Concentrations of cytokinin and the use of agricultural residues in the in vitro propagation media of highbush blueberry

The objective of the research was to evaluate cytokinin concentrations and agricultural residues as physical structuring agents of the culture medium in the in vitro propagation of highbush blueberry (Vaccinium corymbosum ‘Duke’). To that end, three different concentrations of 2-isopentenyladenine (2iP) (0, 5, and 10 mg L-1)in woody plant medium (WPM) and five structuring agents (in natura rice husks, carbonized rice husks, coconut fiber, S-10 Beifort®, and agar)were tested. After 60days of culture, the following parameters were evaluated: survival (%),sprout and shoot lengths (cm),propagation rate, number of sprouts, and shoot dry mass (g). The substrates used as structuring agents were analyzed in terms of pH and electrical conductivity. The in natura rice husk, S-10 Beifort®, and carbonized rice husk did not differ from the agar in terms of the sprout length. The shoots were longer (1.64 cm) in cultures within natura rice husk than in other treatments. In the absence of 2iP, S-10 Beifort® resulted in the highest propagation rate (2.97). Concerning the number of sprouts, S-10 Beifort®, in natura rice husks, and carbonized rice husks did not differ from agar. However, when the regulator was added, the agar performed better in comparison to the other residues. Thus, in natura rice husks, carbonized rice husks, and S-10 Beifort®are potential physical structuring agents of the culture medium that can be used without 2iP.


INTRODUCTION
Native to North America, the high bush blueberry (Vaccinium corymbosum) has fruits appreciated for their exotic flavor and nutraceutical properties, especially in Europe, Asia and some regions of the Pacific, and South America (RETAMALES & HANCOCK, 2018). In Brazil, this fruit tree has been researched and produced in the South Region as an alternative in farm diversification, due to the high economic returns in a short timeframe (DAMIANI & SCHUCH, 2008). Among the cultivars used in the South is Duke, a tetraploid plant that requires an exposure to a long chilling period for 1 Nadal et al. profitable growth and production. Other species, less demanding in chilling requirement, are becoming popular for cultivation in the Southeast Region of the country (CANTUARIAS-AVILES et al., 2014).
The highbush blueberry can be propagated by stem cuttings or micropropagation. Micropropagation is an effective technique, ensuring higher quality seedlings and shorter production time (DAMIANI & SCHUCH, 2008;PELIZZA et al., 2013),as well as better use of physical space. However, compared with stem cuttings, micropropagation is a more expensive process (JUNGHANS& SOUZA, 2013).
In order to reduce the costs of micropropagation, research to replace reagents such as agar with alternative solidifying materials for culture media that also allow for adequate development of the explants has been conducted. For example, GOLLE et al. (2010) experimented with corn starch, vermiculite, filter paper, and hydrophilic cotton for micropropagation of the loblolly pine (Pinus taeda), CURTI & REINIGER (2014) tested vermiculite, Plantmax ® , and fine sand in yellow poinciana (Peltophorum dubium), OLIVEIRA et al. (2015aOLIVEIRA et al. ( , 2015b tested corn starch and cassava starch in pineapple (Ananas comosus), and RODRIGUES et al. (2016) andNADAL et al. (2018) assessed the use of coconut fiber, carbonized rice husk, and in natura rice husk for propagation of golden rain orchid (Oncidium baueri).
The use of agro-industrial residues as substrates can help to reduce the costs of different processes, while eliminating the materials accumulated in the environment (ASSIS et al., 2011;OLIVEIRA et al. 2018). However, there is little information on the use of residues as physical structuring agents of culture media and their possible interactions with the commonly used growth regulators such as cytokinins. The cytokinin 2-isopentenyladenine (2iP) enables a superior development of V. corymbosum . However, there is no information on the effect of this cytokinin on micropropagation with different structuring agents, with the exception of agar.
Therefore, the present research aimed to evaluate concentrations of 2iPand the use of different agricultural residues as physical structuring agents of the culture medium in the in vitro propagation of the highbush blueberry cultivar 'Duke'.

MATERIALS AND METHODS
Sprouting segments of the high bush blueberry 'Duke', were collected from established plants at Federal University of Pelotas, Capão do Leão, Rio Grande do Sul, Brazil. The segments, each with two axillary buds, were used as explants and cultivated in vitro for 90 days in a growth chamber under a photoperiod of 16 h, temperature of 25 ± 2 °C, and light intensity of 27 μmol m -2 s -1 .
Woody plant medium (WPM) (LLOYD & MCCOWN, 1980), containing 0.1 g L -1 of myoinositol and 30 g L -1 of sucrose, was used as culture medium. After adjusting its pH to 5.0, 30 mL of the liquid culture medium was poured into 300 mL glass flaks holding the structuring agents. Flasks were autoclaved at 120 °C and 1.5 atm for 20 min.
After transferring the explants into the flasks, these were sealed with aluminum foil and transparent plastic film and kept in a growth chamber with a 16-hour photoperiod, temperature of 25±2 °C, and light intensity of 27 μmol m -2 s -1 , where they remained for 60 days. After the 60 days, the variables evaluated were: survival (%), sprout length (cm), shoot length (cm), propagationrate, number of sprouts, and dry mass of plants (g).
The explants that were green and presented at least two leaves were considered survivors. The propagation rate was obtained by dividing the number of buds per plant at 60 days of cultivation by the number of buds per explant at the beginning of the experiment. The length was measured with a graduated ruler. The plant dry mass for each treatment was obtained by weighing the plants that were ovendried in an oven at 50 °C until constant mass.
The pH and electrical conductivity of the substrates were analyzed before their transfer into the flasks and the addition of the nutritive solution following the methodology proposed by KÄMPF (2006).
The data were subjected to analysis of variance using the F test (p≤0.05). Statistical significance was further examined by Duncan's test (p≤0.05). Table 1 is constructed from the variables that showed no interactions between the tested treatments.

RESULTS AND DISCUSSION
In all treatments 100% of the plants survived (Table 1). One of the main aspects for the viability of micropropagation as a method of seedling production is the high plant survival (SCHUCH & ERIG 2005). Survival is related to factors such as the type of explant, optimal hormonal balance, and adequate asepsis during the micropropagation process.
The length of sprouts in treatments within natura rice husk, carbonized rice husk, and S-10 Beifort ® did not differ from that in agar cultures. In contrast, the plant shoots were longer in treatment with in natura rice husk than in treatments with the other residues and agar (Table 1). These results showed that agricultural residues can be potentially used as medium structuring agents and be more efficient for the growth of plants compared with agar.
In experiments with other fruit trees, such as the pineapple cultivar 'Vitória', the sprout survival in culture media with different structuring agents, such as pure maize (Zea mays) starch oragar with partial or total addition of starch, did not differ from that in agar media. Thus, plants cultivated in environments with corn starch showed a similar development to those cultivated in agar (OLIVEIRA et al. 2015a).
Among the tested structuring agents not supplemented with a growth regulator, S-10 Beifort ® resulted in the highest propagation rate, 2.97. The propagation rate in treatments with in natura rice husks, (2.32) and carbonized rice husks (2.40) was higher than that of plants in agar medium (Table 2). This trend was observed for the number of sprouts, where the number of 0.60 sprouts per plant obtained in treatments with S-10 Beifort ® was similar to those obtained with agar, in natura rice husk, and carbonized rice husk. However, when these structuring agents were supplemented with 5 and 10 mg L -1 of 2iP, the propagation rate(17.10 and 15.67), sprout formation (3.10 and 4.0 sprouts per plant), and dry mass accumulation (0.034 and 0.039 g) were the highest in treatments with agar (Table 2).
These results can be attributed to the properties of agar as a structuring agent in the culture medium and its ability to control the reactions between the nutrient solution and the medium. Agar is inert and provides pH stability to the medium, thereby improving the availability of nutrients and the absorption of the growth regulator. The other structuring agents present different pH values and electrical conductivity (Table 3). It is known that the interactions of salts with the medium are altered by changes in the pH (KÄMPF, 2006). Thus, even when the nutritive medium was added to the structuring agent at pH and electrical conductivity conditions optimal for the culture, cation exchange may occur, leading to alterations in electrochemical characteristics of the medium.
The better performance of agar media may be contributed to the chemical structure of the growth regulator used in this study. Namely, RODRIGUES et al. (2016) andNADAL et al. (2018), who used another class of cytokinin, the 6-benzylaminopurine (BAP), reported that certain concentrations of BAP added to coconut fiber, in natura rice husks, and carbonized rice husks promote propagation at the same level as that obtained by agar supplemented with the same concentrations of the hormone. This suggested that the regulator in the present study was retained by the structuring agents and thus made unavailable to the explant.
The pH values of the substrates varied from 4.8 to 6.8, while the electrical conductivity varied from 0 to 26 µs m -1 (Table 3). Studies have showed that highbush blueberry cultures develop well in slightly acidic culture media, with pH around 5.0 (PELIZZA et al., 2013;SCHUCH et al., 2008). The in natura rice husk and the carbonized rice husk had a pH of 6.8, the coconut fiber and S-10 Beifort ® had a pH of 4.5 and 5.8 respectively and the pH of agar was adjusted to 5.0. It should be noted that the concentration of salts estimated by the electrical conductivity testing was higher in the residues than in agar (Table 3). This may have helped the development of the plants in the absence of 2iP, and may have affected the availability of the regulator when it was added to the medium.
The use of agar with the growth regulator provided the highest averages for some plant variables, whereas the medium containing S-10 Beifort ® , in natura rice husk, and carbonized rice husk generated plants with larger propagules. This characteristic is fundamental for the subsequent rooting process, because larger propagules generate more leaves and consequently resulted in greater photosynthetic rate, which is fundamental for plant development (TAIZ et al., 2017). CA -in natura rice husks; CAC -carbonized rice husks; FC -coconut fiber; S-10-S-10 Beifort ® .  Altogether, results suggested that the in vitro propagation of highbush blueberry 'Duke' can be conducted using agricultural residues without the supplementation with2iP. In addition, thein natura and carbonized rice husks and S-10 Beifort ® are potential crop medium structuring agents, after the protocol adjustment. Finally, in pursuit of cost reduction in micropropagation, such substrates can be substituted for agar in culture media, especially in regions with abundance of these agricultural residues, thereby contributing to the reduction of their volume in the environment.

CONCLUSION
For the in vitro propagation of highbush blueberry 'Duke', thein natura rice husk, carbonized rice husk, and S-10 Beifort ® are presented as potential physical structuring agents of the WPM culture medium that can be used without 2iP.