In Vitro Plant Regeneration in Gloxinia [Sinningia Speciosa (Lodd.) Hiern.]

Home , Gesnerioideae, Gloxinia (genus), Sinningia, Sinningia speciosa

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 characters: 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

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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 explant, but the shoot lacked roots. In another similar study, adventitious shoot regeneration was compared among various leaf-derived explants cultured on MS medium supplemented with different concentrations of BA, TDZ, and NAA; the goal was to investigate the optimal explants types and size of leaves for obtaining efficient shoot regeneration (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

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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 transferred 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 induction 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. Experiments were performed with five flasks with days (Table 1). GA 3 exhibited a positive effect on seed ge- three explants per replicate, and the experiment was evalu- rmination of S. speciosa . For cv. ‘Brocade’, the percentage ated after 60 days. of germination increased to a maximum of 68.5% when GA 3 was incorporated into the culture media at 10 mg/L concentration, which was significantly higher than the 2.4. Medium and Culture Conditions control (no-GA 3) (~35%). The same results was found in In all experiments, the MS basal medium was supplement- cv. ‘Avanti’, the germination percentage increased to 59.2 ed with 2.0% sucrose, 1.0 mg/L thiamine.HCl, 100 mg/L % when GA 3 was incorporated into the culture media at the myo-inositol and solidified with 0.7% (w/v) agar. Only in same concentration, which was also significantly higher the seed germination treatments the MS basal medium was than the control (~19%) (Table 1). Sucrose at 2.0% and 3.0 supplemented with sucrose (2.0 – 4.0%). % also exhibited a positive effect on seed germination in gloxinia. In both cultivars, seedlings were obtained in all The pH of all media was adjusted to 5.8 ± 0.1, with either treatments tested. In general, seed germination occurred KOH or HCl before adding agar, and then steam-sterilized within 2-3 weeks. Radicles emerged first, and then cotyle- in an autoclave at 121 oC at 105 kPa for 20 min. Thirty mL dons appeared during the following two weeks. Fully dev- of medium was poured into 200 mL tissue culture flasks eloped plantlets were available after 6 to 8 weeks of cult- closed with polypropylene screw-on lids. Seed germination ure. Gibberellins overcome both kinds of seed dormancy and all of the subsequent culture steps were carried out in a and both kinds of bud dormancy in many species (Salisbu- culture room at 24±2 oC under standard cool white fluores- ry & Ross, 1992 and Davies, 1995), for instance, in Cedre- cent tubes with a flux rate of 70 µmol s -1 m-2, a 16-h photo- la montana it was necessary to supplement the culture me- period, and a relative air humidity of approximately 70%. dium with GA 3 1.0 and 2.0 mg/L, increasing the germination in 62% and 72%, respectively (Díaz-Quichimbo et al, 2.5. Acclimatization of Regenerated Plants 2013); likewise unexpectedly, the application of GA3 at a wide range of concentrations exhibited a negative effect of After eight weeks, the in vitro rooted plants were washed seed gemination of galax ( Galax urceolata ) (Yang et al, with tap water to remove agar, transferred to pots containi- 2013) and Pyxidanthera brevifolia (Wall et al, 2010). In ng several types of substrates, and covered with polyethyl- the other hand, the effect of seed maturity and sucrose ene bags for two weeks to maintain high humidity. The concentration (0.0 to 4.0%) were investigated via asymbio- substrates used were (1) earthworm humus, (2) a mixture tic germination of nobile Dendrobium hybrids, observing

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that immature seeds from 4-month old capsules showed 3.2. Shoot Elongation greatest germination rate of tested treatments, whereas 3- month old immature seeds showed the least germination After the gloxinia seedlings were transferred onto appropr- (Udomdee et al, 2014). iate media, shoot proliferation occurred within 3 to 4

Table 1. Effect of Different Concentrations of GA 3 and Sucrose on in vitro Germination of Sinningia speciosa Seeds after 30 Days in Culture

Treatments Seed Germination Time (Days) Germination (%) Plantlet Survival (%) GA 3 (mg/L) Sucrose (%) Brocade Avanti Brocade Avanti Brocade Avanti 0.0 0.0 6.5 9.3 35.4 d 18.6 de 58.0 e 52.5 cd 0.0 2.0 6.8 9.6 41.6 c 25.9 d 62.0 de 54.4 cd 5.0 2.0 6.6 9.2 40.4 c 24.5 d 60.0 de 53.3 cd 7.5 2.0 10.4 14.0 41.3 c 32.6 c 73.3 d 80.0 b 10.0 2.0 12.2 6.6 68.5 a 26.1 d 93.5 b 40.0 e 0.0 3.0 7.1 9.7 45.5 c 38.2 bc 57.0 e 63.7 c 5.0 3.0 10.4 19.2 50.0 bc 40.0 bc 80.0 c 86.8 b 7.5 3.0 8.8 11.4 47.8 bc 25.9 d 66.5 de 53.3 cd 10.0 3.0 15.8 12.2 51.8 bc 59.2 a 80.0 c 86.5 b 0.0 4.0 7.3 9.9 43.2 c 23.1 d 67.9 de 59.1 c 5.0 4.0 12.6 10.4 35.6 d 26.5 d 80.0 c 60.0 c 7.5 4.0 10.0 8.6 54.1 b 46.5 b 100.0 a 93.3 a 10.0 4.0 8.8 6.0 54.8 b 34.3 c 100.0 a 80.0 b

Values with different letters in the same column are significantly different at (P ≤ 0.05)

In Park et al (2012) study, gloxinia seeds were germinated weeks (Figure 1b). The combination of KIN (0.5 and 1.0 on MS basal salts medium solidified with 0.7% agar, in a mg/L) and NAA (0.0 and 0.5 mg/L) did not show signific- growth chamber at 25±1 oC with a photon flux rate of 35 ant affect on shoot elongation, shoot proliferation, or rooti- µmol s -1m-2 and a 16 h photoperiod. In the study of Chae et ng; however, differences in these characters were observed al (2012), shoot organogenesis and plant regeneration in S. between the cultivars ‘Brocade’ and ‘Avanti’. The greatest speciosa were improved using ethylene inhibi-tors such as shoot number (25.0) and survival (98.9%) was obtained in aminoethoxyvinylglycine (AVG), cobalt chloride (CoCl 2), the cv. ‘Avanti’ on medium containing KIN and NAA at a and silver thiosulphate (STS), and the explants were obta- ratio of 1:1 and on medium with a KIN: NAA ratio of 2:1, ined from seedlings grown under similar culture conditi- respectively. However, the greatest shoot elongation leng- ons. These conditions were similar to those described in hts (2.3 and 2.5 cm) were obtained in ‘Brocade’. In both our paper; however, in the studies of Chae et al (2012) and cultivars, the rooting percentage was around 85% (Table Park et al (2012), the gloxinia variety tested and the seed 2). Shoot vitrification and callus formation not was observ- germination percentages observed are not mentioned. ed in these cultures.

In another ornamental species, such as Cyclamen alpinus In gloxinia the combination of 2.0 mg/L BAP and 0.1 (Bürün & Sahin, 2009), Lippia graveolens , commonly mg/L NAA was found to be the most efficient, producing known as Mexican oregano (Castellanos-Hernández the greatest shoot lenghts (1.2 cm); however, the greatest et al, 2013) and Galax urceolata (Yang et al, 2013), shoot lenghts (1.7-1.8 cm) were found when the MS media several factors as seed size and/or weight in wild and was supplemented with 2.0 mg/L BAP, 0.1 mg/L NAA, cultivated populations, seed bury depth, temperature, light, and either 7.0 mg/L AgNO 3 or 50 mg/L putrescine (Park et pre-treatment, seasonal variation, and plant growth al, 2012). Likewise, in another study, the longest shoot regulators affect the germination of seeds on artificial (1.27 cm) was found when the regeneration medium (MS medium (Nikolic et al, 2006 and Bürün & Sahin, 2009). In media with 2.0 mg/L BAP and 0.1 mg/L NAA) was suppl- our study, others factors as the genotype, gibberellins emented with 5.0 mg/L STS (silver thiosulphate) (Chae et concentrations and sucrose levels also affect the seed al, 2013). In our study shoot length were greater than those germination. reported by Park et al (2012) and Chae et al (2013) and

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Table 2. Effect of KIN + NAA Combination on Shoot Elongation and Rooting from Apical Buds of S. speciosa after Two Months of Culture

Treatments Shoot Elongation No. of shoot No. of Leaf Rooting (%) Survival (%) (mg/L) (cm) Proliferation Formation KIN NAA Brc. Avt. Brc. Avt. Brc. Avt. Brc. Avt. Brc. Avt. 0.0 0.0 0.0 b 0.0 b 0.0 b 0.0 b 0.0 0.0 0.0 0.0 0.0 c 0.0 c 0.5 0.0 1.5 b 1.4 b 13.0 b 17.5 b 10.0 14.0 35.0 32.4 88.8 ab 93.2 ab 0.5 0.5 2.3 a 1.9ª 18.0 a 25.0 a 11.0 15.0 85.4 86.2 91.3ª 95.1ª 1.0 0.0 2.2 a 2.1 a 19.0 a 24.1 a 10.0 13.0 28.5 23.1 94.5ª 97.5 a 1.0 0.5 2.5 a 2.0 a 19.0 a 22.0 b 10.0 12.0 86.6 86.5 95.0 a 98.9 a

Values with different letters in the same column are significantly different at (P ≤ 0.05)

KIN was more efficient than BAP; however, the longest the major advantages of callus or cell culture systems over shoots (5.6 cm) were found when the MS media was sup- the conventional cultivation of whole plants is that autom- plemented with 2.0 mg/L BAO (Sharma & Sharma, 2013). ated control of cell growth and the rational regulation of metabolic processes can reduce labor costs and improve 3.3. Callus Induction productivity (Vanisree et al, 2004 and Danelutte et al, 2005). After two weeks of culture, the best callus production occurred on media containing 3.0 and 4.5 mg/L NAA, for both 3.4. Direct Organogenesis cultivars; however, a lower concentration of NAA (1.5 mg/L) resulted in the formation a larger number of shoots, After two months of culture, optimum shoot induction was with 66.7 and 100% of explants in ‘Brocade’ and ‘Avanti’, observed on MS medium containing 6.0 and 9.0 mg/L respectively (Table 3). In all treatments, shoots were not BAP in ‘Brocade’ with around 20 shoots per explant formed on the callus. (Figure 1a), and 9.0 mg/L BAP in ‘Avanti’, with around 30

Table 3. Effect of NAA and BAP on Callus Induction, Shoot Formation (Direct Organogenesis), and Survival in Leaf Cultures of S. speciosa after 60 days in Culture

Treatments (mg/L) Explants with Callus (No./%) Shoot Formation per Explant (No./%) Browning (No/%) NAA Brocade Avanti Brocade Avanti Brocade Avanti 0.0 0.0/0.0 c 0.0/0.0 c 0.0/0.0 d 0.0/0.0 d 0.0/0.0 c 0.0/0.0 d 1.5 1.8/12.0 b 1.3/8.7 b 4.3/66.7 a 5.2/100.0 a 2.4/62.5 b 3.3/50.0 b 3.0 2.6/17.3 a 2.1/14.0 a 3.5/33.3 c 4.4/83.3 b 3.4/100.0 a 4.2/75.0 a 4.5 2.5/16.7 a 1.9/12.7 a 1.0/50.0 b 1.3/50.0 c 3.1/75.0 b 3.3/12.5 c

(No./%), number and percentage; Values with different letters in the same column are significantly different at (P ≤ 0.05)

In the study of Park et al (2012), gloxinia leaves were cult- shoots per explant. In both cultivars, the survival rate was ured on MS media supplemented with 2.0 mg/L BAP and around 90% with no abnormalitie observed (Table 4). 0.1 mg/L NAA. During the initial stage (1-2 weeks) of incubation, there was some expansion and proliferation of In our study, explant size (10x10 mm) did not influence the cells at the cut surface, but callus growth was limited; shoot induction. In another studies leaves of S. speciosa we observed that the epidermal cells proliferated to prod- were taken from in vitro -grown plants and cut aseptically uce the shoots directly, without an intervening callus phase at the ends, into sections of approximately 7x7 mm in size and without supplementation of BAP. In another study, (Park et al, 2012). However, in the studies of Naz et al 0.1% activated charcoal was added to the medium to inhib- (2001), and Sharma & Sharma (2013), it was observed that it the callus formation at the base of the shoot in the rooti- 2x2 cm leaf discs gave the best response for shoot inducti- ng medium supplemented with NAA or IBA (0.5, 1.0, 1.5, on, and when larger explants were used, they did not resp- and 2.0 mg/L) (Sharma & Sharma, 2013), although in our ond even after four months and contamination was also study the highest callus induction was observed at the hig- high in these cultures, while explants smaller than 2x2 cm hest concentrations of NAA tested (3.0 and 4.5 mg/L); ho- died within 10 days after inoculation. Likewise, in the stu- wever, in tissue culture, callus tissue is of great utility and dy of Naz et al (2001) optimum shoot induction was obser-

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ved on MS medium containing 3.0 mg/L BAP, and by dec- ence on shoot induction and regeneration on MS media su- reasing or increasing the level of BAP, a gradual decline in pplemented with 5.0 mg/L IAA (Scaramuzzi et al, 1999). shoot initiation was observed; BAP at 2.0 mg/L induced the maximum shoots number (28 plants produced per cult- 3.5. Rooting ure) and it was also reported that higher levels of BAP cau- sed abnormal shoot formation. Root development was also observed simultaneously with shoot elongation in all of the cultures. The effects of differ- Table 4. Shoot Formation (Direct Organogenesis) in Leaf Cultures ent medium hormone composition are shown in Table 5. of S. speciosa after 60 Days in Culture The results indicate that in all treatments tested roots formation was observed. Treatments No. of Shoot Survival (%) (mg/L) Formation In in vitro propagation of gloxinia of the different concent- BAP Brocade Avanti Brocade Avanti rations of NAA used, only 1.0 mg/L gave the best results, 0.0 0.0 c 0.0 d 0.0 c 0.0 c while comparatively less rooting was observed when the b b b concentration of NAA was increased or decreased, and 3.0 16.6 23.2 71.9 87.5ª IBA did not give good results (Naz et al, 2001). Otherwise, c b b 6.0 20.1ª 19.4 70.0 67.5 Scaramuzzi et al (1999) obtained the best rooting on media 9.0 19.8ª 31.1ª 90.0 a 87.5ª containing IBA. In another studies, the regenerated shoots were initially rooted on MS medium (full- or half-strength) Values with different letters in the same column are significantly without growth hormones, after three weeks, with more different at (P ≤ 0.05) than 90% of the shoots producing roots after 5 weeks (Ch-

On the other hand, gloxinia plants were efficiently regener- ae et al, 2012 and Park et al, 2012). Likewise, successful ated using leaf, petiole and root segments as explants, and rooting of the in vitro -grown shoots was achieved on MS the three types of explants all first formed callus and adve- medium supplemented with 1.0 mg/L NAA (97.7%) or 2.0 ntitious buds on MS medium supplemented with 2.0 mg/L mg/L IBA (94.4%) and in culture medium without growth BA + 0.2 mg/L NAA; of these, 99.04% of the explants pr- hormones the rooting success was 0.0% (Sharma & Shar- oduced buds, there was 5.53 buds per explants on average, ma, 2013), although these results contradict the observati- and 98.81% of the buds formed roots (Xu et al, 2010). Co- ons of Naz et al (2001), Chae et al (2012) and Park et al mparing these results with those obtained in our study, (2012). In our study various combinations of BAP-NAA were observed that both explant size (10x10 mm) and BAP led to 100% of rooted plantlets and fully developed plant- levels (6.0 and 9.0 mg/L) were optimal at higher induction lets were available by two months of culture. of shoots formed per explant. 3.6. Acclimatization According Park et al (2012), MS medium containing BAP (2.0 mg/L) and NAA (0.1 mg/L) resulted in the highest ef- The selection of a suitable substrate can be decisive for ficiency of shoot regeneration per explant (12.3±0.8) and acclimatization. In our study, maximum plant survival was in the greatest shoot growth (1.2±0.1 cm) after 6 weeks; achieved in potting mixtures consisting of cachaza alone however, the addition of silver nitrate (7.0 mg/L), an ethyl- with 70% in ‘Brocade’, and a mixture of cachaza, soil, and ene inhibitor, increased the shoot number (23.9±1.6) and sand (1:1:1), with 55% in ‘Avanti’ (Figure 1b). The lowest shoot length (1.7±0.2 cm), and similarly, putrescine (50 survival rate were observed in a potting mixture consisting mg/L) improved the shoot number (19.2±1.6) and growth of earthworm humus, with 30% and 0% survival in cultiv- (1.8±0.2 cm). In another similar study, the addition of 1.0 ars ‘Brocade’ and ‘Avanti’, respectively (Table 6). mg/L aminoethoxyvinylglycine (AVG), 1.0 mg/L cobalt chloride (CoCl 2), and 5.0 mg/L silver thiosulphate (STS), In another study, rooted plantlets of gloxinia were success- all ethylene inhibitors, significantly improved the regener- fully established in potting mixtures consisting of cocopeat ation frequency, giving higher shoots numbers per explant and sand (1:1), with 79.9% survival (Shar-ma & Sharma, and longer shoots, using leaf explants cultured on initial 2013). In two other similar studies rooted plants were tran- shoot regeneration media containing MS salts, 2.0 mg/L sferred to pots containing autoclaved verminculite after BAP and 0.1 mg/L NAA (Chae et al, 2012). Likewise, a five weeks of culture, and the regenerated plants survived combination of cytokinin (2.0 mg/L BAP) and auxin (0.5 at rates of 80% and 90% and flowered within three months mg/L NAA) resulted not only in the highest shoot regener- (Chae et al, 2012 and Park et al, 2012). However, vermicu- ation efficiency (77.7%) but also gave the maximum num- lite showed a low capacity of ag-gregation with the roots, ber of shoots per explant (6.0) (Sharma & Sharma, 2013). which negatively affected plant development, in agreement These results were, however, not better than those obtained with the results of Hoffmann et al (2001), for apple tree in our study, where only BAP was used as growth regulat- roostocks, Rodrigues et al (2005), for Heliconia bihai , and or. Generally BAP has been shown to have a similar influ- Da Silva et al (2003), for gloxinia plantlets.

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Table 5. Effect of BAP+NAA Combinations on Shoot Elongation and Rooting in S. speciosa after 60 Days in Culture

Treatments (mg/L) Elongation (cm) No. of Leaf Formation Rooting and Root Length (%/cm) BAP NAA Brocade Avanti Brocade Avanti Brocade Avanti 0.0 0.0 0.0 c 0.0 c 0.0 0.0 0.0/0.0 0.0/0.0 1.0 0.5 4.9 b 6.3 b 11.0 12.0 100.0/1.2 100.0/2.4 2.0 0.75 7.4ª 8.0 a 10.0 12.0 100.0/1.8 100.0/2.5 3.0 1.0 6.9ª 9.7ª 11.0 11.0 100.0/1.7 100.0/2.4

Values with different letters in the same column are significantly different at (P ≤ 0.05)

In our study, although the survival rate was relat-ively lo- References wer than that observed in other studies, the use of cachaza, a residual component of sugarcane, significantly lower the APG III (Angiosperm Phylogeny Group) (2009) An update costs of large-scale production. of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botani- Table 6. Effect of Different Potting Mixtures on Hardening of in vitro –Grown Rooted Plantlets of Gloxinia cal Journal of the Linnean Society , 161, pp. 105-121.

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