 μm acetosyringone; in this treatment, induction of transformed roots was 57.5% in the inoculated sites. The activity and presence of the foreign genes in the transformed roots of A. salmianawere verified as follows: 1) histochemical staining for GUS activity was determined in 80% of the tested root; and 2) molecular analysis via PCR was made to verify the presence of nptII gene and rol B gene (both were present in 60% of the tested root). This is the first report of the arbuscular mycorrhizal colonization on wild roots and transformed roots of Agavewith Glomus intraradicesSchenck and Smith. The result of the monoxenic culture was as follows: mother spore germinated 5 days; the colonization of the transformed roots was 70%. Then we proceeded to the recovery of daughter spores, in which we obtained an average 300 daughter spores per petri dish, 6 months after inoculation.