Protocol for in vitro rooting ofPyruscomunnisrootstocks

ABSTRACT: Effective protocols for in vitro rooting for woody fruit trees are still a challenge for in vitro seedling production, especially when there is a need to insert new cultivars or rootstocks. These protocols are essential to accelerate studies in plant breeding programs and for seedling distribution. This study evaluated the use of 6-Benzylaminopurine (IBA) in in vitro rooting of Pyruscomunnis rootstocks, clones ‘OHxF87’ and Pyrodwarf. Explant exposure times (0, 24, 48, 72, and 96 hours) to 20 mg L-1 IBA were tested for in vitro rooting. The exposure to IBA resulted in rooting rates above 80%, surpassing some results reported in the literature. The 24-hour treatment provided 81,81% survival, leading to an average growth of five roots with 19 mm length, for ‘OHxF87’ rootstock. The same exposure time resulted in the highest survival rate (75%) and the highest mean root number, seven roots per plant with 10 mm length, for ‘PDW’ rootstock. Root formation did not occur in the absence of synthetic auxin. Therefore, it can be concluded that a 24-hour exposure at 20 mg L-1 IBA was sufficient to promote in vitro rooting in ‘OHxF87’ and Pyrodwarf rootstocks’.

Pear is among the temperate climate fruits best accepted by the domestic consumer market, being one of the main fruits imported by Brazil.One of the limitations in the cultivation of this fruit tree in Brazil is the lack of genetic material (RUFATO et al., 2012).In this sense, new materials have been studied in Brazilian plant breeding programs, for example, rootstocks 'OHxF87' and Pyrodwarf ('PDW), promising in high density plantations.
The series of clones OHxF (Old Home x Farmingdale) originates from Pyrus communis, helps in the precocity, yield and quality of some European pear cultivars (ERCISLI et al., 2006).'OHxF87' clone is one of the best in the series, of semi-dwarf size and compatible with most European and Asian pear varieties (APAL, 2019)  Effective protocols for in vitro rooting for woody fruit trees are still a challenge for in vitro seedling production, especially when there is a need to insert new cultivars or rootstocks.These protocols are essential to accelerate studies in plant breeding programs and for seedling distribution.Concentrations of this plant growth regulator in the forms of indole-3-acetic acid (AIA), indole-3-butyric acid (IBA), naphthalene-acetic acid (NAA), have guaranteed success in rooting cultivars and genotypes of different pear species, such as P. communis, P. pyrifolia, P. calleryana, P. amygdaliformis, P. pyraster, P. syriaca, P. betulifolia, and P. bretschneideri (BELL & REED, 2002).
The results reported in the recent literature demonstrated that, for different Pyrus species, in vitro rooting does not occur without the use of synthetic hormonal regulators, and rooting efficiency is dependent on genotype.Therefore, this study evaluated the 6-Benzylaminopurine (IBA) in vitro rooting of P. comunnis rootstocks, 'OHxF87' and 'PDW' clones.
The plant material was established in QL medium (LEBLAy et al., 1991) at the Laboratory of Plant Micropropagation at Santa Catarina State University.For the multiplication protocol, MS culture medium was used (MURASHIGE & SKOOG, 1962), containing 30 mg L -1 of sucrose and 5.5 mg L -1 of agar, supplemented with 1.5 mg L -1 BAP and 0.1 mg L -1 IBA, pH adjusted to 5.8.A total of 20 mL of medium solution was added to the test tubes, which were kept in a growth room at 24 °C with a 16-hour photoperiod (40 -56 μmol m -2 s -1 ) for 45 days.
For rooting, the exposure time of the explants to IBA was tested.For this purpose, 20 mg L -1 of IBA were used at 0, 24, 48, 72, and 96 hours.A completely randomized design was used, with 5 treatments, containing 12 replications for each Pyrus comunnis rootstock ('OHxF87' and Pyrodwarf).After the application of the treatments, the plants were exchanged from the tubes and cultivated in the culture medium; growth conditions were described for the multiplication protocol.After 45 days, the percentage of survival and rooting, the number of leaves and roots, shoot length and longest root length were evaluated.
The Shapiro-Wilk test was performed to assess data normality at a significance level of 5%.ANOVA was applied to the data that exhibited a normal distribution, comparing the means by the Scott-Knott test (5%).For data that did not have a normal distribution, the Kruskal-Wallis test was used, and the results were compared using the Nemenyi test.The software R was used for statistical analyses.
The 24-hour treatment provided 81,81% survival for 'OHxF87' rootstock.The same exposure time resulted in the highest survival rate (75%) for 'PDW' rootstock.Root formation did not occur in the absence of synthetic auxin (Figure 1).
Exposure time did not influence shoot length or the number of leaves in 'OHxF87' clone.The 24-, 72-and 96-hour treatments did not differ for length of the longest root.For the number of roots, there was no difference between treatments (Figure 1).
The 72-hour treatment led to the greatest growth in shoot length, differing only from the absence of IBA in 'PDW' clone.Exposure time did not influence number of leaves.Treatments 24 and 72 hours showed greater lengths of the longest root and number of roots, differing only from that with the absence of IBA (Figure 1).
In the literature, the protocols used for rooting selections of P. communis differ in terms of culture medium, type and concentration of plant growth regulator.For the selections 'OHxF' QL medium modified by Leblay yields satisfactory results in all stages of in vitro culture (SILVA et al., 2018).QL, ½MS and MS media for 'PDW' clone (LIZÁRRAGA et al., 2017;RUŽIĆ et al., 2011;SILVA et al., 2018).The forms and concentrations of IAA, IBA and naphthalene-acetic acid (NAA) ensure the rooting of cultivars and genotypes of different pear species (AyGUN & DUMANOGLU, 2015;BELL & REED, 2002;yANG et al., 2017), but with a very variable rooting rate, depending on the concentration of these plant growth regulators.
These results showed that the culture medium does not interfere with the rooting of these clones, but with the concentration of auxins.Auxins induce the formation of embryos from somatic cells, contributing to the formation and maintenance of the root apical meristem (TAIZ et al., 2017).In this study, exposure to IBA resulted in rooting rates above 80%, surpassing some results reported in the literature and confirming that in vitro rooting of clones occurs only in the presence of some type of treatment with a hormonal stimulus.Therefore, it can be concluded that an exposure time of 24 hours at 20 mg L -1 IBA was sufficient to promote 'OHxF87' and Pyrodwarf rootstocks' in vitro rooting.

ACKNOWLEDGMENTS
This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES), finance code 001, Conselho Nacional de Desenvolvimento Científico e Tecnológico -Brasil (CNPq), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).The authors acknowledge the assistance of Laboratory Technician Vantuil Antônio Rodrigues and anonymous referees for their comments and constructive suggestions for improving the quality of the manuscript

Figure 1 -
Figure 1 -Boxplot and statistical test of the rooting of 'OHxF87' and 'PDW' rootstocks exposed to synthetic auxin (IBA) for different times for 45 days of in vitro cultivation.Variable: S -percentage of plant survival; R-percentage of rooting; LAP -shoot length, in mm; NL -number of leaves; LLR -length of longest root, in mm; NR -number of roots.Treatment: Exposure times of 0, 24, 48, 72, and 96 hours.Statistics: SK -means followed by the same letter do not differ by the Scott-Knott test at 5% significance; KN -means followed by the same letter do not differ by the Kruskal-Wallis-Nemenyi test at 5% significance.
Nadal et al. good compatibility with European and Asian pear varieties, in addition to low susceptibility to iron chlorosis (WSU, 2019).