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Influence of culture medium, explant length and genotype on micropropagation of Pinus taeda L.

Influência do meio de cultura, comprimento do explante e genótipo na micropropagação de Pinus taeda L.

Abstracts

This work aimed to establish a micropropagation protocol for Pinus taeda L. Apical shoots from 5-day seedlings, of different genotypes (F27, B05 and PC), were cultured on WV5 medium supplemented with 44 µM 6-benzylaminopurine (BAP) and 0.05 µM α-naphtaleneacetic acid (NAA), for 14 days, followed by subcultures on growth regulator-free medium. Explants were sectioned into apical shoots and nodal segments for multiplication. Different lengths of explants, BAP concentrations and cultivation periods were tested. Rooting was induced on WV5/2, WV3/2, GDm/2 and agar-water culture media supplemented with 2.68 µM NAA and 0.44 µM BAP for nine days, followed by a 4-week subculture on growth regulator-free medium. It was verified that genotype influenced shoot formation and rooting. The length of 1.0 cm is recommended for nodal segment explants to obtain a high number of axillary shoots. For apical shoots, 0.5 cm explant and WV5 medium formulation allowed the best results for elongation. The best period of subculture was eight weeks, both for nodal segments and for apical shoots. The higher percentage of nodal segments with shoots (99.2%) and the higher average number of shoots per explant (4.0) were obtained with F27 genotype in medium culture containing 2.5 µM BAP. For apical shoots, the best result for elongation was observed for the shorter explants (0.5 cm) on WV5 culture medium (218.3%). The maintenance of in vitro clonal micro garden of vigorous shoots was obtained for two years of 8-week subcultures on WV5 culture medium. The best rooting rate (55.6%) was obtained when shoots were inoculated in agar-water medium with 2.68 μM NAA and 0.44 μM BAP for nine days. Plantlets were successfully acclimatized (85% of survival), so a micropropagation protocol was established.

loblolly pine; in vitro culture; forestry species; agar-water


O presente trabalho teve como objetivo estabelecer um protocolo de micropropagação de Pinus taeda L. Brotações apicais de plântulas de cinco dias de germinação, de diferentes genótipos (F27, B05 e PC) foram cultivadas em meio de cultura WV5, acrescido de 44 µM de 6-benzilaminopurina (BAP) e de 0,05 µM de ácido α-naftalenoacético (ANA) durante 14 dias, seguido de quatro subcultivos para meio sem reguladores de crescimento. Explantes foram seccionados em brotações apicais e segmentos nodais para a multiplicação. Foram testados comprimentos diferentes dos explantes, concentrações de BAP e períodos de cultivo. O enraizamento foi induzido em meios de cultura WV5/2, WV3/2, GDm/2 e ágar-água, acrescidos de 2,68 µM de ANA e 0,44 µM de BAP, pelo período de nove dias, seguido de subcultivo para as mesmas formulações, sem reguladores por quatro semanas. Verificou-se que o genótipo influenciou a formação de brotações e enraizamento. O comprimento de 1,0 cm é recomendado para os segmentos nodais produzirem o maior número de brotações axilares. Para brotações apicais, os explantes de 0,5 cm cultivados em meio de cultura WV5 apresentaram melhores resultados de alongamento. O melhor período de subcultivo foi oito semanas, tanto para os segmentos nodais como brotações apicais. A maior percentagem de segmentos nodais com brotações (99,2%) e o maior número médio de brotações por explante (4,0) foram obtidos com o genótipo F27, em meio de cultura contendo 2,5 µM de BAP. Para brotações apicais, o melhor resultado para alongamento foi observado com explantes menores (0.5 cm) em meio de cultura WV5 (218,3%). A manutenção de microjardim clonal in vitro de brotações vigorosas foi obtida por dois anos em meio de cultura WV5, com subcultivos de oito semanas. O melhor resultado de enraizamento (55,6%) ocorreu com indução por nove dias em meio ágar-água com 2,68 μM de NAA e 0,44 μM de BAP. A aclimatização de mudas foi bem sucedida (85% de sobrevivência), de modo que foi estabelecido um protocolo de produção de mudas por micropropagação.

cultura in vitro ; espécie florestal; ágar-água


INTRODUCTION

Pinus taeda L. is one of the main forestry species cultivated worldwide. Its vegetative propagation is often difficult, due to its poor adventitious rooting and response variation with genotype (AMERSON et al., 1985AMERSON, H. V. et al. Loblolly pine tissue culture: laboratory, greenhouse and field studies. In: HENKE, R. R. (ed.) et al. Tissue culture in forestry and agriculture. Plenum Press: New York, p.271-287, 1985.). The two main in vitro techniques used for conifers are somatic embryogenesis and organogenesis. Especially in the genus Pinus, shoots can be induced in vitro by culturing excised cotyledons in medium containing cytokinins as the sole inducing agent, usually 6-benzylaminopurine (CORTIZO et al., 2009CORTIZO, M. et al. Benzyladenine metabolism and temporal competence of Pinus pinea cotyledons to form buds in vitro. Journal of Plant Physiology, v. 166, p. 1069-1076, 2009.). Conifers are considered difficult to root and major influence on production of a well-branched root system include auxin concentration, shoot quality, donor age and clone (JANG; TAINTER, 1991JANG, J. C.; TAINTER, F. H. Micropropagation of shortleaf, Virginia and loblolly pine x shortleaf pine hybrids via organogenesis. Plant Cell Tiss Org Cult, v. 25, p. 61-67, 1991.). Somatic embryogenesis has been described for loblolly pine and could be the most productive propagation method, but it is still unclear how to fully control the embryogenetic process (PULLMAN; JOHNSON, 2002PULLMANN, G.S.; JOHNSON, S. Somatic embryogenesis in loblolly pine (Pinus taeda L.): improving culture initiation rates. Ann For Sci, v. 59, p. 663-668, 2002.; SILVEIRA et al., 2004SILVEIRA, V. et al. Effect of plant growth regulators on the cellular growth and levels of intracellular protein, starch and polyamines in embryogenic suspension cultures of Pinus taeda. Plant Cell Tiss Org Cult, v. 76, n. 1, p. 53-60, 2004.), it has low initiation frequencies and sometimes, limited success in the maturation of embryogenic tissue into cotyledonary somatic embryos (MONTALBÁN et al.2011MONTALBÁN, I. A. et al. Testing novel cytokinins for improved in vitro adventitious shoots formation and subsequent ex vitro performance in Pinus radiate. Forestry, v. 84, n. 4, p. 363-373, 2011.). For these reasons, the production of clonal plants from seeds via organogenesis has been studied for several conifers in the last 30 years, as Pinus pinea (CORTIZO et al., 2009CORTIZO, M. et al. Benzyladenine metabolism and temporal competence of Pinus pinea cotyledons to form buds in vitro. Journal of Plant Physiology, v. 166, p. 1069-1076, 2009.), Pinus radiata (MONTALBÁN et al., 2011MONTALBÁN, I. A. et al. Testing novel cytokinins for improved in vitro adventitious shoots formation and subsequent ex vitro performance in Pinus radiate. Forestry, v. 84, n. 4, p. 363-373, 2011.), Pinus peuce (STOJICIC et al., 2012STOJICIC, D. et al. Micropropagation of Pinus peuce. Biologia Plantarum, v. 56, n. 2, p. 362-364, 2012.), Pinus taeda (OLIVEIRA et al., 2012OLIVEIRA, L. F. et al. Micropropagation of Pinus taeda L. from juvenile material. Tree For Sci Biotech, v. 6, n. 1, p. 96-101, 2012.).

Direct organogenesis does not go through a callus, therefore it could increase the genomic stability of regenerated plants (TANG; GUO, 2001TANG, W.; GUO, Z. In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Regul, v. 33, p. 25-31, 2001.). Direct organogenesis of Pinus taeda was already described by Mehra-Palta et al. (1978)MEHRA-PALTA, A.; SMELTZER, R. H.; MOTT, R. L. Hormonal control of induced organogenesis - Experiments with excised plant parts of loblolly pine. Tappi J, v. 61, n. 1, p. 37-40, 1978., Amerson et al. (1985)AMERSON, H. V. et al. Loblolly pine tissue culture: laboratory, greenhouse and field studies. In: HENKE, R. R. (ed.) et al. Tissue culture in forestry and agriculture. Plenum Press: New York, p.271-287, 1985., Sen et al. (1989)SEN, S.et al. Abscisic acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants. Plant Cell Rep, v. 8, p. 191-194, 1989., Tang and Guo (2001)TANG, W.; GUO, Z. In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Regul, v. 33, p. 25-31, 2001., but an efficient protocol that actually meets commercial production needs has not been established yet. Optimization of factors influencing each step of micropropagation procedure is necessary for successful regeneration through adventitious bud induction (STOJICIC et al., 2012STOJICIC, D. et al. Micropropagation of Pinus peuce. Biologia Plantarum, v. 56, n. 2, p. 362-364, 2012.).

The aim of this study was to test the effect of explant length, genotype, culture medium, plant growth regulators and subculture periods to establish an organogenesis protocol for juvenile material of Pinus taeda.

MATERIAL AND METHOD

Source of explants

Seeds from selected families were provided by Battistella Florestal Company (Rio Negrinho, Santa Catarina state, Brazil). These families were ranked according to shaft quality. Genotype F27 corresponded to a controlled-pollinated family and B05 to an open-pollinated one, being respectively the 2nd and the 41st best families of the company. Genotype PC corresponded to a mixture of open-pollinated seeds from the best 129 families of company. Seeds were collected in January 2009, stored at 8°C for one month and then soaked for 24 hours. Afterwards, seeds were stratified for 25 days at 8°C, in Petri dishes (100 x 20 mm) with soaked paper filter and placed in new moist Petri dishes, for germination in growth room.

Culture conditions

Growth room with 16h photoperiod, white cool fluorescent lamps (40 µmol.m-2.s-1), temperature of 27 ± 2ºC (day) and 18 ± 2ºC (night).

Culture media

WV5 (COKE, 1996aCOKE, J. E. Basal nutrient medium for in vitro cultures of Loblolly pines. US Patent 5534434. http://www.freepatentsonline.com/5534434.html, 1996a.
http://www.freepatentsonline.com/5534434...
), WV3 (COKE, 1996bCOKE, J. E. Basal nutrient medium for in vitro cultures of Loblolly pines. US Patent 5534433. http://www.freepatentsonline.com/5534433.html, 1996b.
http://www.freepatentsonline.com/5534433...
) culture media were used for shoot and root induction. GD (GRESSHOFF; DOY, 1972GRESSHOFF, P. M.; DOY, C. H. Development and differentiation of haploid Lycopersicum esculentum (tomato). Planta, v. 107, p. 473-497, 1972.) was used as modified by Mehra-Palta et al. (1978)MEHRA-PALTA, A.; SMELTZER, R. H.; MOTT, R. L. Hormonal control of induced organogenesis - Experiments with excised plant parts of loblolly pine. Tappi J, v. 61, n. 1, p. 37-40, 1978. and referred as "GDm". Agar-water (AW) was also tested for rooting. The pH was adjusted to 5.8 with NaOH and/or HCl at 0.1 N before autoclaving at 121°C for 20 minutes. 30 g.L-1 sucrose was added to shoot induction and multiplication media. Culture media without sucrose or with 20 g.L-1 of sucrose were used for rooting experiments. All media were supplemented with 5.6 g.L-1 of Himedia(r) agar. Test tubes (150 x 25 mm) with 10 mL culture medium each were used for shoot induction step. Flasks (125 x 65 mm) containing 40 mL culture medium and five explants each were used for multiplication and rooting steps.

In vitro establishment and shoot induction

Five-day germinated seeds had their teguments off and were immersed for 20 min in 30% hydrogen peroxide followed by immersion in a 6% sodium hypochlorite plus 0.1% Tween(r) 20 solution, for 10 min. Three rinses with sterilized distilled water were realized. Germinated seeds were then immersed in 0.05% mercuric chloride plus 0.1% Tween(r) 20 solution during five minutes and finally rinsed six times in sterile distilled water. The excised explants (apical shoots of seedlings) were vertically inoculated in WV5 culture medium (COKE, 1996aCOKE, J. E. Basal nutrient medium for in vitro cultures of Loblolly pines. US Patent 5534434. http://www.freepatentsonline.com/5534434.html, 1996a.
http://www.freepatentsonline.com/5534434...
) with 44 µM 6-benzylaminopurine (BAP) and 0.05 µM α- naphtaleneacetic acid (NAA) (MEHRA-PALTA et al. 1978MEHRA-PALTA, A.; SMELTZER, R. H.; MOTT, R. L. Hormonal control of induced organogenesis - Experiments with excised plant parts of loblolly pine. Tappi J, v. 61, n. 1, p. 37-40, 1978.). Shoot induction lasted 14 days and was followed by four subcultures on WV5 growth regulator free, each subculture during four weeks (Figure 1a). These initial cultures produced elongated adventitious and axillary shoots which were sectioned into nodal segments and apical shoots.

Figure 1.
Micropropagation of Pinus taeda. a-Apical shoot induced on WV5 culture medium with 44µM BAP and 0.05 µM NAA for 14 days, followed by two subcultures on growth regulator-free medium; b- Axillary shoots developed from nodal segment 2.0 to 2.9 cm long, after four weeks on WV5 culture medium with 0.1 µM BA and eight weeks in WV5 medium without plant growth regulators. c- Elongation of apical shoots with initial length of 1.0 cm, after eight weeks on growth regulator free GDm or WV5, respectively. d- Secondary root formation after nine days of induction on GDm/2 medium with 20 g.L-1 of sucrose, 2.68 µM NAA and 0.44 µM of BA, followed by six weeks of subculture on the culture medium of same composition, without growth regulators.

Nodal segment multiplication

Experiment I

Nodal segments (1.0 - 1.9 cm, 2.0 - 2.9 cm or 3.0 - 4.0 cm long) of F27 genotype were cultivated on WV5 medium with 0.1 µM BAP during four weeks and then subcultivated on WV5 medium without regulators, being evaluated after four and eight weeks of subculture. The experimental design was completely randomized in a 3 x 2 factorial arrangement (length x subculture time) with three repetitions and experimental unit of 15 explants.

Experiment II

Nodal segments (1.0 cm long) of F27, B05 and PC genotypes were cultivated on WV5 medium supplemented with 0.25, 0.5 or 2.5 µM BAP. The experimental design was completely randomized in a 3 x 3 factorial arrangement (genotype x BAP concentration) with four repetitions and experimental unit of 10 explants.

Apical shoots multiplication

Apical shoots (0.5,1.0 or 2.0 cm long) of F27 genotype were cultivated on WV5 or GDm without growth regulators for eight weeks. The experimental design was completely randomized in a 3 x 2 factorial arrangement (length x culture medium) with four repetitions and experimental unit of 15 explants.

Rooting

Experiment I

Apical shoots, 1.5 cm long, elongated on WV5 medium without growth regulators for eight weeks were used as explants. Shoots of F27 genotype were inoculated on WV5, WV3 (COKE, 1996bCOKE, J. E. Basal nutrient medium for in vitro cultures of Loblolly pines. US Patent 5534433. http://www.freepatentsonline.com/5534433.html, 1996b.
http://www.freepatentsonline.com/5534433...
), GDm or AW, supplemented with 2.68 µM NAA and 0.44 µM BAP (MEHRA-PALTA et al., 1978MEHRA-PALTA, A.; SMELTZER, R. H.; MOTT, R. L. Hormonal control of induced organogenesis - Experiments with excised plant parts of loblolly pine. Tappi J, v. 61, n. 1, p. 37-40, 1978.) for nine days. Culture media WV5, WV3 e GDm had their salts reduced by half and contained 20 g.L-1 sucrose. Agar-water medium did not contain sucrose. After rooting induction period, explants were transferred to the flasks with culture medium of the same composition, without growth regulators, except explants on agar-water formulation, which were subcultured on GDm/2 with 20 g.L-1 sucrose. Experimental design was completely randomized, with four repetitions and experimental unit of ten explants.

Experiment II

Shoots of genotypes F27, B05 and PC were cultured for nine days on GDm/2 or agar-water, combined with 0 or 20 g .L-1 of sucrose, supplemented with 2.68 µM NAA and 0.44 µM BAP and then transferred to GDm/2 without plant growth regulators. The experimental design was completely randomized in a 2 x 2 factorial arrangement (culture medium x sucrose concentration) with four repetitions and experimental unit of 12 explants.

Evaluations

Percentage of explants with shoots and average number of shoots per explant were evaluated after four and eight weeks for nodal segments multiplication. Percentage of elongation [(final length - initial length/ initial length) x 100] after eight weeks was evaluated for apical shoots. Percentage of rooted shoots and average number of roots per shoot were evaluated after six weeks.

Statistical analysis

The data were submitted to Bartlett's Test and analysis of variance (ANOVA) and means were compared by Tukey's test at P ≤ 0.05. When necessary variables were transformed by log (x + 10) or [log(x+ 10)] 1/2.

RESULTS AND DISCUSSION

Nodal segments and apical shoot multiplication

There were no differences of shoot formation percentage or the average number of shoots per explant among the three groups of nodal segments with different initial length (Table 1; Figure 1b), differing from the results observed for other Pinus species, like Pinus tecunumanii and Pinus oocarpa which showed increased multiplication with longer segments (BAXTER et al., 1989BAXTER, R. et al. Production of clonal plantlets of tropical pine in tissue culture via axillary shoot activation. Can J For Res, v. 19, p. 1338-1342, 1989.). Therefore, the use of shorter nodal segments for Pinus taeda (1.0-1.9 cm) can be more advantageous, since it is possible to obtain more short segments than longer segments from the same initial stock material.

Table 1.
Shoot formation on Pinus taeda nodal segments from F27 family with different lengths, after four weeks on WV5 medium supplemented with 0.1 µM BAP, after subculture on WV5 medium without growth regulators.

A higher percentage of explants with shoots was observed after eight weeks of subculture than after four weeks (Tables 1 and 2) and the shoots were vigorous. This result contrasts with the subculture period of four weeks without losses of vigor suggested by Amerson et al. (1985)AMERSON, H. V. et al. Loblolly pine tissue culture: laboratory, greenhouse and field studies. In: HENKE, R. R. (ed.) et al. Tissue culture in forestry and agriculture. Plenum Press: New York, p.271-287, 1985. for Pinus taeda organogenic cultures. It also suggests that WV5 formulation probably allowed a longer subculture period than the described for other culture media used for Pinus taeda, like GD Gresshoff and Doy's (1972)GRESSHOFF, P. M.; DOY, C. H. Development and differentiation of haploid Lycopersicum esculentum (tomato). Planta, v. 107, p. 473-497, 1972. medium (AMERSON et al., 1985AMERSON, H. V. et al. Loblolly pine tissue culture: laboratory, greenhouse and field studies. In: HENKE, R. R. (ed.) et al. Tissue culture in forestry and agriculture. Plenum Press: New York, p.271-287, 1985.; JANG; TAINTER,1991JANG, J. C.; TAINTER, F. H. Micropropagation of shortleaf, Virginia and loblolly pine x shortleaf pine hybrids via organogenesis. Plant Cell Tiss Org Cult, v. 25, p. 61-67, 1991.) or Litvay's (LITVAY et al., 1981LITVAY, J. D et al. Conifer suspension culture medium development using analytical data from developing seeds. Institute Paper Chemistry, Appleton (U. S.), 1981.) medium (JANG; TAINTER, 1991JANG, J. C.; TAINTER, F. H. Micropropagation of shortleaf, Virginia and loblolly pine x shortleaf pine hybrids via organogenesis. Plant Cell Tiss Org Cult, v. 25, p. 61-67, 1991.).

The simple sectioning and decapitation of shoots allow the production of new axillary shoots and can increase multiplication rate (BAXTER et al., 1989BAXTER, R. et al. Production of clonal plantlets of tropical pine in tissue culture via axillary shoot activation. Can J For Res, v. 19, p. 1338-1342, 1989.; GEORGE et al, 2008), hence nodal segments can be multiplied without using BAP. However, the use of this cytokinin in increasing concentrations raised the average number of shoots per explant after eight weeks (Table 2). Similar results were obtained by Lambardi et al. (1993)LAMBARDI, M.; SHARMA, K. K.; THORPE, T. A. Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.). In vitro Cell Dev Biol Plant, v. 29, p. 189-199, 1993. and Nandwani et al. (2001)NANDWANI, D.; KUMARIA, S.; TANDON, P. Micropropagation of Pinus kesiya Royle ex Gord (Khasi pine). Eur J Hort Sci, v. 66, p. 68-71, 2001. who verified that the addition of cytokinin to culture medium increased proliferation of axillary shoots of Pinus halepensis and Pinus kesiya, respectively. Since long term maintenance on a cytokinin supplemented medium may cause elongation inhibition in subsequent subcultures or rooting difficulties (KAUL, 1990KAUL, K. Factors influencing in vitro micropropagation of Pinus strobus L. Biol Plant, v. 32, n. 4, p. 266-272, 1990.; ZEL, 1993ZEL, J. Micropropagation of Pinus sylvestris. In: Ahuja MR (Ed.) Micropropagation of woody plants. Kluwer: Academic Publishers, Dordrecht, p. 347-365, 1993.), it is suggested that, after an eight week subculture period with BAP, a subculture on BAP-free medium should be done for Pinus taeda. Similar procedure was suggested by Álvarez et al. (2009)ÁLVAREZ, J. M.; MAJADA, J.; ORDÁS, J. An improved micropropagation protocol for maritime pine (Pinus pinaster Ait.) isolated cotyledons. Forestry, v. 82, p. 175-184, 2009. for Pinus pinaster.

Table 2.
Shoots formation on 1.0 cm long nodal segments of different genotypes of Pinus taeda, after four and eight weeks on WV5 medium supplemented with 0.25, 0.5 or 2.5 µM BAP.

F27 genotype explants presented a significantly higher number and percentage of explants with shoots than PC explants after four weeks of culture (Table 2). Since the F27 family was the genotype with best shaft quality a relationship between field and in vitro performances may be inferred and further tests being necessary to prove this hypothesis. In the first studies made by Westvaco, genetically superior families of Pinus taeda showed positive in vitro responses (HANDLEY et al., 1995HANDLEY, L. W et al. Research and development of commercial tissue culture systems in loblolly pine. Tappi J, v. 78, n. 5, p. 169-175,1995.), indicating that family influenced shoot production, though it was not confirmed whether or not the propagation capacity was related to field characteristics, like growth in height or volume.

Percentage of elongation was higher for the shorter initial length explants and for the explants grown on WV5 culture medium (Table 3; Figure 1c), indicating that this medium could be more adequate for this species. Shoots cultivated in GDm culture medium presented shorter internodes, darker green leaves and more lignified explant bases than the ones cultivated in WV5 medium, which contains higher concentrations of N, B, Ca, Mg, S, K, P, Mn, I, Zn, Cl and myo-inositol than the GDm formulation. Comparing the composition of these two culture media with the nutritional requirements of cellular suspension cultures of P. taeda (TEASDALE et al., 1986TEASDALE, R. D.; DAWSON, P. A.; WOOLHOUSE, H. W. Mineral nutrient requirements of a Loblolly Pine (Pinus taeda) cell suspension culture - Evaluation of a medium formulated from seed composition data. Plant Physiol, v. 82, p. 942-945, 1986.), it was verified that GDm medium has lower B and Mg quantities than the minimum required, what does not occur with WV5 salt formulation. Moreover, WV5 culture medium was specifically developed for P. taeda cultures (COKE, 1996aCOKE, J. E. Basal nutrient medium for in vitro cultures of Loblolly pines. US Patent 5534434. http://www.freepatentsonline.com/5534434.html, 1996a.
http://www.freepatentsonline.com/5534434...
), considering the nutritional requirements of the species and it is richer in mineral salts than GD or Litvay's media.

Table 3.
Percentage of elongation of Pinus taeda apical shoots (0.5, 1.0 or 2.0 cm long), from F27 family, after eight weeks of culture on WV5 or GDm media without growth regulators.

In this work, the maintenance of vigorous organogenic cultures of Pinus taeda occurred mostly on WV5 culture medium, for 24 months, with four or eight-week subcultures, with and without BAP, without major symptoms of nutritional deficiency. That is longer than the observed for the same species by Handley et al. (1995)HANDLEY, L. W et al. Research and development of commercial tissue culture systems in loblolly pine. Tappi J, v. 78, n. 5, p. 169-175,1995. and by Jang and Tainter (1991)JANG, J. C.; TAINTER, F. H. Micropropagation of shortleaf, Virginia and loblolly pine x shortleaf pine hybrids via organogenesis. Plant Cell Tiss Org Cult, v. 25, p. 61-67, 1991., who succeeded in maintaining cultures for six and ten months, respectively, on GDm or Litvay's culture media.

Rooting

Spontaneous rooting occurred at low frequency (less than 1%). According to Diaz-Sala et al. (1996)DIAZ-SALA C. et al. Maturation-related loss in rooting competence by loblolly pine stem cuttings: The role of auxin transport, metabolism and tissue sensitivity. Physiol Plant, v. 97, p. 481-490, 1996., Pinus taeda rooting depends on the application of exogenous auxin. In the present work, we used growth regulator concentrations like proposed by Mehra-Palta et al. (1978)MEHRA-PALTA, A.; SMELTZER, R. H.; MOTT, R. L. Hormonal control of induced organogenesis - Experiments with excised plant parts of loblolly pine. Tappi J, v. 61, n. 1, p. 37-40, 1978. i.e., a combination of NAA and BAP for root induction. Mehra-Palta et al. (1978)MEHRA-PALTA, A.; SMELTZER, R. H.; MOTT, R. L. Hormonal control of induced organogenesis - Experiments with excised plant parts of loblolly pine. Tappi J, v. 61, n. 1, p. 37-40, 1978. and Tang and Ouyang (1999)TANG, W.; OUYANG, F. Plant regeneration via organogenesis from six families of loblolly pine. Plant Cell Tiss Org Cult, v. 58, n. 3, p. 223-226, 1999. observed that addition of other regulators (cytokinin or gibberellin), together with an auxin, was necessary to increase rooting rates of shoots of Pinus taeda, rather than auxin alone.

In this work, the best rooting percentages were similar to those obtained by Mehra-Palta et al. (1978)MEHRA-PALTA, A.; SMELTZER, R. H.; MOTT, R. L. Hormonal control of induced organogenesis - Experiments with excised plant parts of loblolly pine. Tappi J, v. 61, n. 1, p. 37-40, 1978. who related 50% of in vitro rooting for Pinus taeda. However, in that work, the first roots appeared after five months in medium with regulators, while in the present study first roots were observed after three weeks, considering the nine-day period of rooting induction with regulators plus two weeks of subculture without regulators. De Klerk et al. (1997) verified that the hormonal stimulation which induces an in vitro physiological reaction could be inhibitory when the new organ is developing. The necessary time for root formation was reduced when compared to the results of Tang and Ouyang (1999)TANG, W.; OUYANG, F. Plant regeneration via organogenesis from six families of loblolly pine. Plant Cell Tiss Org Cult, v. 58, n. 3, p. 223-226, 1999., who observed the first responses of in vitro adventitious rooting of Pinus taeda after six weeks of culture.

Roots formed after two weeks on culture media without regulators were white-colored without secondary roots. They acquired a darker color and presented secondary ramifications after six weeks of culture (Figure 1d). Average length of roots was about 0.2-0.5 cm after two weeks of subculture (Figure 1d) and about 1.0-1.7 cm after six weeks of subculture, without differences among the treatments, in both rooting experiments.

Similarly to what occurred in multiplication experiments, the saline formulation had an effect on rooting induction of Pinus taeda shoots. However, unlike the multiplication experiments, the more concentrated saline formulations, WV3/2 and WV5/2, presented low or no positive results, while the higher rooting percentages were seen in AW and GDm/2 media (Table 4). Webb et al. (1988)WEBB, D. T.; FLINN, B. S.; GEORGIS, W. Micropropagation of eastern white pine (Pinus strobus L.). Can J For Res, v. 18, p. 1570-1580, 1988. obtained greater success for Pinus ponderosa in vitro rooting using agar-water culture medium than using other formulations, similarly to what was observed in this work for Pinus taeda. Oliveira et al. (2012) also observed that P. taeda shoots did not need salts to acquire rooting competence; however they pointed out that absence of salts reduces significantly further root development. Mean number of roots per explants was higher for the explants induced in GDm/2, when compared to AW medium (Table 5). The presence or absence of sucrose in the induction culture medium did not influence adventitious rooting (Table 5).

Table 4.
Pinus taeda rooting from apical shoots of F27 family, induced for nine days on GDm/2, WV5/2, WV3/2 culture media with 20 g.L-1 sucrose or AW medium without sucrose, supplemented with 2.68 µM NAA and 0.44 µM BAP, after six weeks of subculture on culture media without growth regulators.
Table 5.
Pinus taeda rooting from apical shoots of F27, B05 and PC genotypes, induced for nine days on GDm/2 or AW culture medium, supplemented or not with 20 g.L-1 sucrose, 2.68 µM NAA and 0.44 µM BAP, after six weeks of subculture on GDm/2 medium with 20 g. L-1 sucrose and without growth regulators.

Genetic factors could influence rooting results, once similar treatments with GDm and agar-water formulations from the two experiments presented different rooting percentages (Tables 4 and 5). This is in accordance with the results of Tang and Ouyang (1999)TANG, W.; OUYANG, F. Plant regeneration via organogenesis from six families of loblolly pine. Plant Cell Tiss Org Cult, v. 58, n. 3, p. 223-226, 1999. who observed variation of rooting percentage (8.7% to 46.7%) among six families of Pinus taeda. The influence of genotype on in vitro responses of Pinus taeda is often reported in the literature (JANG; TAINTER, 1991JANG, J. C.; TAINTER, F. H. Micropropagation of shortleaf, Virginia and loblolly pine x shortleaf pine hybrids via organogenesis. Plant Cell Tiss Org Cult, v. 25, p. 61-67, 1991.; HANDLEY et al., 1994; TANG et al., 1998TANG, W.; WHETTEN, R.; SEDROFF, R. Genotypic control of high-frequency adventitious shoot regeneration via somatic organogenesis in loblolly pine. Plant Sci, v. 16, p. 167-272, 1998.; TANG; GUO, 2001TANG, W.; GUO, Z. In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Regul, v. 33, p. 25-31, 2001.), therefore being a challenge to establish a reliable protocol for the species, not restricted to only few genotypes.

In the present study, axillary shoots obtained within 12 and 24 months of culture could be induced to root, in contrast to the results of Jang and Tainter (1991)JANG, J. C.; TAINTER, F. H. Micropropagation of shortleaf, Virginia and loblolly pine x shortleaf pine hybrids via organogenesis. Plant Cell Tiss Org Cult, v. 25, p. 61-67, 1991., who observed loss of adventitious rooting capacity of Pinus taeda cultures after eight months of culture on GD or Litvay's media.

Plants were acclimatized by transfer to vermiculite: plantmax (1:1) substrate, in open glass flasks, in growth room conditions, for 20 days, followed by transfer to greenhouse under uncontrolled conditions, with survival rate of 85% after 40 days of acclimatization.

CONCLUSIONS

It was concluded that genotype had influenced shoot formation and rooting. It is recommended the length of 1.0 cm for nodal segment explants to obtain a higher number of axillary shoots. For apical shoots, the 0.5 cm explant length and WV5 medium formulation allowed their bigger elongation, this way they can be used as stocks for new nodal segments sections, increasing multiplication rates. The best period of subculture was eight weeks, both for nodal segments and for apical shoots.

With the use of WV5 culture media, it was possible to maintain cultures of vigorous shoots during two years, allowing the maintenance of an in vitro clonal micro garden.

In vitro, rooting of P. taeda does not need salts during the nine days induction period with 2.68 μM NAA and 0.44 μM BAP and the presence of sucrose in the induction culture medium does not influence adventitious rooting. Plantlets were acclimatized successfully, so a direct organogenesis protocol was established.

ACKNOWLEDGEMENTS

To the Pinus project colleagues, in particular to Fernanda Silveira, for their help and support. To Marguerite Quoirin for the English review. To Battistella Florestal and FUPEF for financing the project. To CAPES for the scholarship to the main author.

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Publication Dates

  • Publication in this collection
    Jan-Mar 2015

History

  • Received
    21 Aug 2012
  • Accepted
    08 July 2013
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