Propagation from axillary buds and anatomical study of adventitious roots of Pinus taeda L .

Micropropagation of Pinus taeda by axillary bud proliferation technique and anatomical study of adventitious roots were studied. Apical and basal segments were established in vitro in Murashige and Skoog (MS) culture medium. For multiple shoot induction, the explants were transferred to WV3 medium with or without 6-benzylaminopurine (BAP) (0.12, 0.25 or 0.50 μM). The effect of the culture medium and combinations of α-naphthalene acetic acid (NAA) with BAP on adventitious rooting were evaluated. The basal segments exhibited higher multiplication rates (3.7 shoots per explant) than apical segments during establishment of in vitro culture. The best rooting rate (40%) was achieved by induction of roots in water-agar (WA) culture medium supplemented with 2.69 μM NAA and 0.44 μM BAP, for 9 days, followed by transfer into GDm/2 medium. Adventitious roots can be derived from the cambium cells, from adventitious meristem, or from a callus developed at the base of the shoots. When formed indirectly, roots can be derived from peripheral cells of the callus. In treatment resulting in the best rooting rate, 58.3% of the roots were formed directly. The callus formation at stem basis did not prevent rooting and did not impair acclimatization. The plantlets were successfully acclimatized with 90% survival.


INTRODUCTION
Pinus taeda L. is an important forest species because of the productivity and quality of its timber, being used primarily at sawmills, rolling mills, manufacturing of particle board, MDF, construction, furniture, crates, pulp and paper (Schultz, 1997).In Brazil, in addition to Eucalyptus, Pinus species provides raw material for products coming from forests, thus reducing the exploitation of native species such as Araucaria angustifolia (Selle et al., 1994;Souza et al., 2008).
Research on vegetative propagation of Pinus is therefore of great importance and has been applied to improve the quality of its wood and plantation.However, it is frequently still propagated from seed, since the rooting of cuttings depends on the season of the year or on the availability of juvenile material (Schestibratov et al., 2003;Alcantara et al., 2007;Cuesta et al., 2008;Andrejow and Higa, 2009).The micropropagation techniques became a necessary tool for large-scale production of P. taeda plants and serve as strategies for tree improvement (Gupta and Durzan, 1991;Govil and Gupta, 1997;Menzies and Aimers-Halliday, 1997).
Research on P. taeda is related to direct (Mehra-Palta et al., 1978;Mott and Amerson, 1981;Jang and Tainter, 1991;Frampton et al., 1998;Tang and Guo, 2001) and indirect organogenesis (Tang et al., 1998;Tang and Ouyang, 1999;Tang, 2000).Given the limitations of clonal propagation during the adult phase of trees, most studies used juvenile material as a source of explant, such as various parts of the zygotic embryo, cotyledons, epicotyls and apical meristems.Moreover, in techniques involving a callus phase, such as indirect organogenesis and embryogenesis, the occurrence of somaclonal variation and phenotypic alterations is common, which does not occur in direct organogenesis (DeVerno et al., 1999;Tang, 2000;Cuesta et al., 2008).Pinus species have pre-existing axillary buds at the base of their needles that can be induced to develop into shoots.Micropropagation via axillary buds has the advantage of not involving callus formation and avoiding the use of high concentrations of plant growth regulators (PGRs), thus promoting higher genetic stability.
Adventitious rooting is an essential step for obtaining the economically efficient production system required by clonal forestry (Foster, 1990;George et al., 2008).For conifers such as in Pinus, it is considered a difficult step and its success may depend on the species, family and clone (Bergmann and Stomp, 1994).
The use of exogenous auxin is usually required for the induction of adventitious roots in difficult-to-root species.For conifers, α-naphthaleneacetic acid (NAA) is the most commonly used, sometimes in combination with cytokinins (Abdullah et al., 1989).Mehra-Palta et al. (1978) achieved higher percentage of rooting (50%) in P. taeda combining 0.54 µM NAA and 0.22 or 0.04 µM BAP in Gresshoff and Doy modified culture medium (GDm), while Mott and Amerson (1981) succeeded in rooting loblolly pine with a combination of 2.69 µM NAA and 0.44 µM BAP added to the same medium.
Depending on the species, the roots may originate from different tissues, either directly or indirectly, which callus can limit rooting and make it difficult transplantation (Hicks, 1987;Apter et al., 1993a, b;Ballester et al., 1999).
In Pinus species, the callogenesis at the base of explants before the emergence of roots is common (Wagley et al., 1987;Hammann, 1998;Álvarez et al., 2009).When roots emerge from callus, the vascular connection between root and stem is of great importance for the functioning of the vascular system and the survival of plants after transplantation in greenhouse (George et al., 2008).In some Pinus species, the high survival rate can be related to vascular connection (Álvarez et al., 2009).
The purpose of this study was to establish an efficient method for micropropagation of P. taeda by axillary buds, comparing the presence and absence of apical buds, combinations of plant growth regulators to induce adventitious rooting, transplantation and performing anatomical studies in order to ascertain the origin of in vitro-formed roots.

Establishment of in vitro cultures
Two to four-month-old seedlings from a commercial clonal orchard were provided by the Battistella Forest Company located at Rio Negrinho, Santa Catarina, Brazil.The seedlings were kept in a greenhouse for 20 days and treated with 1 g L -1 Cercobin ® (a fungicide) every two days, making a total of 10 sprays.
For establishment of in vitro cultures, apical and basal segment, 3.5 cm long, were collected from the apical portion of the plants, one explant per seedling, and 90% of the needles were cut.The explants were disinfested by immersion in 0.05% (w/v) HgCl 2 for 5 min, followed by 0.5% (v/v) NaOCl for 5 min, under constant agitation.Tween 20 (CRQ ® Cromato Produtos Químicos) 0.1% (v/v) was added to the disinfestation solutions.After disinfestation, the explants were rinsed three times in sterile distilled water.
The explants were inoculated vertically in 15.0 × 2.5 cm test tubes containing 10 mL of MS (Murashige and Skoog, 1962) culture medium supplemented with 30 g L -1 sucrose and solidified with 5.6 g L -1 bacteriological Himedia ® agar.
After 6 weeks of culture, microbial contamination, the necrosis and survival rates, the percentage of explants with shoots and the average number of axillary shoots per explant (final number of shoots/initial number of explants) were evaluated.The experimental design was completely randomized.For each explant type, there were 30 replications of 10 explants per replicate and one explant per test tube.

Shoot multiplication
For induction of multiple shoots, apical and basal segments established in vitro were transferred to flasks of 12.5 × 6.2 cm, containing 40 mL of WV3 (Coke, 1996) culture medium, supplemented with BAP (0, 0.12, 0.25 or 0.50 µM), 30 g L -1 sucrose and 5.6 g L -1 bacteriological Himedia ® agar.Apical segments longer than 4 cm were subdivided into two segments, with the upper segment containing the apical bud and the lower segment being devoid of apical buds.Two subcultures were performed on the same culture medium with the same concentrations of BAP.The culture period was four weeks for the initial culture and eight weeks for the two subsequent subcultures.At the end of each subculture, the following parameters were evaluated: percentage of explants with shoots, average number of axillary shoots per explant (final number of shoots / initial number of explants) and percentage of elongation rate.
The experimental design was completely randomized, in split plots with three explants per flask, five flasks per replicate and five replicates per treatment.Four BAP concentrations were compared in the plots and three subcultures in the subplots.The data were analyzed separately for each type of explant.

Adventitious rooting
For adventitious rooting, individual shoots were inoculated into plant growth regulator (PGR)-free WV5 medium supplemented with 2 g L - 1 activated charcoal 20 days before rooting induction.Then, shoots (1.5 to 2.0 cm high) with a double bevel cut at the base were inoculated into GDm/2 medium (Gresshoff and Doy modified by Mehra-Palta et al., 1978) with half-strength salts.The control was a PGRfree medium and the treatments were: 0.54 µM NAA and 0.04 µM BAP, 0.54 µM NAA and 0.22 µM BAP, 0.54 µM NAA and 0.44 µM BAP, 2.69 µM NAA and 0.44 µM BAP.We also used a medium composed of water and agar (WA) supplemented with 2.69 µM NAA and 0.44 µM BAP.In the two later treatments, an induction period of 9 days was tested, followed by transfer of the shoots into PGR-free GDm/2.In the other treatments, the shoots remained in the same culture medium for ten weeks.The GDm/2 medium contained 20 g L -1 sucrose and all media were solidified with 5.6 g L -1 bacteriological Himedia ® agar.
The experimental design was completely randomized, with four explants in each flask, two flasks per replicate and six replicates per treatment.After 10 weeks of culture, the following parameters were evaluated: percentage of rooted shoots, average number of roots per shoot and the percentage of roots developed from a callus or directly from the stem.

Transplanting and acclimatization
Plants 1.5 to 2.5 cm high and with roots longer than 0.6 cm were used for transplanting and acclimatization.The roots were washed in running water and plantlets planted in Plantmax Forests ® substrate in plastic bags of 600 cm³, one plant per bag, for 60 days.The plants were manually irrigated 4 to 5 times daily during the first 20 days.After 90 days, plants were evaluated for the percentage of survival and average number of roots per plant.

Culture conditions
All media had their pH adjusted to 5.8 with NaOH 0.1 N or HCl 0.1 N and were autoclaved for 20 min at 121°C.In vitro cultures were maintained in a growth room with temperatures of 19 ± 2°C (night) and 28 ± 2°C (day), a 16-h photoperiod under white fluorescent light (PFD = 40 μmolm -2 s -1 ).For transplantation and acclimatization, the plants were maintained in a greenhouse under shade, with temperatures ranging from 25 ± 7°C.
The data were submitted to Bartlett's test and analysis of variance (ANOVA) and means compared by Tukey's test (P≤0.05)using the statistical software MSTAT-C ® (Department of Crop and Soil Sciences, Michigan State University).

Anatomical study of adventitious roots
The anatomical study was realized according to de Oliveira et al. (2012).Samples were collected from shoots with roots approxi- de Oliveira et al. 5415 mately 0.2 cm long, developed directly or indirectly from stem basis.The material was fixed in FAA (Berlyn and Miksche, 1976), dehydrated in alcohol series, then infiltrated and embedded in Historesin ® following the manufacturer's instructions (Leica Microsystems, Germany).Longitudinal series sections (7 µm thick) were performed on a rotation microtome RM2145 (Leica Microsystems, Germany) and stained with toluidine blue (O'Brien et al., 1965).The characteristics of the material were recorded on digital equipment connected to a Zeiss microscope ® .

RESULTS AND DISCUSSION
Explants of 2-to 4-month-old seedlings (Figure 1a) were used, because in preliminary tests, the older explants (6month-old) showed slower responses and higher microbial contamination.For conifers and woody plants in general, juvenile explants have shown better responses in vitro (El-Nil, 1982;Lapp et al., 1996;Bonga et al., 2010) and better responses to rooting in mini-cuttings (Majada et al., 2011).Disinfestation treatment allowed a survival of around 60% of the plantlets, with no influence of the explant type.The percentage of necrosis was low for both apical and basal segments (Table 1).
The explant type influenced the formation of axillary shoots.Basal segments showed a higher percentage of explants forming axillary shoots (90 to 100%) and average number of shoots per explant (3.7), compared to apical segments (Table 1; Figure 1b and c).The presence of the apical bud can induce a greater elongation of the shoots, while its absence enables axillary shoots to develop, as observed for some species of Pinus (McKellar et al., 1994;Scaltsoyiannes et al., 1994).A similar response was obtained for P. brutia × P. halepensis, probably due to overcoming apical dominance when the apical bud was removed (Scaltsoyiannes et al., 1994;George et al., 2008).McKellar et al. (1994) also observed better results in basal segments than in apical segments of P. patula, comparing the average number of shoots in both explant types.
In the induction of multiple shoots, there was no interaction between the number of subcultures and the concentrations of BAP tested.However, the number of subcultures influenced the induction of axillary shoots.In the second subculture, basal segments showed a significant decrease in the percentage of explants with axillary shoots, from 98.4% to 41.2% (Table 2) and in the average number of shoots per explant (from 1.4 to 1.2) (Table 3).According to the study of George (1996), in woody plants, it is common to note a decrease in the rate of multiplication during the subcultures, after reaching a maximum value.Apical segments inoculated into culture medium containing BAP showed a higher percentage of explants forming shoots than in the control (Figure 1d), while there was no significant differences between treatments for basal segments (Table 2).
The explant type initially used influenced the response of multiple shoot induction during the subcultures.Unlike the results of establishment of in vitro culture, the average number of shoots per explant from basal segments was not superior to the number obtained with explants from apical segments, with approximately 1.2 shoots per explant being obtained at the end of the two subcultures (Table 3).Although the overcoming apical dominance has led to the formation of up to three axillary shoots per explant during establishment of in vitro culture, at the multiplication step some of these axillary shoots did not elongate or could not be individualized, maintaining the same multiplication rate.Axillary shoots developed along the whole axis of the explant; however, in apical segments, they developed preferably in the middle region and more frequently in the basal region of the segment.This effect may be related to a gradient of juvenility starting near the basal region of the explant (Figure 1d), as it occurs in mature trees or in seedlings (Hartmann et al., 2002;George et al., 2008).In this study, the highest average numbers of axillary shoots per explant (2 to 3) were obtained in apical segments (Figure 1d), with no difference between the control and explants cultured on media containing BAP (0.12, 0.25 and 0.50 µM) (Table 3).A similar result was obtained with P. lambertiana, after 8 weeks in DCR culture medium supplemented with a higher concentration of BAP (2.22 µM) (Gupta and Durzan, 1985).
These results indicate that induction of axillary shoots is feasible on culture medium containing low concentrations of BAP or without BAP.This second alternative is interesting for a commercial protocol because it reduces the cost of the medium.However, the juvenile shoots  Values are means ± SD, n = 5.Means within a column followed by the same lower case letter and means within a line followed by the same upper case letter do not differ significantly according to Tukey's test (P ≤ 0.05).Means ± SD, n = 5.Means within a column followed by the same lower case letter and means within a line followed by the same upper case letter do not differ significantly according to Tukey's test (P ≤ 0.05).
of P. taeda must have an endogenous level of cytokinins sufficient to promote induction.Addition of high concentrations of cytokinins to the culture medium can cause necrosis or even a low rate of development of axillary shoots, as was observed by Lapp et al. (1996) in P. monticola.According to the study of Baxter et al. (1989) for P. oocarpa, P. caribaea and P. tecunumanii, there was multiplication of axillary buds even in low concentrations of cytokinins (0.1 to 5.0 µM BAP) and levels above 1 µM were not recommended, thus avoiding high concentrations such as those used in other micropropagation techniques such as organogenesis.
In general, the low concentrations of BAP tested in this study did not influence elongation (Table 4).The high con-  *Period of induction of 9 days followed by transfer to GDm/2 culture PGR-free medium.Means ± SD, n = 6.Means in the column followed by different letters are different according to Tukey's test (P ≤ 0.05).
concentrations of cytokinins can promote high development of buds, but inhibit shoot elongation (Žel et al., 1988).The highest rooting rate (40%) was observed in WA culture medium supplemented with 2.69 µM NAA and 0.44 µM BAP for 9 days, followed by transfer into GDm/2 medium for 10 weeks, although Tukey's test showed that this average did not differ significantly from those obtained with other combinations of plant growth regulators (Table 5).For P. sylvestris root induction, Žel et al. (1988) also recommended a medium composed of water and agar, but in pulse treatment with 53.8 µM NAA for 24h.For P. virginiana, Chang et al. (1991) obtained lower rooting rates (29.2%)than in this study using the same combination of plant growth regulators.
GDm/2 culture medium supplemented with 0.54 µM NAA and 0.04-0.44µM BAP was not efficient for root induction (14-23% rooting) (Table 5).However, Mehra-Palta et al. (1978) obtained 50% rooting with the same combinations in P. taeda.It is possible that these differences were due to the use of different families or different genotypes.In this work the sources of explants were seedlings from a clonal commercial orchard.Some genotypes tend to have better responses than others and rooting of forest species usually presents a great variability among different selected families (Foster, 1990;Scaltsoyiannes et al., 1994;Tang and Ouyang, 1999;Cuesta et al., 2008).
Within four and five weeks of culture, roots emerged at the base of some shoots while other shoots rooted after ten weeks of culture (Figure 3).This rooting rate in this period of time is relatively good when compared to other studies with Pinus that showed rooting varying between 40 and 50% in the same period (Cuesta et al., 2008;Stojičić et al., 1999;Watt et al., 1998;Gladfelter and Phillips, 1987;Scaltsoyiannes et al., 1994;Stiff et al., 1989).The mean number of roots per shoot was similar for all treatments, around 1.5 (Table 5).
Adventitious rooting occurred directly from the stem (Figure 1e) or indirectly from the callus (Figure 1f).The roots originated either from the cambium cells (Figure 2a), as observed in Castanea sativa (Ballester et al., 1999), from meristematic cells (Figure 2c and d), or from callus developed at the base of the shoots (Figures 2e and f), which seems to be common in Pinus species, as reported in P. monticola (Stiff et al., 1989), P. pinaster (Alvarez et al., 2009) and P. pinea (Cuesta et al., 2008).
The percentage of roots formed directly from the stem or indirectly from a callus (Figure 1e and f) varied according to rooting treatment (Figure 4).In the most efficient treatment, 58.3% of the roots were formed directly and 41.7% indirectly.This can be due to the duration of auxin treatment, which can promote or inhibit rooting for some species or even induce the development of structures that look swollen or calluses at the shoot base (Gladfelter and Phillips, 1987;Lin et al., 1991).Mott and Amerson When the roots formed directly from the stem, cells with meristematic characteristics were observed (Figure 2c  and d).They have prominent nuclei, dense cytoplasm and Attention: horizontal axis is not formatted properly (numbers).There may have been some compatibility issue in the file.Attached I am sending the file with Word and Excel graphic corrected.Above is the picture correctly.
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thin cell walls.The presence of the root cap and apical meristem is also observed.The vascular connection between root and stem is not present in the initial stage of root formation, but only in most developed roots (de Oliveira et al., 2012).A continuous connection of the vascular system between root and shoot is essential for the survival of micropropagated plants, as reported for P. eldarica (Wagley et al., 1987).Roots formed from cambium cells have vascular connections through tracheids arising from the procambium and cambium (Figure 2a and b), unlike roots formed from peripheral cells of the callus, where the tracheids are formed from the differentiation of parenchyma and cells with a high division rate, as described by Hamann (1998) andde Oliveira et al. (2012) in cuttings and microcuttings of P. taeda, respectively.This differentiation of parenchyma cells in tracheids, also observed in P. pinea hypocotyl (Kalev and Aloni, 1998), may be due to the action of auxin.
When formed indirectly (Figure 2e and f), roots can be derived from peripheral cells of the callus, as observed by de Oliveira et al. (2012) or from cambium cells, after emerging from callus, as described by Hamann (1998).
The micropropagated plants showed a high survival rate, irrespective of root origin, with 90% of transplanted seedlings surviving after 60 days in the greenhouse (Figure 1g).The plants showed good adaptation to acclimatization and the results were better than those found by Leach (1979), where 38% of micropropagated plants of P. taeda died after 5 weeks of acclimatization.
The acclimatized plants showed a satisfying number of roots per plant (4 secondary roots larger than 1.5 cm per plant) (Figure 1h), as observed by de Oliveira et al. (2012).According to the study of Anderson et al. (1992), this result is an essential factor for plant survival during the acclimatization, because plants with fine or little branched roots may not absorb nutrients well.
In this study, it can be concluded that the micropropagation of P. taeda by axillary buds is feasible.The explant type affected the establishment of in vitro cultures and induction of multiple shoots.Use of both explant types is recommended for increasing the multiplication rate.In general, the addition of BAP to culture medium did not affect the shoot induction or elongation responses.For adventitious rooting, the culture of shoots for 9 days in a culture medium composed of water and agar supplemented with 2.69 µM NAA and 0.44 µM BAP is recommended, followed by transfer to GDm/2 culture medium for root development.The callus presence did not inhibit rooting as the formation of the roots can occur from various parts of the callus or directly from the shoot base, and it did not affect acclimatization.

Figure 1 .
Figure 1.Micropropagation of Pinus taeda L. by axillary buds.(a) Two to four-month-old seedlings kept in a greenhouse used as explant source; (b) apical segments and (c) basal segments inoculated into MS culture medium, after 6 weeks with axillary shoots (arrows); (d) apical segment with axillary shoots developed in WV3 culture medium with 0.25 µM BAP at the initial culture; (e) rooted shoots with the root derived directly from the stem (arrow) and (f) rooted shoots with the roots derived from a callus at the base (arrow); (g) micropropagated plantlet after 2 months of acclimatization; (h) plantlet with developed roots after 2 months of acclimatization.Bars a, g and h = 5 cm; b-f = 1 cm.

Figure 2 .
Figure 2. Anatomical study of Pinus taeda L. adventitious root formation in vitro.(a) and (b) root developed in vitro formed from cambium cells with vascular connection; (c) and (d) root formed directly from stem with an adventitious meristem that precedes the root; (e) and (f) roots formed indirectly from a callus.Bars a, c, e and f = 1 mm; b and d = 500 µm.am apical meristem, c callus, m meristematic cells, r root, rc root cap, rp root primordium, s stem, vc vascular connection, x xylem Figure 3.

Figure 3 .
Figure 3.Effect of combinations of NAA and BAP and culture medium on adventitious rooting of Pinus taeda shoots.GDm/2, Gresshoff and Doy modified by Mehra-Palta et al. (1978), with salts reduced by half; WA, water and agar 5.6 g L -1 .*Induction period of 9 days followed by transfer to growth-regulator-free GDm/2 medium.

Figure 4 .
Figure 4. Effect of combinations of NAA and BAP and culture medium on the percentage of roots formed from a basal callus or directly from the stem of Pinus taeda shoots.The explants were inoculated into GDm/2, Gresshoff and Doy modified by Mehra-Palta et al.(1978), with salts reduced by half, except ( 1 ), cultured in WA (water and agar 5.6 g L -1 ).*Induction period of 9 days followed by transfer to growthregulator-free GDm/2 medium.

Table 1 .
Effect of explant type on establishment of in vitro culture and axillary shoot development in Pinus taeda, after 6 weeks of culture.

Table 2 .
Percentage of explants with axillary shoots that developed in Pinus taeda in WV3 medium supplemented or not with BAP, after ( a ) four weeks and ( b ) eight of culture.

Table 3 .
Average number of axillary shoots developed per explant of Pinus taeda in WV3 medium supplemented or not with BAP, after ( a ) four weeks and ( b ) eight of culture.

Table 4 .
Percentage of elongation of apical segments of Pinus taeda inoculated into culture medium WV3 supplemented or not with BAP, after ( a ) four weeks and ( b ) eight of culture.

Table 5 .
Effect of culture medium and concentrations of NAA and BAP on adventitious rooting of Pinus taeda shoots after 10 weeks of culture.