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In Vitro Regeneration from Longitudinal Sections of Seedlings

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In Vitro Regeneration from Longitudinal Sections of Seedlings HORTSCIENCE 51(3):279–284. 2016. (Engelmann, 2011); however, it is now recog- nized that these can be efficiently comple- mented by ex situ techniques. Actually the In Vitro Regeneration from latter may represent the only feasible option for conserving certain highly endangered and rare Longitudinal Sections of Seedlings of species (Sarasan et al., 2006). Plant tissue culture has proven to be Beaucarnea purpusii Rose, an Endemic a valuable technique for propagation, conser- vation, and sustainable use of many species, specially of those with sexual and asexual and Endangered Species reproduction limitations, slow growth rate, Mar´ıa del Carmen Vadillo-Pro, Luis Hernandez-Sandoval, low seeds availability, and endangered con- and Guadalupe Malda-Barrera servation status (Pence, 2011; Sarasan et al., 2006), all of which is true for most Beau- Faculty of Natural Sciences, Autonomous University of Queretaro, Boulevard carnea species (Contreras et al., 2008). de las Ciencias S/N, Juriquilla Queretaro 76230, Mexico To initiate in vitro propagation, plant growth regulators (PGRs) can be applied to Mar´ıa Luisa Osorio-Rosales explants following two different methods: 1) Environment and Sustainability Network, Institute of Ecology A.C., Carretera traditionally the explants are placed on nu- Antigua a Coatepec 351, El Haya 91070, Xalapa, Veracruz, Mexico trient medium containing PGRs for 3–4 weeks, transferring them afterward to me- 1 Mart´ın Mata-Rosas dium lacking PGRs. 2) Explants can be pulse Biotechnology Resources Management Network, Instituto de Ecologıa´ A.C., treated with a concentrated liquid solution of Carretera Antigua a Coatepec 351, El Haya 91070, Xalapa, Veracruz, cytokinin and auxin solution for several Mexico hours, after which they are placed on medium free of these PGRs, and some studies report Additional index words. micropropagation, cytokinins, pulses, direct organogenesis improved organogenesis when using the liq- uid pulse method (Goldfarb et al., 1991; Abstract . The present study establishes an efficient protocol for in vitro propagation from Madhulatha et al., 2004; Ram´ırez-Malagon Beaucarnea purpusii longitudinal sections of seedlings of , a threatened and highly et al., 2008). 6 appreciated ornamental species. The effect of three cytokinins: N -benzyladenine (BA), In spite of the botanical and ornamental kinetin (Kin), and thidiazuron, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ), in semi- interest in Beaucarnea species, studies con- solid media and three different concentrations, as well as the effect of BA and TDZ pulses cerning their in vitro propagation are scarce. at higher concentrations in liquid culture medium, were investigated. Adventitious shoot There are some reports for Beaucarnea formation by direct organogenesis was observed from all treatments. Additionally, recurvata (Bettaieb et al., 2008; Osorio- adventitious shoot formation was recorded from the leaves of the new shoots; this Rosales and Mata-Rosas, 2005; Reyes et al., particular response was exclusive to treatments supplemented with TDZ. In the 2013; Sajeva et al., 1994; Samyn, 1997), experiment using semisolid culture media, the highest means of shoots per explant were Beaucarnea gracilis (Osorio-Rosales and m obtained from treatments containing TDZ, particularly at a concentration of 0.45 M Mata-Rosas, 2005), and Beaucarnea inermis (25.8 shoots per explant). For the pulses experiment, the liquid culture media (Guillen et al., 2015), but to the best of our m m supplemented with TDZ at 22.35 M for 24 hours and 136.21 M for 96 hours, induced knowledge, there are no reports on in vitro a mean of 3.9 and 3.3 shoots per explant, respectively. Subculturing individual shoots on propagation of B. purpusii. MS and half-strength MS (1/2MS) media, both supplemented with activated charcoal at In the pursuit of determining an efficient L1 1g·L , induced rooting in 85% to 95% of shoots. A survival rate of 100% under protocol for in vitro propagation of endan- greenhouse conditions was achieved. The results of this study provide an efficient gered species with low growth rates such as B. purpusii alternative for mass propagation of and may also contribute to the B. purpusii, it is necessary to study the effects conservation and sustainable use of this valuable natural resource. of several PGRs at different concentrations on various explant types, with special interest to variables that have not been evaluated The genus Beaucarnea belongs to the individuals, which are highly appreciated as previously. Therefore, the present study at- family Asparagaceae, subfamily Nolinoi- ornamental plants. These circumstances affect tempts to provide an efficient protocol for in deae, and occurs from northeastern Mexico the population size and the sex ratio, causing vitro propagation of B. purpusii from longi- to northern Central America (Bogler, 1998). a low fertilization rate and consequently a re- tudinal sections of seedling using different Mexico harbors 10 of the 11 recognized duced seed production (Contreras et al., 2008; concentrations of BA, Kin, and TDZ on species, of which eight are catalogued as Golubov et al., 2007). semisolid media, as well as to evaluate the threatened, one as endangered (B. purpusii), B. purpusii occurs in small areas of the effects on the shoot formation of explants and one under study to determinate its status Tehuacan-Cuicatlan Biosphere Reserve, Mex- exposed to pulse treatments of higher con- (Hernandez et al., 2012). ico, which encompasses the states of Puebla centrations of BA and TDZ. The critical situation of most of Beaucar- and Oaxaca in Mexico (Fig. 1A), and the main nea species is due to habitat alteration and reasons for this species being endangered are Materials and Methods over-collection of seeds, seedlings, and adult the small size of its populations, its restricted geographic distribution, the lowest growth ratio Mature seeds were collected from a natu- of the 11 species in the genus, and the local use ral population of B. purpusii growing in Received for publication 3 Sept. 2015. Accepted of the inflorescences and infrutescences as Tehuacan-Cuicatlan Biosphere Reserve. The for publication 25 Jan. 2016. small Christmas trees, leading to a decreasing seeds were stored at 4 °C until use. They were Mar´ıa del Carmen Vadillo-Pro thanks CONACyT number of seeds available for natural germina- successively washed with liquid detergent for for the grant offered to conduct her studies. We tion (Hernandez et al., 2009). Because of this 30 min, immersed in a 0.3% fungicide solution thank Felix Krengel for constructive comments and Ò language corrections and Edgar Couttolenc for his situation, it is critical totakeactiontoguarantee (Captan 50, Bayer de Mexico S.A. de C.V., assistance in statistical analysis. its continued survival and sustainable use. Mexico City) for 24 h, rinsed once with sterile 1Corresponding author. E-mail: martin.mata@inecol. For species like B. purpusii, the traditional distilled water, surface sterilized with a 3% mx. conservation approach is in situ conservation hydrogen peroxide solution for 10 min and with HORTSCIENCE VOL. 51(3) MARCH 2016 279 3 months, survival rate and mean of shoots per explant were recorded. Rooting stage. At the end of the growth stage, shoots were individualized, transfer- ring 180 of a uniform size of 0.5 · 5.0 cm to test tubes (25 · 200 mm) containing 20 mL of either MS or 1/2MS (salts and vitamins) semisolid medium, both supplemented with sucrose at 30 g·L–1 and activated charcoal at 0, 0.5, or 1 g·L–1. The overall rooting rates of each treatment were registered after 45 d. Ex vitro culture. Rooted shoots were extracted from the test tubes and washed thoroughly under running tap water. The plantlets were then transferred to propagation trays (Hummert International, Earth City, MO), containing a mixture of worm compost, loam, and pumice (1:1:1) and kept under greenhouse conditions (average temperature 30 °C, relative humidity 80% to 90%). To prevent fungal growth, the plantlets were sprayed with a 0.3% fungicide solution (Cap- tan). The trays were covered with plastic translucent lids the first 15 d, after which, the relative humidity was gradually de- creased to between 50% and 60% by re- moving the lids during increasing amounts of time. Survival rates were recorded after 2 months. Statistical analysis. Shoot formation was analyzed by a Friedman two-way non- parametric analysis of variance, and different treatments were classified using Duncan’s Fig. 1. (A) Adult individual of Beaucarnea purpusii on wild; (B) initial growth of adventitious shoots from test (P # 0.05) (SAS 9.1; SAS Institute, longitudinal section of seedlings cultured in MS medium + TDZ at 0.45 mM;(C) multiple shoot Cary, NC). We tested rooting data for differ- formation from longitudinal section of seedlings cultured in MS medium + TDZ at 0.45 mM;(D) hyperhydrated shoots observed from explants cultured in MS medium + BA at 13.3 mM;(E) ences between treatments using Kruskal– development of adventitious shoots from leaves of shoots previously formed on medium supplemented Wallis test using the NPAR1WAY procedure with TDZ; (F) cross section through the foliar blade (FB) showing the development of an adventitious in SAS ver. 9.2. Multiple comparisons were shoot (AS), it is possible to observe connection of vascular bundles (VB), epidermis (EP), and made with least squares means and Bonfer- parenchyma cells connection between leaf and adventitious shoot; (G) rooting of individual shoot on roni adjustment procedures, P # 0.05. MS medium after 45 d of culture; and (H) plants of B. purpusii after successfully established in soil for 2 months. Bar = 1 cm. Results and Discussion First signs of seed germination were 70% ethanol for 1 min, immersed in a 30% (v/v) avoid the effect of apical dominance, and the observed on the 6th day, and the whole commercial chlorine solution (sodium hy- leaves and roots were trimmed away, each process continued until day 40.
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