SANTA-ROSA S; SOUZA FVD; VIDAL AM; LEDO CAS; SANTANA JRF. 2013. Micropropagation of the ornamental vulnerable bromeliads Aechmea blanchetiana and Aechmea distichantha. Horticultura Brasileira 31: 112-118. Micropropagation of the ornamental vulnerable bromeliads Aechmea blanchetiana and Aechmea distichantha Sandra Santa-Rosa1; Fernanda VD Souza2; Ádila M Vidal3; Carlos Alberto da S Ledo2; José RF de Santana1 1UEFS-Depto. Ciências Biológicas, Av. Universitária km 03 BR116, 44031-460 Feira de Santana-BA; 2Embrapa Mandioca e Fruticultura, C. Postal 007, 44380-000 Cruz das Almas-BA; 3UFRB-Depto. Ciências Agrárias, 44380-000 Cruz das Almas-BA; sandra_santarosa@ yahoo.com.br; [email protected]; [email protected]; [email protected]; [email protected]
ABSTRACT RESUMO Aechmea blanchetiana and Aechmea distichantha are Brazilian Micropropagação das bromélias ornamentais vulneráveis native bromeliads with great ornamental value, although none of the Aechmea blanchetiana e Aechmea distichantha species are produced commercially. Therefore, market demands are Aechmea blanchetiana e Aechmea distichantha são bromélias met through nature predatory exploitation. This study intended to nativas do Brasil, de grande valor ornamental. Não sendo produzidas (1) evaluate the in vitro seed germination of A. blanchetiana and A. comercialmente, ambas sofrem exploração predatória. Este trabalho distichantha and (2) establish micropropagation protocols for both teve como objetivos (1) avaliar a germinação de sementes in vitro species, aiming at producing plantlets for the market, as well as to de A. blanchetiana e A. distichantha e (2) estabelecer protocolos de germplasm in vitro conservation. Germination experiments were micropropagação destas bromélias, visando a produção de mudas carried out in completely randomized designs with two treatments para atender ao mercado e à conservação in vitro. Os experimentos (presence and absence of light) and seven and eight replications de germinação foram conduzidos em delineamento inteiramente respectively for A. blanchetiana and A. distichantha. In vitro casualizado, com dois tratamentos (presença e ausência de luz) e seedlings, produced under light conditions, were used as explants sete e oito repetições respectivamente para A. blanchetiana e A. in the multiplication experiment (MS medium). The experimental distichantha. Plantas in vitro, obtidas em presença de luz, foram uti- design was again completely randomized, with treatments in lizadas como explantes no experimento de multiplicação (meio MS). factorial 2 (NAA concentrations; 0.05 and 0.5 μM) x 2 (cytokines: O delineamento utilizado foi inteiramente casualizado, em fatorial BAP and KIN) x 2 (cytokine levels: 2.2 and 4.4 μM) + 1 (control 2 (concentrações de ANA: 0,05 e 0,5 μM) x 2 (citocininas: BAP e with basal MS, without growth regulators), with ten replications per CIN) x 2 (concentrações de citocininas: 2,2 e 4,4 μM) + 1 (controle treatment. We evaluated fungal and bacterial contamination, total and com meio MS básico, sem reguladores de crescimento), com 10 re- cumulative seed germination and number of shoots per treatment in petições por tratamento. Foram avaliadas as contaminações fúngica each subculture. We observed only fungal contamination and only e bacteriana, germinação total e cumulativa e número de brotos por in A. blanchetiana (14.0%). Light favored in vitro seed germination tratamento em cada subcultivo. Observou-se apenas contaminação in both species (99 and 62% respectively for A. blanchetiana and A. fúngica e somente em A. blanchetiana (14,0%). A presença de luz distichantha). MS medium + 0.5 μM NAA + 2.2 μM BAP resulted favoreceu a germinação de sementes in vitro de ambas as espécies in the highest multiplication rate for both species, with significant (99 e 62% para respectivamente A. blanchetiana e A. distichantha). contrasts between this treatment and the control in both species Meio MS + 0,5 μM ANA + 2,2 μM BAP promoveu a maior taxa de (105.62 more shoots in A. blanchetiana; 223.80 in A. distichantha). multiplicação para ambas as espécies, com contrastes significativos BAP promoted the formation of rootless shoots, while kinetin favored entre este tratamento e o controle em ambas as espécies (105,62 brotos the formation of roots rather than shoots. A. blanchetiana and A. a mais em A. blanchetiana; 223,80 em A. distichantha). BAP promo- distichantha showed 90 and 97% of survival respectively in plantlet veu a formação de brotos sem raízes, enquanto cinetina favoreceu a acclimatization. formação de raízes em detrimento de brotos. Na aclimatização das plântulas, A. blanchetiana e A. distichantha apresentaram 90 e 97% de sobrevivência respectivamente.
Keywords: Bromeliaceae, tissue culture, in vitro multiplication rate, Palavras-chave: Bromeliaceae, cultura de tecidos, taxa de growth regulators. multiplicação, reguladores de crescimento.
(Recebido para publicação em 13 de abril de 2012; aceito em 5 de novembro de 2012) (Received on April 13, 2012; accepted on November 5, 2012)
enus Aechmea is one of the in the Atlantic Rainy Forest (Smith Brazilian native bromeliad, terrestrial, Glargest in family Bromeliaceae. & Downs, 1979). Most bromeliads occasionally epiphytic, perennial (Bert Aechmea belongs to the subfamily within the genus have high ornamental & Luther, 2005) and frequent among Bromelioideae and comprises about potential, such as Aechmea blanchetiana sand coastal plants and in the Atlantic 240 species (Luther, 2006), from which and Aechmea distichantha, and thus are Rainy Forest in states of Bahia and 70% are estimated to be distributed in target of predatory exploitation. Espírito Santo (Martinelli et al., 2008). Brazil, having its center of diversity A. blanchetiana (Figure 1A) is The inflorescence consists of a set of red
112 Hortic. bras., v. 31, n. 1, jan. - mar. 2013 Micropropagation of the ornamental vulnerable bromeliads Aechmea blanchetiana and Aechmea distichantha
various concentrations, according to A B the particularities of each step of the micropropagation process (Souza et al., 2003; Droste et al., 2005; Bellintani et al., 2008; Silveira et al., 2009). As in other species, the need to develop specific in vitro protocols for Aechmea blanchetiana and A. distichantha comes mainly from the possibility that a fortuitous genotype-specific dependency appears, which would require optimization procedures to avoid compromising the results. On the other hand, in spite of all the benefits ofin vitro propagation, it is important to bring into the spot that the cloning of poorly studied species may jeopardize the preservation of genetic Figure 1. Flowering plants of Aechmea blanchetiana (A) and Aechmea distichantha (B) variability in natural populations and, in (plantas em florescimento deAechmea blanchetiana (A) e Aechemea distichantha (B)). Cruz the long run, germplasm conservation, das Almas, Embrapa Mandioca e Fruticultura, 2009. resulting in irreversible genetic erosion. Given this, seed production, seems to suit best as propagation strategy for bracts that can last for several months. Micropropagation can guarantee not species like Aechmea at this point. This characteristic adds to the large only plantlet production in large scale, Sexual reproduction results in offspring ornamental potential the species already but also the means for establishing in that ensures the preservation of genetic has, a remarkably long postharvest life vitro banks for both species, expanding variability and avoids gene loss (Mercier (Lorenzi & Souza, 1998). The growing the conservation strategies of this & Nievola, 2003; Carneiro & Mansur, employment of A. blanchetiana in germplasm, currently under threat. 2004; Silveira et al., 2009). landscaping and gardens using plants In vitro propagation has been Taking into account what was supplied by predatory exploitation, showing great potential when compared exposed so far, the objective of this places the species in constant threat. to conventional propagation methods study was to develop protocols for Kanashiro et al. (2007) stated that the in bromeliads. In vitro propagation micropropagation of the ornamental lack of plant commercial production to allows for a quick genetically safe bromeliads Aechmea blanchetiana meet market demands resulted in the multiplication of a high number of and A. distichantha using plantlets presence of A. blanchetiana in the list plants, which matches with the large originated from in vitro germination of endangered species. market demand. Studies involving of seeds. A. distichantha (Figure 1B), typical in vitro propagation resulted in the from the Brazilian savannah, has development of multiplication protocols MATERIAL AND METHODS epiphytic, lithophytic or terrestrial habit, for several species (Mendes et al., 2007; arched leaves reaching up to 90 cm, Silva et al., 2008; Silveira et al., 2009; In vitro seed germination and an erect rosette which lasts up to a Santos et al., 2010; Huang et al., 2011), of Aechmea blanchetiana and A. month (Bert & Luther, 2005). Similarly including A. blanchetiana (Kanashiro et distichantha - Seeds from both species to the A. blanchetiana, the species is not al., 2007; Galvanesi et al., 2007; Chu et were taken from ripe fruits; but while grown commercially and has been used al., 2010). for A. blanchetiana fruits were collected as an ornamental plant due to predatory Several factors can influence the at the farm Flor de Brotas, in the supply, which led A. distichantha to efficiency of a micropropagation city of Irará, state of Bahia, for A. the category of species vulnerable to protocol. Culture medium is one of distichantha, they were collected in extinction (Martinelli et al., 2008). the major factors, as it paces plant the Pineapple Active Germplasm Bank Taking this framework into growth and development (Grattapaglia of Embrapa Cassava & Fruits. We perspective, plantlet production is the & Machado, 1998). The MS basal sterilized the seeds in aseptic conditions. way out to simultaneously meet market medium (Murashige & Skoog, 1962) In a laminar flow hood, we washed them demands and minimize extractive is the most used for micropropagation and removed residues, followed by 5 activities. Therefore, information about in bromeliads, using supplementation minutes immersion in 70% ethanol, the propagation system of these species with auxins and cytokines, for example 30 minutes immersion in 100 mL of is required to subsidize the development naphthaleneacetic acid (NAA), kinetin aqueous sodium hypochlorite solution, of efficient micropropagation protocols. (KIN) or benzylaminopurine (BAP) at containing 2% active chlorine with
Hortic. bras., v. 31, n. 1, jan. - mar. 2013 113 S Santa-Rosa et al. three drops of Tweem-20®, and three in growing explants of each species in sterilization methods. On the contrary, rinses using autoclaved distilled water. the same medium, but without growth bacteria incidence, especially when After, we placed the seeds in Petri regulators. We incubated the explants endophytic, implies a much more dishes containing 15 mL of autoclaved in a growth chamber (25±2oC, 16 complex control. (121oC, 20 min) MS medium, plus 30 h photoperiod, photon flow density Despite the common presence of g L-1 sucrose, solidified with 2.4 g -1L of 30 mmol m-2s-1) and performed endophytes in bromeliad axillary and Phytagel®, pH adjusted to 5.8. Then, we three successive subcultures at 45-day apical buds leading to contamination in incubated the plates in a growth chamber intervals, cutting shoots longitudinally the early stages of in vitro establishment, (25±2oC, 16 h photoperiod, photon flow whenever possible. there are few reports of contamination density of 30 mmol m-2s-1). We carried out the experiment rates when seeds are used as initial We counted the number of in a completely randomized design, explants (Pereira et al., 2003). Most germinated seeds (radicle protrusion) with treatments in factorial 2 (NAA protocols which use seeds as explant daily until it became stable for five days. concentrations) x 2 (cytokines) x 2 source in bromeliads (Pickens et al., Following, we evaluated fungal and (levels of cytokines) + 1 (control), with 2006; Bellintani et al., 2008; Silveira bacterial contamination and calculated ten 1-plant flask replications. We counted et al., 2009; Souza et al., 2009) do not the total percentage of germination the number of shoots developed per report contamination. and the cumulative germination. For explant in each subculture. We used the Seed germination rate was always analysis, we transformed the figures Tukey’s test (p>0.05) to compare means higher in A. blanchetiana than in A. of percentage of germination to among treatments and subsequently the distichantha. In addition, the total arcsin ( x /100 ) and compared them Dunnett’s test (p>0.05) to contrast each germination rate was significantly by Tukey’s test (p>0,05), using the mean against its respective control. We affected by light conditions: in both SAS statistical software (SAS Institute, transformed the means into log (x + species, germination rates were higher in 2004). 10) to correct data deviations from the the presence than in the absence of light normal distribution. All analyses were We carried out the experiments (99 and 94% in presence and absence of performed using the SAS statistical independently, one for each species, light, respectively, for A. blanchetiana; software (SAS Institute, 2004). although both in a completely and 62 and 47% for A. distichantha), randomized design with two treatments We acclimatized plantlets in a indisputably showing that light favored (presence and absence of light) and greenhouse, using the commercial seed in vitro germination in both species. ® 15-seed plates as replications. We used substrate Plantmax , for 60 days. Seed germination in A. blanchetiana seven and eight replications respectively started five days after sowing, regardless for A. blanchetiana and A. distichantha. RESULTS AND DISCUSSION of the presence of light, and continued until the 10th day after sowing for seeds In vitro multiplication of axillary In vitro germination of Aechmea placed under light and up to 15th day buds of Aechmea blanchetiana and A. blanchetiana and A. distichantha - distichantha - We cleaned (removal of Fungal contamination occurred only in roots and residues of seed coat) 60-day seeds of A. blanchetiana (14.0%) with no Table 1. Average number of shoots produced old plants derived from the previous records of bacterial contamination in the by Aechmea distichantha in vitro explants experiments, produced in the treatment experiments. Seeds of A. distichantha depending on ANA concentrations, after with light, and placed them in test tubes showed no contamination at all. In the third subculture (número médio de (150x25 mm) containing 15 mL of MS, spite of the fungal growth recorded brotos produzidos por explantes in vitro plus 30 g L-1 sucrose, solidified with in A. blanchetiana, the disinfestation de Aechmea distichantha em função da 2.4 g L-1 Phytagel®, for 60 days, for treatment we used can still be considered concentração de ANA, após o terceiro plant development. After this period, efficient. subcultivo). Cruz das Almas, Embrapa Mandioca e Fruticultura, 2009. we extracted 1.0 cm-long stems from Our results on seed contamination agreed with those of Galvanesi et ANA concentration these plants to be used as explants Shoots (no)1 in this experiment. We distributed al. (2007), which indicate that, in (µM) the explants in flasks containing MS general, seed sterilization should not 0.05 98.62 b -1 medium supplemented with 30 g L be an issue for those involved with 0.5 104.59 a sucrose and all combinations among bromeliad in vitro propagation. The CV (%) 15.69 the following factors: 0.05 and 0.5 µM fungal contamination we observed in 1 NAA (naphthalene acetic acid); 2.2 and A. blanchetiana is very likely related to Analysis performed over values transformed to log (x + 10) [análise ralizada sobre valores 4.4 µM BAP (6-benzylaminopurine); seed handling and sterilization during transformados em log (x + 10)]; Means inoculation. Fortes et al. (2004) stated and KIN (6-furfurylaminopurine). We followed by the same letter do not differ -1 ® used 2.4 g L Phytagel to solidify the that, despite the losses, fungi occurrence significantly from each other, Tukey’s test, culture medium, adjusted its pH to 5.8 in in vitro conditions is not as important p>0.05 (médias seguidas de mesma letra and autoclaved it (121oC, 20 min.). We as bacteria, since preventing fungi não diferem estatisticamente entre si, teste also set control treatments that consisted occurrence depends only on adjusting de Tukey, p>0,05).
114 Hortic. bras., v. 31, n. 1, jan. - mar. 2013 Micropropagation of the ornamental vulnerable bromeliads Aechmea blanchetiana and Aechmea distichantha
Table 2. Average number of in vitro shoots produced by explants of Aechmea distichantha germination happens exclusively under depending on cytokinine type and concentration, after the third subculture (número médio light (Socolowski & Takaki, 2004). de brotos produzidos por explantes in vitro de Aechmea distichantha em função do tipo e Therefore, it is plausible to infer that A. concentração de citocinina, após o terceiro subcultivo). Cruz das Almas, Embrapa Mandioca blanchetiana and A. distichantha have e Fruticultura, 2009. phytochrome A, since seed germination Concentration (µM)1 Cytokinin occurred with or without light, although 2.2 4.4 quicker when seeds were exposed to BAP 237.30 aA 108.50 aB light. KIN 20.89 bA 24.22 bA It is known that water and CV (%) 15.69 temperature, as well as other factors, interact with each other and may interfere 1Analysis performed over values transformed to log (x + 10) [análise realizada sobre valores with the metabolic processes that result transformados em log (x + 10)]; Means followed by the same capital letter in the line and small letter in the column do not differ significantly from each other, Tukey’s test, p>0.05 in seed germination. Furthermore, (médias seguidas de mesma letra maiúscula na linha e minúscula na coluna não diferem differences in germination may be estatisticamente entre si, teste de Tukey, p>0,05). characteristic to the genetic background of the species. Our results demonstrated that in vitro seed germination is effective Table 3. Difference in number of shoots between A. blanchetiana e A. distichantha explants in A. blanchetiana and A. distichantha, grown in MS1 medium modified with growth regulators and in basal MS1 (diferença em once starting from seeds, we obtained número de brotos entre explantes de A. blanchetiana e A. distichantha cultivados em meio functional plants of A. blanchetiana 1 1 MS adicionado de reguladores de crescimento e em meio MS básico). Cruz das Almas, and A. distichantha respectively 25 and Embrapa Mandioca e Fruticultura, 2009. 45 days after seed sowing in culture MS1 medium + growth regulartors x Difference in number of shoots medium. Basal MS1 A. blanchetiana A. distichantha NAA2 (0,05 µM) + BAP3 (2,2 µM) x Basal 63.51ns 237.40* In vitro multiplication of axillary NAA2 (0,05 µM) + BAP3 (4,4 µM) x Basal 89.71* 92.10* buds of Aechmea blanchetiana NAA2 (0,05 µM) + KIN4 (2,2 µM) x Basal 45.11ns 3.52ns and A. distichantha - Although we NAA2 (0,05 µM) + KIN4 (4,4 µM) x Basal 51.63ns 9.30ns observed significant differences among treatments, all treatments induced NAA2 (0,5 µM) + BAP3 (2,2 µM) x Basal 105.62* 223.80* the development of axillary buds. In 2 3 ns NAA (0,5 µM) + BAP (4,4 µM) x Basal 61.43 111.50* A. blanchetiana, the cytokine type NAA2 (0,5 µM) + KIN4 (2,2 µM) x Basal 46.62ns 24.10* influenced significantly the number NAA2 (0,5 µM) + KIN4 (4,4 µM) x Basal 52.51ns 24.86* of shoots after the third subculture, *Significant, Dunnet’s test, p>0.05 (significativo, Teste de Dunnet, p>0,05);ns non significant with BAP, regardless of concentration, (não significativo);1 MS: Murashige & Skoog. producing the best response (38.50 shoots). In A. distichantha, in its turn, it were NAA concentration (CA), cytokine type (CT) and concentration (CC), and for those in the dark. In the case of A. (Bellintani et al., 2007; Bencke & the CC x CT interaction that significantly distichantha, although germination also Droste, 2008; Silva et al., 2008). These altered the multiplication rate of axillary began on the fifth day after sowing, results, along with ours, demonstrate that buds. The divergence of results makes the process went on until the 35th and light not only favors seed germination it clear that the morphogenetic response 40th day for seeds kept under light and rates in different bromeliads, but also to changes in explant growth conditions darkness, respectively. speeds up the process. was species-specific. Silveira et al. (2009), working with Some species react to light by Shoot production in A. distichantha Neoglaziovia variegate in the same increasing seed germination (positive reached the maximum (104.59) at 0.5 light conditions we report here, also photoblastism), while in others it is µM NAA (Table 1). When we unfold the observed differences in germination darkness that triggers germination interaction CC x CT, we saw that shoot rate when studying seeds collected in (negative photoblastism) (Rose & yield (237.30) reached the highest at 2.2 ripe and immature fruits. These authors Ferreira, 2001). In positive photoblastic µM BAP, decreasing to 108.50 shoots reported that germination was favored plants, as most of the bromeliads when we doubled BAP concentration. by light, observing 100% and 80% of (Nievola & Mercier, 2003), phytochrome These results suggest that BAP had th th radicle protrusion at the 10 and 15 molecules are quickly activated by an inhibitory effect when used in the days for mature and immature seeds, light. When seeds have phytochrome highest concentration, unlike what we respectively. Several studies have A, germination takes place both in observed with KIN. Nevertheless, it is reported germination rates between 90 the presence and absence of light; worth mentioning that the number of and 100% within one week after sowing while, when phytochrome B is present, shoots obtained with KIN was much
Hortic. bras., v. 31, n. 1, jan. - mar. 2013 115 S Santa-Rosa et al. lower than those observed with BAP and 0.05 µM NAA + 4.4 µM KIN (Table Despite the significant differences (Table 2). 3). Treatments 0.05 µM NAA + 2.2 µM between the control and the treatments In A. blanchetiana, the contrasts BAP and 0.5 µM NAA + 2.2 µM BAP containing NAA and cytokines, the between the control and treatments 0.05 had the largest differences in number interaction NAA X cytokine was not µM NAA + 4.4 µM and 0.5 µM NAA of shoots in relation to the control, significant for neither of the species. BAP + 2.2 µM BAP were significant, the respectively 237.40 and 223.80. This Thus, the auxin-cytokinine balance, in latter showing the largest difference for result stressed once more the difference the concentrations used in this study, number of shoots (105.62) (Table 3). In between species in relation to their in did not alter the number of shoots A. distichantha, all treatments contrasted vitro morphogenetic responses and significantly. Other concentrations of significantly from the control, except also the efficacy of growth regulators these regulators, as well as different treatments 0.05 µM NAA + 2.2 µM KIN in boosting in vitro multiplication rates. balances, should be tried in future
Figure 2. Aechmea blanchetiana (A and B) and Aechmea distichantha (C and D) shoots after the third subculture on medium containing BAP (A and C) and KIN (B and D) (brotos de Aechmea blanchetiana (A e B) e Aechmea distichantha (C e D) após o terceiro subcultivo, em meio contendo BAP (A e C) e CIN (B e D)). Cruz das Almas, Embrapa Mandioca e Fruticultura, 2009.
116 Hortic. bras., v. 31, n. 1, jan. - mar. 2013 Micropropagation of the ornamental vulnerable bromeliads Aechmea blanchetiana and Aechmea distichantha experiments, since the increase in BAP which promotes the development of Bromeliaceae. Vidalia 2: 12-20. concentration in the medium apparently a large number of lateral buds; while CHU EP; TAVARES AR; KANASHIRO S; inhibits shoot formation (Table 2). kinetin apparently favors root rather GIAMPAOLI P; YOKOTA ES. 2010. Aechmea blanchetiana (Bromeliaceae) cultured in vitro. Galvanesi et al. (2007) counted than shoot development. However, Scientia Horticulturae 125: 451-455. an average of 138.71 shoots when there is need for further studies with DROSTE A; SILVA AM; MATOS AV; ALMEIDA A. blanchetiana was grown in liquid higher doses of kinetin to confirm these JW. 2005. In vitro culture of Vriesea gigantea inferences. and Vriesea philippocoburgii: two vulnerable medium supplemented with 5.0 mg Bromeliads native to southern Brazil. Brazilian -1 -1 L NAA (25.0 µM) + 5.0 mg L BAP Plantlet survival reached 90 and 97% Archives of Biology and Technology 48: 717- (22.0 µM) and 62.00 shoots in semisolid for A. blanchetiana and A. distichantha, 722. medium containing 1.0 mg L-1 NAA respectively, 60 days after starting the FORTES GL; FREITAS FO; FORTES MA; (5.0 µM) + 1.0 mg L-1 BAP (4.4 µM), acclimatization in greenhouse. VALLS JFM. 2004. Sobrevivência de acessos de amendoim (Arachis hipogaea) através in 180 days old cultures. Besides the use In conclusion, our results indicated de germinação in vitro. Brasília: Embrapa of liquid and semisolid medium, while that light improved the in vitro Recursos Genéticos e Biotecnologia. 15p. we used solid medium, our work differs germination of A. blanchetiana and A. (Boletim de Pesquisa e Desenvolvimento, 57). from Galvanesi et al. (2007) due to the distichantha on MS medium, as well as GALVANESI MS; TAVARES AR; AGUIAR FFA; much lower concentration of growth KANASHIRO S; CHU EP; STANCATO GC; the use of growth regulators enhanced HARDER ICF. 2007. Efeito de ANA, 6-BA regulators we employed. These suggest the multiplication rate of both species. e ágar na propagação in vitro de Aechmea that A. blanchetiana may present a The highest multiplication rates for blanchetiana (Baker) L. B. Smith, bromélia direct relationship between cytokine these two species were obtained on MS nativa da Mata Atlântica. Revista Ceres 54: concentration in the growth medium 63-67. medium supplemented with 0.5 µM GRATTAPAGLIA D; MACHADO MA. 1998. and multiplication rate. In Billbergia NAA + 2.2 µM BAP, with BAP inducing Micropropagação. In: TORRES AC; CALDAS distachia, Mendes et al. (2007) obtained the formation of rootless shoots and LS; BUSO JA (eds). Cultura de tecidos e 5.1 and 5.3 shoots per stem segment kinetin promoting root formation at the transformação genética de plantas. Brasília: in medium containing respectively the Embrapa Informação Tecnológica/Embrapa expense of the number of shoots. These Hortaliças. p.183-260. combinations 1.5 µM NAA + 5 µM results, along with the high plantlet HUANG PL; LIAO LJ; TSAI CC; LIU ZH. 2011. BAP and 3.0 µM NAA + 5 µM BAP, survival in acclimatization, clearly show Micropropagation of bromeliad Aechmea after 90 days of growth. Silveira et that it is possible to produce plantlets fasciata via floral organ segments and effects al. (2009) in their turn reported 60.57 for both species in large scale and of acclimatization on plantlet growth. Plant shoots of Neoglaziovia variegata in Cell Tissue and Organ Cult 105: 73-78. thus mitigate the predatory extraction KANASHIRO S; RIBEIRO RCS; GONÇÁLVES MS medium supplemented with 0.5 A. blanchetiana and A. distichantha AN; DIAS CTS; JOCYS T. 2007. 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