Link/Page Citation
Introduction
Plantlets from a large number of conifer species have beensuccessfully propagated via organogenesis using various explants[1,15,23]. Araucaria excelsa R. Br. var. glauca has a high ornamentalvalue, due to a good specimen and symmetrical branches. The uniqueaspect of species belonging to the family araucariaceae is that theorthotropic stems with their bud traces, and the plagiotropic brancheswith their bud traces, remain permanently orthotropic and plagiotropic,respectively, regardless of any orientation or treatment imposed on them[17]. Consequently conventional propagation by cutting had limitedsuccess and the grafting showed a high incompatibility rate [22,17].Tips of secondary and tertiary lateral branches that taken from 14 yearsold Araucaria columnaris Hook. had normal orthotropic growth in thesituation of tissue culture [24]. Boulay [4] showed orthotropic behaviorof in vitro shoots derived from stump sprouts of mature sequoia (Sequoiasp.) trees. The objective of this study was investigating the potentialof plagiotropic branches to produce axillary shoots and their growthhabit in tissue culture condition.
Materials and Methods
Three years old plants were used for all experiments. Explants weretaken from primary plagiotropic stem (PPS), secondary plagiotropic stem(apical) (SPSA) and secondary plagiotropic stem (sub-apical) (SPSSA) ofAraucaria excelsa R. Br. (Fig. 1). Four cm long pieces were soaked inrunning tap water for 2 hr. These pieces were then treated for 2 minwith 70% ethanol and were surface sterilized by shaking for 15 min in15% Clorox (containing 5.25% sodium hypochlorite). They were finallyrinsed three times with sterile, distilled water. The explants used weresegments of about 8 to 9 mm long, cut from different parts ofplagiotropic branches. Explants were cultured on MS [21] basal medium.The pH was adjusted to 5.8 prior to autoclaving for 12 min at121[degrees] C and cultures were kept under 16 hr photoperiod under 20[micro]mol [m.sup.-2] [s.sup.-1] illuminance provided by cool whitefluorescent tubes at approximately 24[degrees]C.
[FIGURE 1 OMITTED]
Secondary plagiotropic stem (apical) (SPSA):
The effect of BA concentration on proliferation rate of SPSA wasinvestigated using basal MS medium with different concentrations of 0,3, 6, 12 and 24 [micro]mol in combination with 1, 2 and 3 [micro]molNAA. In another series of experiments, NAA was replaced with IBA, withthe same concentration and a medium was used without auxins. Half, 1.5and 2 strength MS medium salts was used for SPSA. In one experimentstems were prechilled (4 - 7[degrees] C) for 1.5 month before culturingin the medium.
Secondary plagiotropic stem (sub-apical) (SPSSA):
In another experiment all explants taken from SPSSA were culturedon basal MS medium supplemented with 0, 0.3, 0.6, 1.2, and 2.4 umol TDZin combination with 1 and 2 [micro]mol NAA. In another experimentcombination of 3, 6, 9 and 12 [micro]mol BA with 0.3, 0.6, 1.2, and 2.4[micro]mol TDZ and 1 and 2 [micro]mol NAA were used.
In the stages of rooting, etiolated or non-etiolated shoots of A.excelsa R. Br. cultured in either solid or liquid MS medium moved to aliquid or solid medium containing 2 and 3 mg [l.sup.-1] IBA for 2 weeksbefore moving to half- strength MS medium without or with 0.25%activated charcoal.
All the experiments were conducted in a Randomized Complete BlockDesign. Values represent the means of 5 replications with 4 explants ineach replication. Means were separated using Least SignificantDifferences (LSD) test at P=0.05.
Results
Secondary plagiotropic stem (apical) (SPSA):
In some cases SPSAs were bent into the culture medium and had apositive geotropism. Using 0.5, 1.5 and 2 strength MS salts had not anyeffect on curvature of explant tips. SPSA can produce axillary shoots(Table 1). However a high concentration of BA had positive effects oninduction of axillary shoots especially in concentrations more than 6[micro]mol. Twelve [micro]mol BA in combination with 3 [micro]mol NAAcaused 0.6 proliferated shoots.
Secondary plagiotropic stem (sub-apical) (SPSSA):
Results indicated that SPSSA did not show any response to plantgrowth regulators and prechilling treatments. Unfortunately, in asystematic study using over 400 explants taken from 2 years old A.excelsa R. Br. seedlings, only one axillary shoot produced after 3months in culture medium that finaly died (Fig. 2). Prechillingtreatment had not any effect on proliferation rate as well.
Rooting:
Recut of explant had not any effect on rooting, liquid and solidmedium as well. Some abnormal explants with high vegetative growthproduced root in MS medium after 2 months. Using IBA in the culturemedium caused high shoot growth in SPSA (Fig. 3).
Discussion:
A major problem in the propagation of woody plants is that mostsuccess is achieved with juvenile tissue and not from proven maturetrees. Tissue-culture technology is extensively used for the vegetativepropagation of selected plants in agriculture and horticulture and, to alesser extent, in forestry [18]. Micropropagation of most of theconifers has not been successful [24]. There are general tissue culturerelated challenges such as the production of chimeras, somaclonalvariation and endogenous bacterial contamination; the regeneration ofwoody plant species is still considered recalcitrant because of theeffects related to ontogenetic aging [16]. There are some reports on invitro culture of Araucaria [17,20,6,10]. Only one study in this area wasconducted by Sehgal et al. [24] that used tips of secondary and tertiarylateral branches. Most conifers have few axillary buds instead of havinglarge number of leaves. Consequently most conifers have a limitedability to coppice or to produce epicormic shoot after disturbance[14,2,3,12]. In contrast Araucaria possesses an apparently uniqueaxillary structure consisting of undifferentiated axillary meristemsthat have neither leaf primordial nor vascular connections[13,5,6,7,8,9]. These meristems are initially formed in an exogenousposition but are transferred to an endogenous position by the activityof a localized phellogen [6,5] and are consequently not abscised whenwidespread bark formation occurs [6,7]. Our results showed thatplagiotrpic growth was reminded in the in vitro derived shoots of A.excelsa R. Br. that was in contrast with Sehgal et al. [24] results inA. columnaris Hook. Genotype differences may be the cause of theseobservations. Increase in sucrose and salt concentration did not affectthe curvature of explants; inference of possible occurrence thisphenomenon in apical shoot was highly due to low carbohydrate contentand natural characters of plagiotropic branches in this species.Production of axillary buds in apical plagiotropic shoot is better thansub apical and thicker parts of plagiotropic stems due to highregeneration rate in apical meristem. Decapitation of apicalplagiotropic stems in situ had not any effect on production of axillarybuds. However, in vitro decapitation caused the production of axillarybuds that demonstrated plagiotropic stem contained concealed bud traces.However, further studies are needed to demonstrate if sub apicalorthotropic branches have bud traces. Twelve to 24 [micro]mol BA isneeded for proliferation in plagiotropic stems but orthotropic stems arevery sensitive to these concentrations (data not shown). Induction ofbuds in sub-apical branches was not too easy. Microcuttings of sidebranches will root but will produce horizontally growing plantlets.These results are of valuable importance to propagate this species,however further studies are needed to increase our knowledge andprospect for applied propagation of this plant.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
References
[1.] Abdullah, A. and J. Grace, 1987. Regeneration of calabrianpine from juvenile needles. Plant Sci., 53: 147-155.
[2.] Bellingham, P.J. and A.D. Sparrow, 2000. Resprouting as a lifehistory strategy in woody plant communities. Oikos., 89: 409-416.
[3.] Bond, W.J. and J.J. Midgley, 2001. Ecology of Sprouting inwoody plants: the persistence niche. Trends Ecol. Evol., 16: 45-51.
[4.] Boulay, M., 1979. Multiplication et clonage rapid du sequoiasempervirens par la culture in vitro. In: Etudes et Recherches, AFOCEL,Domaine-de-lEtanson, 77270 Nangis, France, 12: 49-55.
[5.] Burrows, G.E., 1986. Axillary meristem ontogeny in Araucariacunninghamii Aiton ex D. Don. Aust. J. Bot., 34: 357-375.
[6.] Burrows, G.E., 1987. Leaf axil anatomy in the Araucariaceae.Aust. J. Bot., 35: 631-640.
[7.] Burrows, G.E., 1990. The role of axillary meristems in coppiceand epicormic bud initiation in Araucaria cunninghamii. Bot. Gaz., 151:293-301.
[8.] Burrows, G.E., 1991. A scanning and transmission electronmicroscope study of leaf axil structure in Araucaria cunninghamii. Aust.J. Bot., 39: 67-76.
[9.] Burrows, G.E., 1999. Wollemi pine (Wollemia nobilis,Araucariaceae) possesses the same unusual leaf axil anatomy as the otherinvestigated members of the family. Aust. J. Bot., 47: 61-68.
[10.] Burrows, G.E., 1983. In vitro culture of hoop pine (Araucariacunnighamii Aiton ex D, Don). Silvicult., 30: 250-251.
[11.] Burrows, G.E., D.D. Doley, R.J. Haines and D.G. Nikles, 1988.In vitro propagation of Araucaria cunninghamii and other species of theAraucariaceae via axillary meristems, Aust. J. Bot., 36: 665-676.
[12.] Del Tredici, P., 2001. Sprouting in temperate trees: amorphological and ecological review. Bot. Rev., 67: 121-140.
[13.] Fink, S., 1983. The occurrence of adventitious and preventousbuds within the bark of some temperate and tropical trees. Amer. J.Bot., 70: 532-542.
[14.] Fink, S., 1984. Some cases of delayed or induced developmentof axillary buds from persisting detached meristems in conifers. Amer.J. Bot., 71: 44-51.
[15.] Garcia-Ferriz, L., L. Serrano and J. Pardos, 1994. In vitroshoot organogenesis from excised immature cotyledons and microcuttingsproduction in stone pine. Plant Cell, Tiss. Org. Cult., 36: 135-140.
[16.] Giri, C.C., B. Sshyamkmar and C. Anjaneylnu, 2004. Progressesin tissue culture, genetic transformation and application ofbiotechnology to trees: an overview. Trees, 18: 115-135.
[17.] Haines, R.J. and R.A. de Fossard, 1977. Propagation of hooppine (Araucaria cunninghamii AIT.). Acta Hortic., 78: 297-302.
[18.] Hall, R.D., 1999 . Plant cell culture protocols. Humana PressInc., U.S.A., pp: 421.
[19.] Handro, W., 1986. Araucaria. In: Biotechnology in agriculture& forestry (Bajaj P.S., ed.), vol. 1, Trees, Springer-Verlag,Berlin, pp: 310-315.
[20.] Maene, L. and P. Debergh, 1986. Araucaria. In: Cell andtissue culture in forestry. (Bonga J.M. and Durzan D.J., eds.), vol. 3,Cell histories, gymnosperms, angiosperms and palms. Martinus Nijhoff,Dordrecht, pp: 176-184.
[21.] Murashige, T. and F. Skoog, 1962. A revised medium for rapidgrowyh snd bioassays with tobacco tissue culture, Physiol. Plant., 15:473-497.
[22.] Nikles, D.G., 1961. The development of a new method forgrafting hoop and kauri pines. Qld Forest Serv. Res. Note, No. 10.
[23.] Saborio, F., W. Dvorak, J. Donahue and T. Thorpe, 1997. Invitro regeneration of plantlets from mature embryos of Pinus ayacahuite.Tree physiol., 17: 787-796.
[24.] Sehgal, L., O.P. Sehgal and P.K. Khosla, 1989.micropropagation of Araucaria columnaris Hook. Ann. Sci. Forest., 46:158-160.
Department of Horticultural Science, College of Agriculture, ShirazUniversity, Shiraz, Iran
M.K. Sarmast, H. Salehi and M. Khosh-Khui; Using Plagiotropic ShootExplants in Tissue Culture of Araucaria excelsa R. Br. var. glauca, Adv.Environ. Biol., 3(2): 191-194, 2009
Corresponding Author
Hassan Salehi, Department of Horticultural Science, College ofAgriculture, Shiraz University, Shiraz, Iran E-mail:[emailprotected]
Table 1: Effects of BA and NAA on shoot production in A. excelsaR. Br. var. glauca apical plagiotropic stems.BA NAA Shoot length Number of([micro]mol) ([micro]mol) (cm) proliferated shoots0 0 1.32 abc ([dagger]) 0 b 1 1.04 c 0 b0 2 1.25 abc 0 b 3 1.46 abc 0 b 1 1.28 abc 0 b3 2 1.26 abc 0 b 3 1.64 abc 0 b 1 1.80 a 0 b6 2 1.27 abc 0 b 3 1.34 abc 0.20 ab 1 1.14 bc 0 b12 2 1.27 abc 0.40 ab 3 1.24 abc 0.60 a 1 1.40 abc 0.40 ab24 2 1.28 abc 0 b 3 1.70 ab 0.20 abBA NAA Length of proliferated([micro]mol) ([micro]mol) shoots (mm)0 0 -- 1 --0 2 -- 3 -- 1 --3 2 -- 3 -- 1 --6 2 -- 3 0.60 a 1 0 a12 2 1.60 a 3 2.00 a 1 1.40 a24 2 0 a 3 0.40 a([dagger]) In each column, means followed by the same letters arenot significantly different using LSD test at P=0.05 (-noproliferation).
COPYRIGHT 2009 American-Eurasian Network for Scientific Information
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2009 Gale, Cengage Learning. All rights reserved.