Synthetic seeds production and the induction of organogenesis in blackberry (Rubus glaucus Benth)

Romanian Biotechnological Letters Vol. 20, No. 1, 2015 Copyright 2015 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Synthetic seeds production and the induction of organogenesis in blackberry (Rubus glaucus Benth) Received for publication, November 17, 2014 Accepted, December 15, 2014 JADÁN, M. 1*, RUIZ, J. 1, SORIA, N. 1, MIHAI, R.A. 1,2 1 Universidad de las Fuerzas Armadas ESPE, Av. General Rumiñahui, Sangolquí Ecuador, P.O.BOX 171-5-231B, Departamento de Ciencias de la Vida y Agricultura, 3989400 ext 2122 2 Institute of Biology, Bucharest, Rumanian Academy, Av. Splaiul Independenţei 296, 060031, OP-CP 56-53, Romania *Corresponding author: mbjadan@espe.edu.ec Abstract The present research consists in the production of artificial seeds and the induction of organogenesis in blackberry (Rubus glaucus Benth) starting from explants represented by apical and nodal segments. The disinfection process was achieved using different concentrations of sodium hypochlorite (0.5, 1 and 1.5%) with various immersion times (10 and 15 minutes), the best treatment being represented by the concentration of 1.5%. Shoots were induced from nodal and apical segments on MS basal medium containing various concentrations of TDZ (0.25, 0.5 0.75 and 1 mg.l-1), the treatment with 0.5 mg.l -1 TDZ having the best response. The explants were induced during the multiplication stage on MS basal medium containing various concentrations of AIA (0.5, 0.75 and 1 mg.l -1 ) associated with BAP (2 and 3 mg.l -1 ). The increased number of internodes and higher plants were achieved using the concentrations of 2 mg.l -1 BAP and 0.75 mg.l -1 AIA. The optimal explants obtained were encapsulated in sodium alginate (3%) and calcium chloride (1%) dissolved on MS using different concentrations of brassinolide (0.1, 0.5 and 1 mg.l-1) and activated charcoal (0 and 1 g L-1) followed by the in vitro germination of blackberries using the treatment of BRA 1 mg.l -1 and 1 g L -1 activated charcoal. Keywords: synthetic seed, TDZ, AIA, BRA, activated charcoal 1. Introduction Blackberry is one of the most valuable commercially fruit in the world and the most cultivated in regions ranging from 1,200 to 3,000 m altitude. It is a highly perishable fruit, rich in vitamin C and with high water content, native to the tropical American highlands mainly Colombia, Ecuador, Panama, Guatemala, Honduras, Mexico and El Salvador. The genus Rubus contains one of the largest numbers of species in the plant kingdom spread on almost all the landscapes except the desert areas. In vitro propagation becomes necessary due to the increasing demand worldwide for these fruit. In recent years, in vitro growth becomes very important since the plant material obtained through this technique guaranties quality and safety compared to the traditional production which involves the transmission and contamination with pests and diseases. Furthermore, this methodology will facilitate the multiplication of some plants with desirable agronomic traits that today are only found in small towns and have a great potential for research purposes and for small farms production. According to J. QUIALA [1], the propagation 10134 Romanian Biotechnological Letters, Vol. 20, No. 1, 2015

Synthetic seeds production and the induction of organogenesis in blackberry (Rubus glaucus Benth) of blackberry (Rubus glaucus Benth) through synthetic seeds (encapsulated buds) will be cheap enough to compete with the sexual seed, this system being able to break the barriers that has limited in vitro propagation of blackberry (Rubus glaucus Benth), restricted only to those species where the unit price is so high to justify the use of this technique. The direct planting of encapsulated buds on field using automatic planter could make more competitive prices compared to the use of seeds. [2] Therefore the present study consists in establishing a protocol for obtaining viable propagules of blackberry (Rubus glaucus Benth) using the encapsulation technique. The obtained results may provide an important basis for future investigations regarding in vitro propagation techniques with application not only for species of agronomic interest but also for the endangered ones. [3] 2. Materials and Methods A. Sample Collection Young plants with good morphological characteristics (structure and uniform coloration in leaf area, absence of injuries or necrotic stems) were selected and disinfected with 70% alcohol; the branches of about 50-60 cm in length were placed in a sterilized bag container for transport in the laboratory. The samples were collected from the province of Tungurahua, Ecuador. B. Disinfection process of the plant material Apical and axillary buds were selected and were subjected to a sterilization process of eight treatments (Table 1), consisting in washing with sterile distilled water and 5 g.l -1 powdered detergent for 10 minutes, followed by the immersion in sterile water (10 minutes) and in a fungicide solution of 0.1% Phyton during 15 minutes. Further, the explants were washed with sterile water for 10 minutes and immersed in 70% ethanol solution during 1 minute, in sodium hypochlorite solutions of different concentrations (0.5, 1, and 1.5%) (10 and 15 minutes), adding Tween 20 to maximize the effect. Finally, three immersions in sterile water were performed inside the laminar flow cabinet prior to culture initiation. Table 1. Disinfection treatments Treatments NaClO % Immersion Time (minutes) 1 2 3 4 5 6 0.5 0.5 1 1 1.5 1.5 10 15 10 15 10 15 C. Shoot induction For the shoot induction the basal medium MS [4] was used and were tested four concentrations of Thidiazuron (TDZ). A total of five treatments as shown in Table 2 were tested and evaluated regarding the presence/absence of outbreaks (shoots) from the explants. Romanian Biotechnological Letters, Vol. 20, No. 1, 2015 10135

JADÁN M., RUIZ J., SORIA N., MIHAI R.A. Table 2. Treatments for shoot induction. Treatments TDZ mg.l -1 1 0.25 2 0.5 3 0.75 4 1 Control 0 D. Shoot Multiplication After four weeks of growth the development of shoots on the explants was observed. These were transferred on other seven multiplication media, as shown in Table 3. Table 3. Multiplication treatments. Treatments BAP mg.l -1 AIA mg.l -1 1 2 0.5 2 2 0.75 3 2 1 4 3 0.5 5 3 0.75 6 3 1 Control 0 0 E. Synthetic seeds Once viable explants were obtained in the multiplication stage, we proceeded to encapsulate them in sodium alginate 3% diluted in MS medium. To perform this technique, a solution of 1% CaCl 2 was prepared, in which the explant was absorbed with a pipette from the solution of sodium alginate and immersed in the chloride solution. The formation of artificial seed occurred due to the ion exchange between Na + and Cl -. (Figure 1) Figure 1. Synthetic seed process. F. Statistical Processing Statistical software InfoStat / Student version 2011 was used for this investigation. 10136 Romanian Biotechnological Letters, Vol. 20, No. 1, 2015

Synthetic seeds production and the induction of organogenesis in blackberry (Rubus glaucus Benth) 3. Results and Discussions A. In vitro Establishment During the disinfection process of blackberry plants, the contamination and viability variables were evaluated. On table 4 the applied disinfection treatments are presented and respectively the percentage of viable explants after 30 days of cultivation. Similarly, the figure 2 displays the corresponding percentages for each disinfection protocol. The treatment with 1.5% NaClO - 10 min showed the highest percentage of success represented by 80% uncontamination and 90% of explant survival, overcoming the problem of oxidation. The effect of the reduced oxidation can be attributed to the use of polyvinylpyrrolidone (PVP). According to A. ANGARITA & A. RAMIREZ [5], blackberry tissues are easily oxidized, so the use of the antioxidant citric acid in the culture medium and incubation of the explants in dark conditions allowed to avoid the oxidation process. Thus, in the Chi-square made for the three studied variables, presented in Table 5, it is shown that there is statistical evidence proving that contamination, oxidation and viability of the explants depend on the concentration of NaClO used. Table 4. The percentages of the uncontaminated and viable explants Explant NaClO (%) Immersion time (min) Uncontaminated Viable explants 0,5 10 60% 100% 1 10 70% 90% 1,5 10 80% 90% Buds 0,5 15 65% 70% 1 15 70% 70% 1,5 15 90% 60% Table 5. Chi-square values for the variables analyzed Contamination Oxidation Viability Chi-square values NaClO 0.0201 Immersion Time 0.3428 NaClO 0.008 Immersion Time 0.1138 NaClO <0.001 Immersion Time 0.0285 Figure 2. Summary of the in vitro culture initiation in Rubus glaucus B. Romanian Biotechnological Letters, Vol. 20, No. 1, 2015 10137

JADÁN M., RUIZ J., SORIA N., MIHAI R.A. B. Shoot induction For micropropagation of fruit species, the cytokinin TDZ is widely used because of the great ability to stimulate the proliferation of buds [6]. For many woody species the common in vitro used cytokinins are not enough to perform their function, so the thidiazuron became a real alternative. The studies performed by B. LANDI and L.MEZZETI [7] point out that the use of TDZ independently provide greater percentage of shoot regeneration on different genotypes of strawberry, being similar with the results obtained in our experiment. According to C. HUETMANN and J. PREECE the use of thidiazuron at low concentrations (0.1 to 1 mg L -1 ) stimulates proliferation of axillary shoots in many woody species, especially fruit species. In our experiment for inducing shoot formation from axillary and apical buds of blackberry (Rubus glaucus Benth), low concentrations of TDZ were used (0.25 mg L -1, 0.5 mg L -1, 0.75 mg L- 1, 1 mg L -1 as seen in figure 3, with a 100% shoots formation at a concentration of 0.5 mg L -1 ), corresponding to treatment 3. The results indicate that the addition of this hormone to the culture medium, acts positively on the induction process, which is correlated with the chi-square (p <0.001), showing the statistical dependence of the induction of outbreaks of blackberry shoots on this citokinin. 10138 Figure 3. Percentages of shoot presence (A) Explant without shoots (B) Explant with shoots. C. Shoot ellongation and number of internodes For in vitro multiplication of plants the use of cytokinins in the growth medium is very important, this hormone promoting cell division and in vitro organ formation by proliferation of axillary buds. The cytokinin TDZ was used in the shoot induction phase, but according to C. HUETTMAN & J. PREECE it may inhibit shoot elongation in in vitro conditions, so the studies of J. AZCON et al. [8] on blackberry suggest the use of 6-benzyl-aminopurine (BAP). Studies conducted by M. BARCELO et al. [9] on strawberry showed good results for the regeneration Romanian Biotechnological Letters, Vol. 20, No. 1, 2015

Synthetic seeds production and the induction of organogenesis in blackberry (Rubus glaucus Benth) and elongation of shoots using the basal medium MS supplemented with different concentrations of BAP and IAA. This was correlated in our study with seven treatments performed by using different concentrations of BAP (2 and 3 mg L -1 ) and IAA (0.5, 0.75 and 1 mg L -1 ). The best results were obtained by using 2 mg L -1 BAP and 0.75 mg L -1 AIA (treatment 3) which induced the regeneration of shoots with seven and three internodes and few buds (Figure 4), and a greater number of elongated shoots (Figure 5). These demonstrate the statistical correlation between using the hormones BAP and IAA and the number of internodes obtained. 12 10 Number of the internodes 8 6 4 2 0 1 2 3 4 5 6 7 #3 #4 #5 #6 #7 Treatments for the shoot multiplication Figure 4. The treatment influence on the number of obtained internodes. Number of internodes The shoots length Romanian Biotechnological Letters, Vol. 20, No. 1, 2015 10139

JADÁN M., RUIZ J., SORIA N., MIHAI R.A. The level of the shoots length Treatments for the shoot multiplication Level 1 Level 2 Level 3 Level 4 Figure 5. The influence of the hormones BAP and AIA on the shoot elongation. D. Synthetic seeds production. The propagation of blackberry (Rubus glaucus Benth) can be performed both sexually (seed) and asexually. According to MONTOYA et al. [10] the sexual propagation is not recommended for this crop, due to the low number of fertile seeds in each fruit, the long period of germination and slow seedling development. For this reason the use of synthetic seed greatly help solving this problem. Some of the factors that are limiting the development of synthetic seed into whole plants are represented by the concentration of nutrients within the sphere, being necessary to build a reservoir of nutrients to promote an adequate optimal conversion rate. [11]. Different concentrations of brassinolide (0.1, 0.5 and 1 mg L -1 ) and activated charcoal (0 and 1 mg L -1 ), where used for seven encapsulation treatments. The statistically evidenced analysis of variance (ANOVA) proved that the germination of the capsules is closely related to the presence of activated charcoal (P = 1.77%) and not related of the concentration of brassinolide (P = 46.21%), although this hormone has the ability to promote growth and cell division. This is similar to studies by S. SAIPRASAD [12], where the addition of activated charcoal to the encapsulation matrix promotes the conversion of the encapsulated explants. This is due to the influence of this compound on the alginate breaks that increases the respiration rate of the encapsulated outbreak and to the ability to retain nutrients for the explant, providing what is necessary for the germination process. (Figure 6) Figure 6. In vitro germination process. A) Capsules with activated charcoal. B) Capsules without the addition of activated charcoal. 10140 Romanian Biotechnological Letters, Vol. 20, No. 1, 2015

Synthetic seeds production and the induction of organogenesis in blackberry (Rubus glaucus Benth) It was observed a higher germination percentage in apical buds (57%) comparing to the axillary ones (28%) (Figure 7), in accordance with the results obtained by R. KAVYASHREE et al. [13], where the encapsulation of apical buds in Morus indicates better results than the axillary buds. Figure 7. The percentage of germinated capsules with apical and axillary shoots. The treatment 7 (1 mg L -1 of brassinolide and 1 gl -1 activated charcoal) has the best results (90%), similar to the results obtained by BAPAT & RAO [14] in Rubus spp, which obtained more than 50% of viability. 4. Conclusions The best treatment for disinfection of blackberry explants (Rubus glaucus Benth) included a concentration of 1.5% sodium hypochlorite and an immersion time for 10 minutes, representing the best results concerning the percentages of the lack of contamination and oxidation as well as the percentage of explant viability. The concentration of thidiazuron (TDZ) that generated the best results in the induction stage of bud explants in blackberry was 0.5 mg L -1. The concentration of 2 mg.l -1 BAP and 0.75 mg.l -1 IAA represented the best combination of cytokinin and auxin for the elongation and number of internodes of blackberry shoots. Synthetic seed production technique is helpful for species exhibiting erratic or slow germination as the case of blackberry, due to the high volume production of plant clones in a small space and for the cost that compete with the use of sexual seed. Sodium alginate (3%) and calcium chloride (1%) generated favorable results regarding the hardness of the capsule that allow in vitro germination of organogenic shoots of blackberry. The encapsulation matrix with the presence of 1 mgl -1 of brassinolide and 1 gl -1 of activated charcoal generate the highest percentage of in vitro synthetic seed germination. Use the apical vs axillary buds in the encapsulation process produces a higher percentage of in vitro germination and the use of activated charcoal breaks the alginate, increasing in this way the respiration rate and promoting the conversion of encapsulated explants. Romanian Biotechnological Letters, Vol. 20, No. 1, 2015 10141

JADÁN M., RUIZ J., SORIA N., MIHAI R.A. 5. Acknowledgements We are gratefully to the Plant Tissue Culture Laboratory and its staff as well as to Universidad de Las Fuerzas Armadas for supporting financially the project in the laboratory mentioned. References [1]. J. QUIALA. Propagación y Mejora Genética de plantas por Biotecnología. Ediciones GEO. Cuba. 225-231 (1998). [2]. J. PEREZ, Propagación y Mejora Genética de plantas por Biotecnología., Ediciones GEO. Cuba. 57-63 (1998). [3]. A. MANOLE PAUNESCU, Biotechnology for endangered plant conservation. In M.R. Ahuja & K.G. Ramawat (Eds) Biodiversity and Biotechnology. Springer International Publishing, Switzerland, ISBN 978-3-319-09380-2:181-202 (2014). [4]. T. MURASHIGE and F. SKOOG. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15(3), 473-497 (1962). [5]. A. RAMIREZ, A. ANGARITA. Estudios preliminares para la propagacion clonal "in vitro"de mora (Rubus glaucus l.) (1990). [6]. C. HUETTMAN, J. PREECEE. Thidiazuron a potent cytokinin for woody plant tissue culture. Plant Cell, Tissue and Organ Culture, 48, 105-119 (1993). [7]. L. LANDI, B. MEZZETTI. Auxin and genotype effects on leaf organogenesis in Fragaria. Plant Cell. Rep. 25 (4): 281-8 (2006). [8]. J. AZCON, M. TALON. Fundamentos de fisiología vegetal (E.U. Barcelona, Ed.) Barcelona: McGraw-Hill (2000). [9]. M. BARCELÓ, I. EL-MANSOURI, J.A. MERCADO, M.A. QUESADA, F. ALFARO, Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler. Plant Cell, Tissue and Organ Culture 54, 29-36 (1998). [10]. C. MONTOYA, L. HINCAPIE, V. URIBE,.Principales Enfermedades y Plagas en el cultivo de la mora. Boletín Técnico. Nariño: Quinchia (1997). [11]. K. REDENBAUGH, J. FUJII, D. SLADE, P. VISS, M. KOSSLER, Artifi-cial seeds - encapsulated somatic embryos. In: Bajaj Y. P. S. (Ed.)., High Technology and Micropropagation I. Biotechnology in Agriculture and Forestry, Springer, Heidelberg Berlin New York, 17: 395-416, (1991). [12]. S. SAISPRASAD. Artificial Seeds and their Applications. http://www.ias.ac.in/resonance/may2001/ pdf/may2001p39-47.pdf, (2001). [13]. R. KAVYASHREE, M. C. GAYATRY, H. M. REVANASIDDAIAH, Propagation of mulberry Propagation of mulberry variety S54 by synseeds of axillary bud. Plant Cell Tissue, Org. Cult. 84: 245-249 (2006). [14]. V. A BAPAT and P. S. RAO, In-vivo growth of encapsulated axillary buds of mulberry, Plant Cell, Tissue and Organ Culture, 20 (1), 69-70 (1990). 10142 Romanian Biotechnological Letters, Vol. 20, No. 1, 2015