Macas -Palacios et al / Journal of Biology ( 2015), Vol. 03, Issue 01, pp . 6-14 ISSN 2052-0751 Research Paper In vitro Plant Regeneration in Gloxinia [Sinningia speciosa (Lodd.) Hiern.] G. del Cisne Macas-Palacios 1, Elsa E. Pucha-Pauta 1, G.E. Delgado-Paredes 2*, C. Rojas-Idrogo 2 and J. Minchala-Patiño 1 1Facultad de Ingeniería Agronómica, Universidad Nacional de Loja, Ciudadela Guillermo Falconí, Loja, ECUADOR 2Facultad de Ciencias Biológicas, Universidad Nacional Pedro Ruiz Gallo, Ciudad Uni., Juan XXIII N o 391, Lambayeque, PERÚ *E-Mail: [email protected] Abstract The objective of this study was to develop an efficient and reproducible protocol for in vitro micropropagation of gloxinia (Sinningia speciosa ), cultivars ‘Brocade’ and ‘Avanti’ from shoot apices and by direct organogenesis. The results indicated that gibberellic acid (GA 3) (0.0, 5.0, 7.5, and 10.0 mg/L) and sucrose (0.0, 2.0, 3.0, and 4.0%) promoted significantly higher seed germination percentages (18.6 to 68.5%). Maximum elongation in apical shoots of seedlings (1.9 to 2.5 cm), was observed in MS medium supplemented with 0.5 KIN alone or 1.0 mg/L KIN + 0.5 mg/L NAA within 60 days, and the number of shoots per explant ranged from 13 to 25. From leaf explants (10x10 mm), maximum shoot proliferation was obtained on MS medium containing 9.0 mg/L BAP with 19.8 to 31.3 shoots per explant. The shoots were 100% rooted in medium supplemented with 1.0 to 3.0 mg/L BAP + 0.5 to 1.0 mg/L NAA. Rooted plants were successfully established in potting mixtures of cachaza and cachaza: soil and sand (1:1:1) with survival rates of 70% and 55% for cultivars ‘Brocade’ and ‘Avanti’, respectively. Keywords: Acclimatization, Gloxinia, Micropropagation, Shoot Regeneration, Sinningia speciosa 1. Introduction The order Scrophulariales consists of 12 families and more variation in the New World subfamily Gesnerioideae is than 11,000 species. Approximately three-fourths of the pronounced, particularly in terms of flower appearance. In species belong to only three large families, the Scrophular- the past decade, phylogenetic relationships in the Gesneri- iaceae (4000), Acanthaceae (2500), and Gesneriaceae oideae have been based on analysis of the nr ITS (nuclear (3000) (Cronquist, 1988). In the system proposed by the ribosomal internal transcribed spacer) and cpDNA trn L-F Angiosperm Phylogeny Group, the family Gesneriaceae is and trn E-T spacer region sequences (Zimmer et al, 2001), placed with Acanthaceae, Bignoniaceae, Lamiaceae, Scro- and nuclear genome size estimates for 10 species of phulariaceae, Verbenaceae, and others families in the order Sinningia , and intraspecific genome size variation and gen- Lamiales, Euasterids I clade (APG III, 2009). ome characterization in S. speciosa was recently reported (Zaitlin & Pierce, 2010). In addition, many new species of The Gesneriaceae is a moderately large tropical family of Sinningia have been, and are continuing to be, described; flowering plants comprising ~133 genera and >3000 spec- an example is S. nordestina , species endemic to northeast- ies (Wenstai et al, 1988), and is distinguished from other ern Brazil (Chautems et al, 2000). members of the order Lamiales by a particular suite of cha- racters: five-lobed corollas, parietal placentation, unilocu- The gloxinia (Sinningia speciosa ), native to Brazil, is a lar bicarpellate ovaries, a pair-flowered cyme inflorescen- familiar commercial ornamental plant. The species produc- ce, minute seeds, and presence of endosperm in the seeds es single or double flowers in a variety of colors and corol- of many taxa (Smith et al, 1997). However, morphological la patterns. Generally, gloxinias are propagated by seed for Available online at www.scientific-journals.co.uk Page 1 Macas -Palacios et al / Journal of Biology ( 2015), Vol. 03, Issue 01, pp . 6-14 ISSN 2052-0751 commercial purposes and seedlings are raised in mass by Co.) is a compact, early-flowering cultivar that produces this method, but seed propagation can be difficult, showing 10 to 15 bell-shaped flowers/plant (Figure 1a). Seeds of high mortality of the small seedlings, and growing plants these cultivars were surface sterilized in a laminar flow from seed can become very expensive (Naz et al, 2001). chamber with 70% (v/v) ethanol for 1 min and then 5% Gloxinias can also be propagated by other means such as (v/v) sodium hypochlorite solution (Clorox ®) supplement- leaf, stem, and crown cuttings taken from mature plants ed with 1-2 drops of polyoxyethylene sorbitan monolaurate after flowering. The commercial production of a blooming (Tween 80 ®) for 10 min, followed by rinsing three times gloxinia takes approximately 6 to 7 months (Chautems et with sterile distilled water. al, 2000 and Zaitlin & Pierce, 2010). The regeneration of gloxinia plants from tissue culture using leaf explants was previously reported by Scaramuzzi et al (1999). In this work, the regeneration media consisted of MS salts supplemented with IAA combined with KIN, 80% of explants produced green callus and 25 to 30 shoots with roots per explant, and with IAA + BA, 80% of the explants produced green callus and 40 to 50 shoots per ex- plant, but the shoot lacked roots. In another similar study, adventitious shoot regeneration was compared among various leaf-derived explants cultured on MS medium sup- plemented with different concentrations of BA, TDZ, and NAA; the goal was to investigate the optimal explants typ- es and size of leaves for obtaining efficient shoot regenera- tion (Nhut et al, 2007). Similarily, in the regeneration of S. speciosa , two protocols were developed for leaf explants by Xu et al (2009): the first method involved producing callus and buds, followed by subsequent root growth on culture medium with NAA (1.0-5.0 mg/L), and the second method involved producing callus and roots, followed by subsequent buds induction on culture medium containing 2.0 mg/L BA and 0.2 mg/L NAA. Recently, an efficient and reproducible protocol for in vitro multiplication of gloxinia has been developed using leaf discs as explants on MS medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA (Sharma & Sharma, 2013). In addition, direct regeneration protocols of floral buds from sepal segments (Pang et al, 2006) and petal segments (Pang et al, 2012) have also been reported. These results seem to confirm that gloxinia plants that develop through tissue culture remain true to type, are inexpensive to produce, and are disease free. In the study of Scaramuzzi et al (1999) chromosome numbers of root tip cells of the mother plant and all in vitro -regenerated plants remained constant at 2n=26. In this study, we report the establishment of an improved method for large-scale propagation of S. speciosa using apical shoots and leaf as explants. 2. Materials and Methods 2.1. Plant Materials Plant material used in this study was Sinningia speciosa cultivars ‘Brocade’ and ‘Avanti’. ‘Brocade’ (Sakata ®) is a showy, double-flowered series that is ideal for gifts and indoor floral sales, and ‘Avanti’ (Mountain Valley Seed Available online at www.scientific-journals.co.uk Page 7 Macas -Palacios et al / Journal of Biology ( 2015), Vol. 03, Issue 01, pp . 6-14 ISSN 2052-0751 Figure 1. In vitro Plant Propagation of Gloxinia ( Sinningia speciosa ). of earthworm humus, soil and sand (1:1:1, w/w), (3) Cach- a. Adult plant of cv. ‘Brocade’; b. Shoot Proliferation aza (sugarcane waste), and (4) a mixture of cachaza, soil, from a Leaf Explant Four Weeks after Cultivation on MS Media Supplemented with 6.0 mg/L BAP; c. Rooting and sand (1:1:1, w/w) sterilized with hot steam for two on MS Media Supplemented with 2.0 mg/L BAP and hours. During the initial period, 90% relative humidity was 0.75 mg/L NAA, and d. Plants Six Months after Transfer maintained, and it was gradually reduced to 50% over a to Pots Containing Cachaza period of 1 month. These hardened plants were then transf- erred to the greenhouse. For acclimatizating, 50 plantlets 2.2. Shoot Elongation and Rooting in Apical Buds of were transferred to soil in each treatment, and the numbers Seedlings of surviving plants were recorded 6 months after transfer. Shoot apical buds (~1.0 cm long) excised from young see- 2.6. Statistical Analysis dlings were placed on MS medium (Murashige & Skoog, 1962) supplemented with 0.5 mg/L KIN or 1.0 mg/L and Results were processed and analyzed by analysis of varia- 0.5 mg/L NAA. Each treatment consisted of 50 explants nce (ANOVA) and the Tukey HSD multiple range tests with five explants per flask, and the experiments were eva- (p ≤0.05) in order to compare treatment means. All statist- luated after 60 days (Figure 1). ical analyses were carried out with Statgraphics Plus 5.0 software (StatPoint, Warrenton, Virgina, USA). 2.3. Shoot Organogenesis Young leaves were taken from 8-week-old in vitro grown 3. Results and Discussion plants. Leaves were cut into sections of approximately 10x 10 mm, and the explants were placed on MS medium sup- 3.1. Seed Germination plemented with NAA (1.5, 3.0, and 4.5 mg/L) for callus in- duction and BAP (3.0, 6.0, and 9.0 mg/L) for bud formati- Seeds of S. speciosa cultivars ‘Brocade’ and ‘Avanti’ were on. In a separate experiment, apical buds were transferred placed on germination medium containing MS salts suppl- to MS medium supplemented with BAP (1.0, 2.0, and 3.0 emented with differents concentrations of GA 3 associated mg/L) and NAA (0.5, 0.75, and 1.0 mg/L) to enhance sho- with 2.0, 3.0 and 4.0% of sucrose, and the percentages of ot elongation and rooting for further growth and develop- germination and plantlets survival were scored after 30 ent.