Use of micropropagation in the vegetative rescue of adult trees of Cedrela odorata L.

: The objective of this work was the vegetative rescue of Cedrela odorata L. adult trees from forest areas by micropropagation using shoot regeneration from axillary buds in vitro . Nodal segments (0.5 and 1.0 cm) of shoots formed from cuttings taken from canopy sprouts were used. The explants were successfully established in vitro on MS medium supplemented with 2.22 μM BAP, 0.49 μM AIB, 0.28 μM GA3 and PPM (0 and 8.6 μM). Shoot multiplication on MS with combinations of GA3 (0.28 and 1.12 μM) and BAP (2.2, 3.3, 4.4, 6.6, and 8.8 μM) and shoot rooting on medium with 4.9 μM AIB, 5.7 μM AIA or 5.37 μM ANA were evaluated. The MS medium supplemented with 0.28 μM GA3 and 8.8 μM BAP generated 4.16 shoots per explant. The best rooting induction was observed on medium containing 4.9 μM IBA, resulting in 60% of shoot rooting. The plantlets rooted were acclimatized and showed normal development with 97% survival rate. The use of canopy sprouts as explants is feasible in the rescue of C. odorata and the combination of BAP and GA3 favors in vitro multiplication.


Introduction
Cedrela odorata L. Meliaceae is distributed in subtropical, tropical, and seasonal forests extending from Mexico to Argentina (Cervi et al., 2008). Due to the economic importance of the wood of C. odorata, the extractive exploitation, which added to the low regeneration capacity, has taken the species to the category of endangered (Brasil, 2014). Another limiting factor to the establishment of commercial C. odorata forests is the presence of the insect Hypsipyla grandella (Zeller, 1848) (Lepidoptera: Pyralidae) which damages the tree's growing points, compromising the upright development of the stem as well as the development and growth of young trees (Castro et al., 2018).
In general, the propagation of cedar plants is done through seeds (Oliveira et al., 2013). However, this form of propagation is dependent on seed viability that is influenced by the seasonality of fruit dispersal and the low quality of diversity of productive matrices (Lesher-Gordillo et al., 2018). The C. odorata species presents allogamous pollination with great genetic diversity among the remaining populations (Martins et al., 2008). Propagation by woody cuttings has been studied, but presents low rooting capacity (Peña-Ramírez et al., 2010). The rescue of forest species by vegetative propagation can be influenced by the ontogenetic age of the tissue, and various techniques are applied to rejuvenate and/or invigorate plants (Stuepp et al., 2018). According to Alvim et al. (2020), in forest species with low propagative rate by seeds, micropropagation enables in vitro conservation of invigorated material, mass multiplication and the selection of genetically superior matrices.
Among the main factors that limit the in vitro regeneration of some species is the explant source, definition of the appropriate culture medium and concentration of growth regulators (Bidabadi & Jain, 2020). Micropropagation is carried out through the stages of establishment, selection of explants and disinfestation, multiplication, rooting, and acclimatization (Cançado et al., 2013). Other limitations in establishment are the high rate of contamination by unwanted microorganisms and the oxidation of explants, which is quite common in the micropropagation of forest species (Salles et al., 2017).
The micropropagation of C. odorata showed satisfactory results using explants from seedlings originated from the in vitro germination of seeds (Pérez et al., 2002;Valverde-Cerdas et al., 2008). The use of sprouts from C. odorata cuttings has enabled the rescue and rooting of explants (García-Gonzáles et al., 2011). The in vitro establishment of woody species explants is one of the main stages for the success of micropropagation, since it involves overcoming oxidation and fungal and bacterial contamination (Almeida et al., 2020). Moreover, the definition of adequate concentrations of growth regulators is determinant in the in vitro multiplication phase of C. odorata (Peña-Ramírez et al., 2010).
Thus, this research aimed to evaluate the vegetative rescue of adult trees of C. odorata L. by micropropagation using nodal segments from budded cuttings collected from crown branches.

Induction of bud sprouts from cuttings and production of explants
The research was conducted at the Laboratório de Biotecnologia of the Empresa de Pesquisa e Extensão Rural do Estado de Santa Catarina -EPAGRI, Lages, Santa Catarina, Brazil. Branches were taken from the crown of three matrix plants of C. odorata with 15-20 years of age and DBH of 10-17.8 cm, located in an area of natural regeneration of Mixed Ombrophylous Forest (27°42'55.52" S, 50°30'24.29" O, 876 m altitude), municipality of São José do Cerrito, state of Santa Catarina, Brazil.
The species identification was carried out with the help of literature and confirmed by botanical experts. Exsiccata were deposited in the Herbário Lages of the Universidade do Estado de Santa Catarina (LUSC). The branches were fractioned into 15 cm cuttings, and kept in plastic trays containing carbonized rice husk, in a controlled environment at 24±2 ºC, relative humidity between 67-71% and 8 h photoperiod.
In vitro establishment treatments consisted of a combination of nodal segment size (0.5 or 1.0 cm) and use of PPM® (0 or 8.6 μM). The explants were kept in a completely randomized design with 25 repetitions per treatment, with one tube per plot, each tube containing one explant. After 30 days of in vitro establishment, survival rates (%), regeneration percentage (shoots longer than 1 mm and presence of leaves), and oxidation rates (%), fungal (%) and bacterial (%) contaminations were evaluated.

In vitro multiplication
In vitro regenerated shoots were subcultured and provided material for the in vitro multiplication experiment.

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In culture medium containing MS salts, combinations of five concentrations of BAP (2.2, 3.3, 4.4, 6.6, and 8.8 μM) and two concentrations of GA3 (0.28 and 1.12 μM). The culture media were supplemented with 30 g L-1 sucrose and gelled with 8 g L-1 agar, and the pH was adjusted to 5.8. The experiment was conducted in a randomized block design, factorial arrangement 2 x 5 (two doses of GA3 x five doses of BAP), with four repetitions of 16 explants per treatment. After 30 days of cultivation, the multiplication rate (%), number of sprouts per explant, length of sprouts (mm) and oxidation rate (%) were evaluated.

Rooting and acclimatization
The sprouts from the multiplication experiment were used for rooting using MS medium containing 30 g L-1 sucrose, 8 g L-1 agar and pH 5.8 plus three auxin-type growth regulators: 4.9 μM of indolbutyric acid (AIB); 5.7 μM indol-3-acetic acid (AIA); 5.37 μM of naphthaleneacetic acid (ANA). Basal medium without a regulator was the control. The treatments were defined based on previous studies (Pérez et al., 2002;Valverde-Cerdas et al., 2008;García-Gonzáles et al., 2011). The experiment was in a randomized block design with four replications of 16 explants. After 30 days, rooting rate (%), number of roots and root length (cm) were evaluated.
Cultures with and without root formation were acclimated ex vitro in expanded polypropylene honeycombed trays containing Organo Plus® commercial substrate, sand and carbonized rice husk (1:1:1, v/v/v), kept inside a 25 L polyethylene plastic tray and covered with transparent plastic film to form a humid chamber. The seedlings were irrigated with 250 mL of water every 3 days, using a sprayer. The trays were kept in a growth room at 25±3 ºC, relative humidity 80%, 16h photoperiod. Rooted and unrooted seedlings constituted the treatments and were conducted in a completely randomized design with 40 replicates per treatment. After 30 days survival (%) was evaluated.

Statistical analysis
For the in vitro multiplication and rooting experiments, the data were submitted to analysis of variance after verification of normality (Shapiro-Wilk) and homogeneity (Bartlett). In cases where the model assumptions were not met, Box-Cox transformation was performed. To evaluate the establishment and acclimatization experiments, generalized linear models (GLM) were used, since the variables presented a binomial probability distribution. Differences between the means of the treatments were compared by the Tukey test (p ≤ 0.05). All analyses were conducted in the R environment (R Core Team, 2018).

Induction and regeneration of shoots in vitro
Regeneration of shoots under in vitro conditions was possible using material rescued from C. odorata matrices. The treatments of explant size and PPM® concentration analyzed in the experiment showed no differences between the variables survival rates (p = 0.3508), fungal contamination (p = 0.0404) and oxidation (p = 0.0446) ( Table 1). The use of fungicides in asepsis of explants can be toxic, increasing the mortality of explants; García-Gonzáles et al. (2011) obtained in C. odorata disinfection of 100% of explants and establishment of 60% of explants from cuttings sprouts, using in asepsis Propiconazole CE 25 5% for 3 minutes. The use of PPM® in the medium showed no effect in suppressing fungal contamination. However, Silveira et al. (2016) showed that using 0.4 and 0.8% PPM in WPM culture medium eliminated bacterial contamination and the 0.8% dose reduced fungal contamination to 2% in nodal segments obtained from 1-2 year old seedlings of Calophyllum brasiliense.
Nodal segments with the size of 1.0 cm without addition of PPM® to the culture medium showed higher regeneration values than nodal segments of 0.5 cm (p = 0.0012). According to Moura et al. (2012), larger nodal segments have relatively larger reserves of hormones and nutrients, providing better development of explants. However, Robert et al. (2020), in vegetative rescue of C. odorata using nodal segments obtained from grafted material observed higher regeneration compared to the use of cuttings buds under in vitro conditions.

In vitro multiplication
The multiplication rate (p = 0.0492), number of sprouts (p < 0.001) and explant length (p = 0.0016) were regulated by the interaction of BAP and GA3 concentrations in the culture medium ( Table 2)
BAP is a cytokinin-type growth regulator, inducer of cell division and widely used in the multiplication phase of C. odorata (Pérez et al., 2002;Valverde-Cerdas et al., 2008). In the in vitro multiplication phase, this growth regulator promotes the development of the aerial part and the number of shoots that capture nutrient reserves from the culture medium, causing a decrease in shoot size in in vitro plants (Almeida et al., 2020). The adequate concentration of GA3 combined with BAP in the in vitro multiplication phase of C. odorata interfered with the number of shoots per explant. This growth regulator is important in cell elongation and promoted the increase of Cordia trichotoma sprouts (Mantovani et al., 2001).
The results of the number of shoots obtained per explant were higher than those found in C. odorata multiplication by Pérez et al. (2002). These authors used seedling material and cultivated the species in culture medium supplemented with BAP, KIN and 2-iP, and found the best result (4.06 sprouts per explant) with 9.76 μM of BAP. On the other hand, in Table 2 In the media containing 0.28 μM GA3 under different doses of BAP no oxidation of the explants was observed. The media with 1.12 μM GA3 at the concentration of 4.4 μM, 6.6 μM and 8.8 μM of BAP showed 13, 62, and 65% oxidized explants, respectively ( Table 2). The oxidation observed can be attributed to regulator levels or phenolic oxidation. High concentrations of growth regulators can cause intoxication and oxidation of explants, indicative of inadequate concentrations (Almeida et al., 2020), as well as modify or inhibit explant growth . The browning resulting from phenol oxidation is due to polyphenol oxidase activity, which results in the production of quinones, which are melanic compounds that contribute to browning and death of explants (Huh et al., 2017). Table 2. Multiplication rate (%), number of shoots per explant, shoot length (mm) and oxidation (%) of C. odorata explants multiplied in vitro under different concentrations of 6-benzylaminopurine (BAP) added to doses of gibberellic acid (GA3). * Not evaluated due to the absence of oxidation. Averages followed by the same lower case letters in the columns and upper case letters in the rows are not significantly different by Tukey test (p ≥ 0.05). CV (%): Coefficient of variation.

Root induction and acclimatization
In the rooting induction phase, callogenesis was observed in explants grown on medium with the auxins AIB and ANA (Figure 1). Callogenesis in some forest species precedes rooting 5/7 (Navroski et al., 2015). Plants have the ability to regenerate their tissues, this can occur by cell dedifferentiation, which under the action of phytohormones generates a mass of cell proliferation, called callus (Fehér, 2019).
The first adventitious roots of C. odorata sprouts appeared in the culture medium containing AIB after 15 days from the start of rooting induction. The culture medium plus 4.90 μM of AIB showed rooting rate of 60% of explants and root length with 2.76 cm, being different from the medium containing 5.37 μM of ANA, treatment that reached 26% rooting. Explants maintained on medium with 5.70 μM EIA and control did not show rooting in the experiment (Table 3). In the ex vitro acclimatization, the seedlings that formed roots had a survival percentage of 97%, while in the sprouts without roots the survival was only 24%, demonstrating the need for rooting phase in the formation of seedlings and success in obtaining micropropagated and acclimatized seedlings ( Table 4).
The use of AIB is indicated in the in vitro rooting of C. odorata, as proven by Rodríguez et al. (2003), who using MS culture medium plus 4.4 μM AIB, obtained 4.18 roots per explant with 3.9 cm in length. Valverde-Cerdas et al. (2008), using 9.8 μM of AIB obtained 6.5 roots per explant. The work of García-Gonzáles et al. (2011) demonstrated that using MS culture medium supplemented with 8.8 μM of BAP and 16.1 μM of NAA obtained the formation of 3.9 roots per plant after six weeks.
The low number and length of C. odorata roots found in our study, compared to other studies, may be related to the evaluation time and the residual effect of GA3 and BAP from the multiplication phase. Using MS culture medium, with activated charcoal and combinations of 2.2 μM of BAP and 2.68 μM of NAA, Huamán et al. (2012) obtained, after five weeks of C. odorata culture, 100% rooting, 8.13 roots per explant and 10.43 cm in length. Activated carbon can be used in the composition of the rooting medium, for its capacity to absorb inhibiting and toxic residual substances from the previous phase of micropropagation, promoting better rooting rates .
The interaction between the concentrations of growth regulators BAP and GA3 may serve as an indication for future evaluation of new protocols for micropropagation of C. odorata, considering the genetic diversity and the importance of the species, and thus may contribute to increase the rate of multiplication and rooting of explants, as well as the rescue and production of seedlings from selected matrices.

Conclusions
The use of explants from nodal segments formed from cuttings taken from crown branches used in micropropagation is feasible in the vegetative rescue of adult C. odorata trees, enabling the selection and multiplication of superior genotypes.
The use of nodal segments 1 cm long presents a higher percentage of in vitro regeneration.
Association of 8.8 μM of BAP and 0.28 μM of GA3 in culture medium with MS promoted production of 4.16 shoots per explant after 30 days of culture.
The explants maintained in medium with the addition of 4.9 μM AIB showed 60% rooting. The rooted and acclimatized seedlings showed 97% survival. Table 4. Survival (%) of acclimatized explants with and without C. odorata root. * Not evaluated due to lack of rooting. Means followed by the same letter in the column do not differ significantly by Tukey test (p < 0.05). ns: Treatments were not significantly different by Tukey test (p ≤ 0.05). CV (%): Coefficient of variation. Table 3. Rooting rate (%), number of roots and root length (cm) of C. odorata under different auxin concentrations.