Acclimatization of Tapeinochilos ananassae plantlets in association with arbuscular mycorrhizal fungi João Ricardo Gonçalves de Oliveira(1), Thiago Alberto de Lima Morais(2), Natoniel Franklin de Melo(3) and Adriana Mayumi Yano‑Melo(4) (1)Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Departamento de Micologia, Rua Nelson Chaves, s/no, CEP 50670‑420 Recife, PE, Brazil. E‑mail: [email protected] (2)Companhia Vale do Rio Doce, Avenida Vicinal Picadão, Km 22, CEP 68390‑000 Ourilândia do Norte, PA, Brazil. E‑mail: [email protected] (3)Embrapa Semiárido, Caixa Postal 23, CEP 56304‑970 Petrolina, PE, Brazil. E‑mail: [email protected] (4)Universidade Federal do Vale do São Francisco, Avenida José de Sá Maniçoba, s/no, Centro, CEP 56304‑917 Petrolina, PE, Brazil. E‑mail: [email protected] Abstract – The objective of this work was to assess the potential of three isolates of arbuscular mycorrhizal fungi to promote growth of micropropagated plantlets of Tapeinochilos ananassae during acclimatization. The experiment was carried out in greenhouse, in a completely randomized block design, with four inoculation treatments: non-inoculated control and plants inoculated with Glomus etunicatum, Acaulospora longula or Gigaspora albida, with ten replicates. After 90 days, the following parameters were evaluated: survival rate, height, leaf and tiller number, leaf area, fresh and dry biomass, contents of macro- and micronutrients in the root and shoot, glomerospore number, and mycorrhizal colonization. The survival percentage was 100%, except for plants inoculated with G. albida (80%). The isolate G. etunicatum is more suitable for plant development, since it improves survival, growth, dry matter production, nutritional status, and vigor of T. ananassae micropropagated plants. Index terms: Glomeromycota, growth promotion, micropropagation, mineral nutrition, tropical flowers. Aclimatização de plântulas de Tapeinochilos ananassae em associação com fungos micorrízicos arbusculares Resumo – O objetivo deste trabalho foi avaliar o potencial de três isolados de fungos micorrízicos arbusculares na promoção do crescimento de plântulas micropropagadas de Tapeinochilos ananassae durante a fase de aclimatização. O experimento foi realizado em casa de vegetação, tendo-se utilizado o delineamento inteiramente casualizado, com quatro tratamentos de inoculação: controle não inoculado e plantas inoculadas com Glomus etunicatum, Acaulospora longula ou Gigaspora albida, com dez repetições. Após 90 dias, foram avaliados os seguintes parâmetros: percentual de sobrevivência, altura, número de folhas e de perfilhos, área foliar, biomassa fresca e seca, conteúdo de macro e micronutrientes nas partes aérea e radicular, número de glomerosporos e colonização micorrízica. O percentual de sobrevivência foi de 100%, exceto para as plantas inoculadas com G. albida (80%). O isolado G. etunicatum é o mais adequado para o desenvolvimento das plantas, pois aumenta a sobrevivência, o crescimento, a produção de matéria seca, o conteúdo nutricional e o vigor de plantas micropropagadas de T. ananassae. Termos para indexação: Glomeromycota, promoção do crescimento, micropropagação, nutrição mineral, flores tropicais. Introduction poorly known in Brazil, but has excellent prospects for acceptance by cultivators and consumers. The great diversity of climates and soils in Commercial production of ornamental plants has Brazil allow the cultivation of numerous species evolved greatly to become an extremely competitive of ornamental plants and flowers, with potential to activity, but it requires technology, advanced compete in the international market (Cançado Júnior knowledge, and efficient marketing (Pasqual et al., et al., 2005). Among the tropical ornamental plants, the 2008). Consequently, vegetative, in vitro propagation genus Tapeinochilos comprises 16 species, of which (micropropagation) has been widely applied to produce, 80% are endemic to the island of New Guinea, and in a short time and at any time of the year, a large scale is characterized by the formation of inflorescences of high quality plantlets, ensuring varietal authenticity composed of bright red bracts of great beauty and (Rout et al., 2006). post-harvest durability (Specht & Stevenson, 2006). Efficiency of micropropagation involves an Tapeinochilos ananassae (Hassk.) K. Shum. is still acclimatization step, which represents the transition Pesq. agropec. bras., Brasília, v.46, n.9, p.1099-1104, set. 2011 1100 J.R.G. de Oliveira et al. -3 -1 -1 from the heterotrophic to the autotrophic phase, capacity of 5.69 cmolc dm , 93 g kg of P, 0.44 g kg of when plantlets must increase photosynthetic rate K, 1.5 g kg-1 of Ca, 1.3 g kg-1 of Mg, 0.84 mg kg-1 of Cu, and absorption of water and minerals (Grattapaglia 68.9 mg kg-1 of Fe, 34.8 mg kg-1 of Mn, 2.10 mg kg-1 & Machado, 1998). Micropropagation techniques of Zn, and 0.14 mg kg-1 of Na. The plantlets were produce plantlets without pathogens, but eliminate irrigated with about 50 mL of distilled water, daily, arbuscular mycorrhizal fungi (AMF), which could bring without supplement of nutrient solution. great benefits to the hosts, whether in the production Micropropagated plantlets of T. ananassae, provided of seedlings in nurseries or in the acclimatization by the Laboratório de Biotecnologia of Embrapa of micropropagated plants (Kapoor et al., 2008). Semiárido, Petrolina, PE, Brazil, were multiplied in Mycorrhization expands the absorption zone around MS medium (Murashige & Skoog, 1962). the root, increasing the contact surface with the soil Isolates of the AMF Glomus etunicatum Becker & and favoring an increased uptake of minerals, such Gerd., Acaulospora longula Spain & Schenck, and as phosphorus, zinc, copper, nitrogen, and potassium, Gigaspora albida Schenck & Smith were propagated resulting in increased plant tolerance to environmental in greenhouse in pots containing previously stresses (Smith & Read, 2008). disinfected sand:soil (1:1 v v-1), with sorghum Although plant‑fungus association is not specific, [Sorghum bicolor (L.) Moench.] as host. The the natural occurrence of mycorrhizal associations inoculum produced was evaluated for the number of in representatives of the family Costaceae has been glomerospores by wet sieving and decanting documented (Santos et al., 2000). Specific favorable (Gerdemann & Nicolson, 1963), followed by combinations between AMF and ornamental plant centrifugation in water and sucrose (40% w v-1) genotypes have been observed in anthurium (Anthurium (Jenkins, 1964). Counting was done in channeled plates, andraeanum Lindl.) (Stancato & Silveira, 2006), using a stereomicroscope (40×). After counting the chrysanthemum (Chrysanthemum morifolium Ramat.) glomerospores, soil inoculum was prepared to contain (Sohn et al., 2003), and gerbera daisy (Gerbera sp.) approximately 200 glomerospores, in addition to (Sato et al., 1999). However, lack of plant growth fragments of mycorrhizal-colonized root and mycelium. promotion has also been reported in associations of The following treatments were established: AMF with heliconia (Heliconia sp.) (Sato et al., 1999), non-inoculated control, and plants inoculated with red ginger [Alpinia purpurata (Vieill.) K. Shum.], G. etunicatum, A. longula, and G. albida. A completely and beehive ginger (Zingiber spectabile Griff.) (Silva randomized block design with ten replicates was used. et al., 2006). Therefore, the selection of mycorrhizal The micropropagated plantlets of T. ananassae were inocula that are effective in plantlet acclimatization removed from growth flasks and washed in running and development is desirable for the improvement of water until the culture medium was completely propagation technology. removed. Specimens were selected in order to achieve The objective of this work was to assess the uniformity of size and number of leaves and roots. At potential of three AMF species to promote growth the moment of transplanting to 2-kg bags containing -1 of micropropagated plantlets of T. ananassae during substrate (soil and vermiculite, 2:1 v v ), plantlets were acclimatization. inoculated or not, according to the treatment. In the control treatment, 2 mL of filtrate were added, which Materials and Methods derived from the screening (45 μm) of all inoculum tested, in order to standardize microbiota. The experiment was carried out in greenhouse, During the 90 days of acclimatization, height and under controlled environmental conditions number of leaves and tillers were assessed. At the (27±2ºC; 75% relative humidity; light intensity of end of the experiment, survival rates, leaf area, fresh 250–560 μmol m-2 s-1). The substrate used was and dry biomass of shoot and root, root colonization, soil (Oxisol) and expanded vermiculite (2:1 v v-1), number of glomerospores, and mineral contents in previously sterilized in autoclave for two periods shoots and roots were determined. of 1 hour at 120°C. The substrate had the following To determine dry biomass, T. ananassae leaf and root characteristics: 6.41 g kg-1 of organic matter, pH 5.6, were placed in oven (65°C), until constant weight. After electrical conductivity of 0.54 dS m-1, cation-exchange weighing, shoot samples were ground in a Wiley mill, Pesq. agropec. bras., Brasília, v.46, n.9, p.1099-1104, set. 2011 Acclimatization of Tapeinochilos ananassae plantlets 1101 in which 0.5-g portions of sample were mineralized Acaulospora sp. in acclimatization have also been by nitric perchloric acid digestion for subsequent