Micropropagation on multiplication of native greenberries in South of Brazil

Rubus brasiliensis and Rubus erythroclados are commonly known as greenberries. This work aimed to test in vitro micropropagation protocols to greenberries. The multiplication experiment was conducted using BAP at concentrations 1 mg L, 2 mg L and 3 mg L. The rooting experiment was carried out in 1⁄2 MS medium without the addition of hormones, using 0.1 mg L NAA or 1.0 mg L IBA. The highest rate of viability of explants was obtained with R. brasiliensis propagation using 1.0 mg L of BAP. Rooting explants showed survival rates higher than 85%. Rooting rates in R. erythroclados was 87% while in R. brasiliensis it was 38%, The highest rates were obtained using 1.0 mg L 1 IBA in R. erythroclados.The acclimatization reached survival rates of 75% when rooted using NAA and 1⁄2 MS and 86% when rooted with IBA. In vitro culture shows to be a viable method to propagate greenberries.


Introduction
In Brazil, 11 species of the genus Rubus are described, six of these occur in the Santa Catarina state and are associated with the Atlantic Forest Biome (Reflora, 2019;Reitz, 1996). The species Rubus brasiliensis Mart. and Rubus erythroclados Mart. are commonly known as whiteberries or greenberries, alluding to the color of the fruits that remain green even when ripe (Bueno, 2015). The fruits have high levels of sugar and a pleasant peculiar flavor, which gives market potential compared to other small fruits (Bueno, Biasi, & Tofanelli, 2018). Information about the ethno-knowledge of these species in the Santa Catarina South Plateau indicates that although they are much appreciated for their sweet taste and were less acidity than blackberries their consumption is made only in fresh fruits mode, when the fruits are found at random, without the habit of collection for processing (Couto Waltrich, Boff & Boff, 2017).
Greenberries are not domesticated or cultivated commercially yet, although their potential as an alternative for cultivation and economic exploitation by family farmers, like blackberries. Currently, around 500 ha of blackberries originating from genetic material native from United States are cultivated in the south and southeast of Brazil (Antunes, et al., 2014). This fruit consumption is associated with nutraceutical properties, such as anticarcinogenic compounds (Magalhães, Maciel & Orsolin, 2017), which are present also in greenberries.
Blackberries are multiplied by rooting steam cuttings, root cuttings and tissue culture using in vitro micropropagation (Antunes & Raseira 2004;Villa, et al., 2008;Villa, et al., 2010). For greenberries, vegetative propagation studies indicate that multiplication via cuttings is inefficient, whereas in vitro cultivation requires adjustments and further studies (Bueno, 2015). The in vitro propagation of R. erythroclados was evaluated by Bueno et al. (2018) who found low rooting rates and paralyzed studies, which highlights the necessity of retake work on development of specific protocols for these species. Micropropagation can provide better seedling uniformity and vigor, increased mass production and better health (Dutra, et al., 2010), in addition to allowing molecular biology work to conserve plant genetic resources (Bakhtiar, Mirjalili, & Sonboli, 2016). Success in berry tissue culture involves adjusting the types and concentrations of growth regulators in the culture medium, especially the cytokinin and auxin types (Villa, et al., 2006). The objective of this work was to define an in vitro micropropagation protocol for greenberries in order to contribute to the domestication of R. brasiliensis and R. erythroclados.

Methodology
This study was based on the quantitative research method (Pereira, et al., 2018) and was developed at the EPAGRI Lages Biotechnology Laboratory, based on the in vitro culture methodology described by George (1993). The vegetative material used in this study came from parent plants of Rubus brasiliensis and R. erythroclados kept in a greenhouse (26 ± 2° C) with intermittent irrigation ( Figure 1A). The explants, consisting of nodal segments containing an axillary bud, without leaf and approximately 3 cm, were collected and immediately immersed in water. Asepsis was performed in a laminar flow chamber with 70 % alcohol for 1 minute, followed by 30 minutes in 2 % sodium hypochlorite with drops of Tween® 20 and under constant stirring. After triple washings in water, the nodal segments were sectioned at 1 cm and transferred to paper bridges in test tubes containing 5 ml of liquid culture medium ( Figure 1B). The culture medium consisted of MS medium (Murashige & Skoog, 1962) supplemented with 1.0 mg L -1 benzylaminopurine (BAP), 0.125 mg L -1 salicylic acid (AAS), 0.25 mg L -1 gibberellic acid (GA3), 30 g L -1 sucrose, 0.30 g L -1 of reduced glutathione, pH 5.8. The segments remained in the dark for 72 hours in a growth room and afterwards a 16h photoperiod (24 ± 2° C, 50 µmol m 2 ). After 40 days, cultures were subcultured in test tubes containing basal medium (MS, 30 g L -1 of sucrose, 0.25 mg L -1 GA3, 2.6 g of phytagel and pH 5.8) supplemented with 1.0 mg L -1 of BAP. Source: Authors.
The multiplication experiment was carried out in culture flasks containing 30 ml of basal medium in concentrations of BAP at 1 mg L -1 ; 2 mg L -1 and 3 mg L -1 , which constituted the treatments. After 60 days, survival and multiplication rates ( Figure   1C) were analyzed. The rooting experiment was carried out in culture flasks containing 30 ml of MS medium with half the concentration of salts (½ MS), supplemented with 20 g L -1 of sucrose, 2.6 g L -1 of Phytagel and pH adjusted to 5.8. Treatments were established with addition of 0.1 mg L -1 of a-naphthalene acetic acid (NAA) or 1.0 mg L -1 of indolbutyric acid (IBA) to the medium without growth regulator. After 38 days, survival rates, rooting, length and number of roots were analyzed ( Figure 1D). The experiments were carried out independently for each species of greenberries (R. brasiliensis and R. erythroclados), in a randomized block design, with three replicates of 25 replicates each. The results were submitted to analysis of variance and test of separation of means (Tukey, p <0.05) using the R environment (R Core Team, 2015).

Results and Discussion
The explant survival of in vitro cultures of greenberries did not differ between the tested BAP concentrations (Table 1).
The highest rate of viability of explants in R. brasiliensis was obtained using 1.0 mg L -1 of BAP. In the species R. erythroclados, the treatments did not differ from each other (Table 1). ns: not significant; averages followed by the same letter do not differ (Tukey, p≤0,05). Source: Authors.
The rate of in vitro rooting obtained in this work for the species R. brasiliensis was higher than that obtained by Bueno (2015), which was 16 % and 5 % of rooting using stem and root cuttings, respectively.
The present results point to 75 % survival rates for greenberries on acclimatization when rooted using NAA and ½ MS and 86 % when rooted with IBA. The facility on acclimatization of blackberries was described by Toledo & Biasi (2018) in work with cultivar Xavante.

Conclusion
Greenberries of the species Rubus brasiliensis and Rubus erythroclados can be propagated through in vitro micropropagation using MS medium with addition of 1.0 mg L -1 of BAP and rooted using ½MS culture medium plus 1.0 mg L -1 IBA. It is necessary to improve the in vitro propagation protocol of Greenberries, especially by testing different concentrations of hormones in the culture medium. Thus, this protocol can contribute to the domestication process and the consequent preservation of these species forgotten until now.