Vegetative propagation of patagonian cypress, a vulnerable species
Contents
1. Results obtained in this work indicated that compatibility between scion and rootstock from different populations i e genetically unrelated of A chilensis would be wide which enhances the probabilities of ob taining good results with low costs for breeding programs Genotype SR8 used as one of the scions donors in this work probably deserves special genetic study because of its behavior in failing all the practiced grafts Several genetically determined mechanisms are known to cause incompatibilities such as poor phloem differentiation from callus cells due to lack of hormones or carbohydrates The success of grafting is primarily determined by the capacity of producing parenchymatous callus tissue in response to wounding This process is dependent on the genetics and on a number of environmental factors among which the season of grafting in relation to scion and rootstock activity and to growth regulators action is one of the most relevant According to Hartmann ef al 2002 many conifers are grafted successfully during late winter to spring In this work early spring was found to be the most appropriate time for scion collection and grafting with activity of both scion and rootstock recently initiated Callus proliferation process that is essential for graft union was observed in all the 42 grafts practiced in the early spring experiment in which 81 of successful grafts were obtained On the contrary none of the grafts practic2. Rovere AE AC Premoli JC Aravena A Lara 2004 Variaci n en Pilgerodendron uviferum D Don Florin Cipr s de las Guaitecas In Donoso C A Premoli L Gallo R Ipinza eds Variaci n intraespecifica en las especies arb reas de los bosques templados de Chile y Argentina Santiago Chile Editorial Universitaria p 253 275 SAGPyA 2008 Boletin precios forestales Marzo de 2008 Accessed Nov 25 2008 Available in http www sagpya mecon gov ar Schmaltz J 1992 La reconquista de la estepa por el bosque de cipr s Descripci n de un caso estudiado primer in forme Esquel Argentina CIEFAP GTZ 6 p Publicaci n T cnica 11 Shoemake LJ MA Arnold Davies FTJr 2004 Provenance impacts transplant establishment and adventitious root regeneration of Sycamore Journal of the American Society of Horticultural Science 129 3 360 367 Stankova T K Panetsos 1997 Vegetative propagation of Cupressus sempervirens L of Cretan origin by softwood cuttings Silvae Genetica 46 2 3 137 144 Tchoundjeu Z ML Ngo Mpecka E Asaaha A Amougoub 2004 The role of vegetative propagation in the domestication of Pausinystalia johimbe K Schum a highly threatened me dicinal species of West and Central Africa Forest Ecology and Management 188 175 183 Veblen TT DH Ashton 1982 The regeneration status of Fitroya cupressoides in the Cordillera Pelada Chile Biological Conservation 23 141 161 25 BOSQUE 30 1 18 26 2009 Vegetative
3. ability would be higher Further research to precisely test this hypothesis is needed Results found for the juvenile cuttings of P12 progeny multi stemmed mother genotype 33 of rooted cut tings in mid summer experiment suggest that there are genotypes with a relatively high intrinsic rooting capacity which can be improved given the appropriate conditions for rooting Environmental factors and ontogenetic stage affecting rooting ability According to Hartmann et al 2002 when rooting narrow leaved evergreens best results are expected if cut tings are collected during late fall to late winter dormant or cessation periods of growth Nevertheless the results could be variable even among closely related species Scarce antecedents have been reported for species genetically related to A chilensis in relation to the optimal season of BOSQUE 30 1 18 26 2009 Vegetative propagation patagonian cypress cutting collection The related Cupressaceae species from Oceania Libocedrus spp and Papuacedrus papuana F Muell H L Li can eventually be propagated by cuttings taken during summer For P uviferum the most closely related to A chilensis species a relatively good rooting ability was found when using vegetative material collected between fall and spring Rovere et al 2004 although no contrasting summer experiments were reported Despite the poor general performance for rooting ob tained in this work an apparent tende
4. latent root Table 3 Provenances used in the rooting and grafting experiments m a p mean annual precipitation Procedencias utilizadas en los ensayos de enraizado e injertado m a p precipitaci n media anual Provenance Forest type Latitude S Longitude W m a p mm Material Puerto Blest Humid 41 01 71 49 2 650 Rootstock cuttings R o Traful Mesic 40 39 71 15 955 Scions Cerro Leones Mesic 41 04 71 09 800 Rootstock Cerro Otto Mesic 41 05 71 19 900 Scions cuttings Huinganco Xeric 37 08 70 36 604 Cuttings San Ram n Xeric 41 00 71 05 500 Scions Pilca eu Xeric 41 13 70 42 330 Rootstock cuttings El Mait n Xeric 42 02 71 12 586 Cuttings 22 initials epicormic buds root sprouting stem adventitious roots to propagate by vegetative means in their evolutive habitats The three genetically related Cupressaceae species from the subantarctic forest of South America Fitzroya cupressoides Molina I M Johnst Pilgerodendron uviferum D Don Florin and Austrocedrus chilensis D Don Pic Ser ef Bizzarri have evolved propagation strategies that as in most conifers rely almost exclusively on sexual reproduction Notwithstanding the three spe cies have also developed complementary mechanisms of vegetative propagation with more or less incidence in their stand regeneration dynamics Probably these adap tive clonal mechanisms allow the species to occupy
5. Single 1 3 SR2 San Ram n Adult Male Single 1 3 SR3 San Ramon Adult Male Multi stemmed 1 3 SR4 San Ram n Adult Male Multi stemmed 1 2 3 4 SR5 San Ram n Adult Female Multi stemmed 1 3 4 SR6 San Ram n Adult Female Multi stemmed 1 3 Ol Cerro Otto Adult Female Single 1 2 3 4 02 Cerro Otto Adult Male Single 1 3 4 04 Cerro Otto Adult Female Single 1 3 4 P12 progeny 10 plants Pilca eu Juvenile Multi stemmed mother 2 4 018 Cerro Otto Juvenile Single 2 M10 progeny 14 plants El Mait n Juvenile Single mother 2 H15 progeny 14 plants Huinganco Juvenile Single mother 4 B provenance 10 plants Puerto Blest Juvenile Single mothers 4 Scions grafting experiments Three different seasons were Table 2 Genotypes used as scions donors in the grafting tried for grafting experiments experiment 5 early spring experiments between 21 and 26 September experiment 6 mid spring on 21 October and experiment 7 late summer between 7 and 14 March Completely exposed partial reiterations were collected of adult trees from the same two populations sampled for rooting experiments O and SR The collected branches were conditioned in humid chamber at 4 C and grafts were practiced within 48 hours after the collections Scions consisted of third order hardwood or semi hardwood stems of 4 5 to 6 5 mm in diameter 12 to 15 cm in length and a remaining leaf area of about 40 cm in the distal third They were classified according to stem and foli
6. propagation by stem rooting although it could be strongly dependent on the genotype The genetic entity at different levels i e provenance family individual is largely relevant in determining the rooting behavior within a species Ying and Bagley 1977 Greenwood and Weir 1994 Dick et al 1996 Stankova and Panetsos 1997 Shoemake et al 2004 As an example individual differences in the rooting capacity of Cupressus sempervirens L ranged from 7 to 81 for one same set of experimental conditions Stankova and Panetsos 1997 Therefore it has to be considered that the propagation protocol to be used in breeding and conservation programs could be not unique within a species Grafting techniques are useful when difficulties are found to propagate trees by stem rooting being also the success of grafting strongly dependent on an intrinsic genetic capacity and on the com patibility between genotypes Copes 1999 Hartmann et al 19 BOSQUE 30 1 18 26 2009 Vegetative propagation patagonian cypress 2002 Besides the intrinsic genetic abilities a multiplicity of environmental factors affects the success of both root ing and grafting processes in trees e g ontogenetic stage type of vegetative material season of collection hormonal treatments propagation environment techniques Although general guidelines are provided in literature in relation to the most important environmental effects Hartmann et al 2002 the results when a
7. propagation patagonian cypress Wollemi Pine International 2004 Accessed Jun 25 2007 Available in http www wollemipine com Wright JW 1976 Introduction to Forest Genetics New York USA Academic Press 463 p Ying CH WT Bagley 1977 Variation in rooting capability of Populus deltoides Silvae Genetica 26 5 6 204 207 26 Zobel DB 1990 Chamaecyparis lawsoniana A Murr Parl Port Orford Cedar In Burns RM BH Honkala eds Silvics of North America 1 Conifers Agriculture Handbook 654 Washington DC USA USDA Forest Service p 155 170 Zobel B J Talbert 1984 Applied forest tree improvement Illinois USA Waveland Press 505 p Recibido 25 01 08 Aceptado 02 01 09
8. rooting and scion grafting The influence of exogenous auxins season of cutting collection ontogenetic stage of the donor plants grafting season and type of scions was analyzed Stem rooting ability was found to be poor both for adult average 0 27 and juvenile ortets average 2 10 Only one treatment a juvenile progeny from a xeric provenance showed remarkable results with 33 33 of rooting capacity Grafting showed good results in early spring experiment using vigorous semi hardwood scions with an average of 80 95 of successful grafts Compatibility between genotypes from different provenances used as scion and rootstock was found to be wide These results indicate that although A chilensis is a difficult to root species its vegetative propagation by means of scion grafting is feasible using low cost technology Key words Austrocedrus Cupressaceae stem rooting scion grafting RESUMEN Los crecientes riesgos de p rdida de recursos gen ticos forestales y la necesidad de una producci n sostenible de madera de calidad hacen necesario orientar la investigaci n hacia especies arb reas nativas no domesticadas Para rescatar la diversidad gen tica ame nazada y como una importante herramienta para el mejoramiento gen tico resulta necesario estudiar la capacidad de las especies para propagarse vegetativamente as como desarrollar t cnicas para obtener copias gen ticas El cipr s de la cordillera Austrocedrus chilensis es una c
9. rooting by hypocotyl cuttings Tree Physiology 15 41 45 Hartmann HT DE Kester FT Davies Jr RL Geneve 2002 Plant Propagation Principles and Practices New Jersey USA Prentice Hall 880 p Havrylenko D 1965 Contribuci n dendrol gica al conocimiento del cipr s de la cordillera Austrocedrus chilensis Don Florin et Boutel Revista Forestal Argentina 9 3 78 80 Hechenleitner VP MF Gardner PI Thomas C Echeverr a B Escobar P Brownless AC Mart nez 2005 Plantas Amenazadas del Centro Sur de Chile Distribuci n Conservaci n y Propagaci n Valdivia Chile Universidad Austral de Chile y Real Jard n Bot nico de Edimburgo 188 p Hinesley LE FA Blazich LK Snelling 1994 Propagation of Atlantic white cedar by stem cuttings Horticultural Science 29 217 219 IPCC 2008 Climate Change and Water Intergovernmental Panel on Climate Change technical paper VI In Bates Kundzewicz BC ZW Wu S Palutikof JP eds IPCC Secretariat Geneva Switzerland 210 p Leakey RRB Z Tchoundjeu K Schreckenberg SE Shackleton CM Shackleton 2005 Agroforestry tree products AFTPs targeting poverty reduction and enhanced livelihoods nt J Agric Sustain 3 1 23 BOSQUE 30 1 18 26 2009 Vegetative propagation patagonian cypress Littell RC GA Milliken WW Stroup RD Wolfinger O Schabenberger 2006 Chapter 8 Random Coefficient Models In SAS for Mixed Models Second Edition SAS Institute Inc Cary NC USA p 317 341
10. the higher the hormone doses applied should be Hartmann et al 2002 In this work the cuttings taken from San Ramon population ca 100 year old trees were in general harder than those from Cerro Otto ca 40 year old trees Nevertheless the only positive results among the adult genotypes were from San Ramon In future experiments juvenile seedlings or rejuvenated grafted or hedged plants material should be used in order to precisely study the genetic variation in rooting ability for A chilensis Studies with hypocotyl cuttings have been developed in conifers Greenwood and Weir 1994 and could be a useful approach in order to avoid the effect of age using progenies as cutting donors More research is necessary to study also the influence of the environmen tal factors on the rooting process and to adjust practical methodologies Higher doses of auxins should be assayed in experiments with larger plot sizes Trevor Hinchliffe 2007 Vegetative propagation of Cupressaceae species from Oceania Terra Flora Landscape Services NSW Australia pers com electronic mail 23 BOSQUE 30 1 18 26 2009 Vegetative propagation patagonian cypress Grafting It is widely accepted that in plants grafts made between genetically related individuals are more likely to be successful than those between unrelated genotypes In conifers notably in Pseudotsuga menziesii Mirb Franco Copes 1969 1999 incompatibility problems are common
11. very specific and stable environmental niches without the need of genetic variation Geneve 2006 In F cupressoides Veblen and Ashton 1982 and P uviferum Rovere et al 2004 vegetative reproduction by branch layering is an important adaptation to humid and bad drained soils in which clonal stand structures are common In A chilensis natural vegetative propagation by branch layering is also observed Gallo et al 2004 Although there is no specific environmental situation in which it becomes the predominant way of reproduction the rela tive frequency of clonal phenotypes is higher in the xeric marginal extreme populations suggesting an adaptive value for natural rooting capacity Although no statistical analyses could be performed for the genetic factors assayed in this work it must be pointed out that 91 6 11 on 12 of the positive responses corresponded to xeric genotypes Despite the limitations of our experimental design one derived hypothesis from these results is that in the ex tremely adverse xeric environment of the ecotone between the subantarctic forest and the patagonian steppe clonal reproduction is an adaptation that complements a highly fluctuating and climate dependent seed regeneration The competitive advantage of the multi stemmed phenotypes Pastorino and Gallo 1997 would be larger in the xeric than in the mesic and humid provenances and therefore the genotypic frequencies of individuals with natural rooting
12. Argentina Santiago Chile Editorial Universitaria p 23 37 Donoso C B Escobar M Pastorino L Gallo J Aguayo 2006 Autoecolog a de las especies Con feras chilenas Gimnospermas In Donoso Zegers C ed Las especies arb reas de los bosques templados de Chile y Argentina Autoecolog a Valdivia Chile Marisa Cuneo Ediciones p 54 67 Gallo LA MJ Pastorino C Donoso 2004 Variaci n en Austrocedrus chilensis D Don Pic Ser ef Bizzarri Cipr s de la Cordillera Jn Donoso C A Premoli L Gallo R Ipinza eds Variaci n intraespec fica en las especies arb reas de los bosques templados de Chile y Argentina Santiago Chile Editorial Universitaria p 233 251 Geneve RL 2006 Alternative strategies for clonal plant repro duction Combined Proceedings of the International Plant Propagators Society 56 269 273 G mez Hern ndez CP 1994 Propagaci n vegetativa de Austrocedrus chilensis D Don Pic Ser et Bizz cipr s de la cordillera Prumnopitys andina Poepp ex Endl de Laub lleuque Eucryphia glutinosa Poepp et Endl Baillon guindo santo y Orites myrtoidea P et E B et H ex Sleumer radal enano mediante enraizamiento de estacas Professional practice report Biologist degree Concepci n Chile Facultad de Ciencias Naturales y Oceanogr ficas Universidad de Concepci n 77 p Greenwood MS RJ Weir 1994 Genetic variation in rooting ability of loblolly pine cuttings effects of auxin and family on
13. BOSQUE 30 1 18 26 2009 Vegetative propagation of patagonian cypress a vulnerable species from the subantarctic forest of South America Propagaci n vegetativa del cipr s de la cordillera una especie vulnerable del bosque subant rtico de Sudam rica Alejandro Aparicio Mario Pastorino Alejandro Martinez Meier Leonardo Gallo Corresponding author Instituto Nacional de Tecnolog a Agropecuaria Unidad de Gen tica Forestal EEA Bariloche CC 277 8400 Bariloche Rio Negro Argentina tel 54 02944 422731 fax 54 02944 424991 aaparicioObariloche inta gov ar bCONICET Consejo Nacional de Investigaciones Cient ficas y T cnicas Argentina SUMMARY Increasing risks of loosing forest genetic resources and the need for a sustainable quality wood supply make focusing research efforts on undomesticated tree species necessary The study of their genetic variation in vegetative propagation ability and the development of practical methodologies to obtain genetic copies are essential tools for conservation and breeding programs patagonian cypress Austrocedrus chilensis is a vulnerable conifer native to the subantarctic forest of South America with high ecological importance and economical potential Its genetic diversity mostly sheltered within unprotected populations is seriously threatened This work examined the ability of different genotypes of A chilensis from xeric mesic and humid populations to propagate asexually by stem
14. Mart nez Crovetto R 1948 Notas sobre plantas ind genas culti vadas en la Argentina Revista de Investigaciones Agr colas 2 3 105 116 Mes n F 1998 Enraizamiento de estacas juveniles de especies forestales uso de propagadores de sub irrigaci n Turrialba Costa Rica Serie T cnica 36 p Manual T cnico CATIE n 30 Park YS JM Bonga TJ Mullin 1989 Clonal Forestry In Mandal AK GL Gibson eds Forest Genetics and Tree Breeding New Delhi India CBS Publishers and Distributors p 143 167 Pastorino MJ 2001 Genetic Variation and Reproduction System of Austrocedrus chilensis D Don Florin et Boutelje a cypress endemic to the Andean Patagonian Forest Ph D Thesis G ttingen Germany University of Gottingen 165 p Pastorino MJ LA Gallo 1997 Natural vegetative propagation of Austrocedrus chilensis Cupressaceae in marginal popula tions II Southern Connection Congress Southern temperate biota and ecosystems past present and future proceedings Valdivia Chile Universidad de Chile y Universidad Austral de Chile p 178 Pastorino MJ LA Gallo HH Hattemer 2004 Genetic variation in natural populations of Austrocedrus chilensis a cypress of the Andean Patagonian Forest Biochemical Systematics and Ecology 32 998 1008 Pastorino MJ MM Farifia D Bran LA Gallo 2006 Extremos geogr ficos de la distribuci n natural de Austrocedrus chilensis Cupressaceae Bol Soc Argentina de Bot nica 41 3 4 307 311
15. age tissues consistence and vigor type 1 hardwood stems with small hard and compact foliage type 2 semi hardwood stems with remarkable growth from the current season and type 3 semi herbaceous shoots with long vigorous internodes from the current year and big leaves Characteristics of scions donors are shown in table 2 In experiments 5 and 7 three secions replicates from each donor genotype were assayed N 42 Each genotype was grafted over three seedlings rootstock corresponding each one to humid mesic and xeric provenances 1 Puerto Blest 2 Cerro Leones CL and 3 Pilca eu Rootstock consisted of three to six year old seedlings with a mean height of 50 10 cm In spring trials they were condi tioned inside the greenhouse 30 days before grafting in order to promote activity In experiment 6 the ortets with the best and worst performances in experiment 5 early evaluated by visual signs of decay were assayed Additionally two adult genotypes corresponding to an extremely rare fastigiated Genotipos utilizados como dadores de p as en los experi mentos de injertado Denomination Provenance Sex Experiment Ol Cerro Otto Female 5 7 02 Cerro Otto Male SET 03 Cerro Otto Juvenile 5 7 04 Cerro Otto Female 5 7 O5 Cerro Otto Female 5 7 06 Cerro Otto Juvenile 5 7 O7 Cerro Otto Female 5 7 SR10 San Ram n Female 57 SR11 San Ram n Male Sol SR12 San Ram n Female 5 7 6 SR13 San Ram n Female SeT SR7 San Ram n M
16. ale 5 7 SR8 San Ram n Male 5 7 6 SR9 San Ramon Male 5 7 FIAV Rio Traful Male 6 F2LM Cerro Otto Male 6 variety of patagonian cypress denominated A chilensis f stricta nov form Havrylenko 1965 were included An individual plot layout was used with 18 ortet SR12 21 ortet SR8 11 ortet FIAV and 42 ortet F2LM replicates All grafts were practiced using provenance P seedlings as rootstock 21 BOSQUE 30 1 18 26 2009 Vegetative propagation patagonian cypress Apical cleft grafting was applied in all three experi ments Scion and rootstock were tied together using rubber grafting tapes and a contact sealing fungicide was applied covering the unions Plastic bags were placed over the grafts to keep up humidity Systemic fungicide was applied to the rootstock each 15 days starting three weeks after grafting Plastic bags were partially opened two months after graft ing and completely removed one month later Survival was evaluated during the growing season following each experiment Scions vegetative growth resume was the criteria to determine graft success Data were analyzed using the generalized linear model with GENMOD procedure in SAS 8 0 SAS Institute Inc Cary N C USA testing the null hypothesis that the response variable graft success binomial response was independent of scion and rootstock provenances and of scion type when corresponding The provenances used as scions and rootstock were selected en
17. ation capacity Although some isolated attempts have been made to asexually propagate A chilensis G mez Hern ndez 1994 or have been mentioned in literature Mat nez Crovetto 1948 Havrylenko 1965 Hechenleitner et al 2005 not enough scientific and systematic research has been devel oped in order to elucidate the genetic physiological and ecological factors involved in macro propagation processes such as stem rooting or scion grafting Currently a clear vegetative propagation protocol does not exist Several adaptive and ecological characteristics of A chilensis suggest that its natural rooting ability would be poor As most conifers it has no adaptations to asexually persist after severe disturbances such as preformed root initials allowing rooting of dislodged stems or the ability of sprouting from stumps or roots Although with fluctua tions in time A chilensis produces large seed outputs with high viability Donoso et al 2006 propagating efficiently by sexual means even in the adverse xeric limits of its distribution Nevertheless adventitious roots formation eventually occurs in natural populations Compact groups of apparently individual trees more frequently observed in the xeric marginal populations have been found to be clones multi stemmed phenotypes formed by branch lay ering Pastorino and Gallo 1997 Gallo et al 2004 This natural process is probably a good signal for the success of artificial vegetative
18. compassing the entire rain gradient and the combinations between scion and rootstock were selected in order to avoid genetic relationships between both grafted genotypes thus augmenting the probability of incompatibilities The main geographic characteristics of the populations used for all the rooting and grafting experiments are shown in table 3 RESULTS Stem cuttings Percentage of rooting was very low in all the four experiments and therefore no statistical analysis on the results was applicable On a total of 1 469 assayed cut tings from adult genotypes only four produced adventitious roots 0 27 and corresponded to the xeric provenance genotypes SR1 late summer experiment 5 600 mg L IBA level SR4 late spring experiment 4 200 and 7 000 mg L IBA levels and SRS late spring experiment 7 000 mg L IBA level For the juvenile genetic entities percent of rooting was also very low with eight rooted cuttings on a total of 370 2 10 Only one treatment P12 progeny multi stemmed mother produced remarkable results in mid summer experiment 33 33 5 on 15 of rooting was obtained at 5 000 mg L IBA level The same progeny produced also two rooted cuttings 13 33 with the NAA gel treatment in the same experiment Only one cutting corresponding to a non xeric provenance juvenile seedlings from B provenance rooted late winter experiment 7 000 mg L IBA level No positive responses were obtained in the control pl
19. ed in the late summer experiment produced callus These extreme responses to wounding early versus late growing season could be indicating that the auxin gradients that are involved in the callusing process Hartmann ef al 2002 diminish rapidly during the activity period in A chilensis Scion stem tissue consistence in relation to season of grafting was found to be an important factor affecting grafting success Scions with better performances were semi hardwood stems with vigorous shoots and long in ternodes from the current growing season The higher rate of failure of hardwood scions with compact foliage and short internodes can be attributable in part to anatomical mismatching due to sinuosity and hardness of the stems Although no other grafting method was assayed in this work the apical cleft grafting was found to be adequate in relation to the growth activity of both scions and rootstock in early spring Grafting methods in which the top of the rootstock is completely removed are usually dependent on a moderate activity of the rootstock Hartmann et al 2002 and the phenological stage of the scion donor plant is equally determinant of success In the adult male individuals used as ortet in our early spring experiment pollen aments were recently developed at the moment of scions collection This phenological stage could be used 24 as a practical indicator of a favorable time for grafting in A chilensis CONCLUSIONS Au
20. financial support of the Agencia Nacional de Promoci n Cient fica y Tecnol gica ANPCyT through the project PICT 12422 Mejoramiento Gen tico de Cipr s de la Cordillera como alternativa productiva para la Precordillera Nordpatag nica REFERENCES Atangana AR Z Tchoundjeu EK Asaah AJ Simons DP Khasa 2006 Domestication of Allanblackia floribunda Amenability to vegetative propagation Forest Ecology and Management 237 246 251 Bran D A P rez D Barrios M Pastorino J Ayesa 2003 Mapa de distribuci n del Cipr s de la Cordillera Fundaci n Vida Silvestre Argentina Bosques y Selvas Accessed Jul 10 2005 Available in http www vidasilvstre org ar Conifer Specialist Group 1998 Austrocedrus chilensis In IUCN 2007 2007 IUCN Red List of Threatened Species Accessed Nov 25 2007 Available in http www iucnredlist org Copes DL 1969 Graft union formation in Douglas fir American Journal of Botany 56 3 285 289 Copes DL 1999 Breeding graft compatible Douglas fir rootstocks Pseudotsuga menziesii Mirb Franco Silvae Genetica 48 3 4 188 193 Dick JMcP H Bisset C McBeath 1996 Provenance variation in rooting ability of Calliandra calothyrsus Forest Ecology and Management 87 175 184 Donoso C L Gallo 2004 Aspectos conceptuales y metodol gicos In Donoso C A Premoli L Gallo R Ipinza eds Variaci n intraespec fica en las especies arb reas de los bosques templados de Chile y
21. ings experiments Rooting experiments were conducted at four different seasons experiment 1 late spring starting on 16 November experiment 2 mid summer starting on 4 February experiment 3 late summer starting on 12 March and experiment 4 late winter starting on 18 August Plot sizes were 8 15 8 and 10 respectively Each experiment was finished after 60 days The adult donor trees ortets were selected within two contrasting provenances 1 Cerro Otto O a mesic population and 2 San Ram n SR a xeric population The average ages of the sampled stands were 40 5 and 100 20 years respectively In SR population both single and multi stemmed genotypes were selected Cuttings were collected from partial reiterations from the lower parts of the crowns Third order semi hardwood stems of 7 to 9 cm long were prepared leaving a remaining leaf area of ca 5 cm in the distal extreme Cuttings from juvenile individuals corresponding to three xeric H Huinganco M El Mait n P Pilca eu one mesic O and one humid B Puerto Blest provenance were collected from seedlings bred in our experimental nursery Complete softwood or semi hardwood second order stems were used Size and leaf area were similar to those of the adult cuttings Characteristics of both adult and juvenile ortets are sum marized in table 1 All four experiments were designed in order to evaluate the variation in rooting ability as a function of the genetic e
22. ipitations regime molds the physiognomy of A chilensis forests that grow along a narrow strip bordering the Cordillera de Los Andes from 37 OT to 43 44 S Pastorino et al 2006 Across this strip in a west east direction mean annual precipitation m a p ranges from ca 3 000 to less than 400 mm This extremely abrupt gradient determines the occurrence of drastic changes in vegetation in distances not larger than 100 km being five A chilensis forest types recognized Gallo et al 2004 The most xeric populations extreme marginal forest type consisting of small isolated patches BOSQUE 30 1 18 26 2009 Vegetative propagation patagonian cypress located into the patagonian steppe were found to be the most genetically diverse Pastorino 2001 Pastorino et al 2004 Despite the evolutionary and adaptive relevance of these xeric extreme marginal populations they still lack of any national or international legal protection policy Their location in private lands the extreme environmental conditions in which they grow their reduced number of individuals and a context of rapid climate changes threaten the genetic diversity they shelter as well as their dynamic evolutionary processes being the rescue and ex situ in vivo conservation of endangered genotypes or even entire populations necessary These extreme marginal populations have to be included in research works oriented to study adaptive traits such as vegetative propag
23. n and domestication programs for A chilensis METHODS Propagation environment Four stem rooting and three scion grafting trials were conducted in an experimen tal greenhouse located at INTA Instituto Nacional de Tecnologia Agropecuaria in San Carlos de Bariloche city latitude 41 07 S longitude 71 15 W altitude 810 m asl Argentina The greenhouse was equipped with intermittent mist run four times per day during the maxi mum temperature hours Relative air humidity inside the greenhouse was maintained between 55 and 95 while mean minimum and maximum temperatures were 10 3 and 25 4 5 C in spring experiments and 7 4 and 25 5 C in late summer experiments These last environmental parameters were relevant for the grafting experiments For stem cuttings experiments a rooting bed with bottom heat and a plastic tunnel was used Bed substrate consisted of inert volcanic sand kept at field capacity humidity level by means of sub irrigation Substrate temperature was regulated between 21 and 28 C Air humidity inside the 20 tunnel was maintained between 75 and 100 In late spring and winter experiments the mean daily air temperature inside the propagation tunnel was 19 3 C while in summer and late summer it was 21 2 5 C Substrate was disinfected with soil fungicide before the installation of the experiments and a systemic fungicide was applied to the cuttings each 15 days Rooting of stem cutt
24. nalyzing a particular group of plants e g narrow leaved evergreens are species specific As an example stem cuttings of Chamaecyparis lawsoniana A Murr Parl root better if collected during the dormant period Zobel 1990 while Chamaecyparis thyoides L roots easily during the activity period Hinesley et al 1994 In the case of A chilensis as a slow growing and long lived conifer the main problem facing its vegetative propagation could be related to the ontogenetic stage of the donor plants The most endangered and genetically variable populations consist mostly of mature and over mature trees what a priori supposes a great disadvantage particularly for stem rooting Related to the ontogeny of plants several characteristics of the vegetative material such as tissue consistence physiological conditions vigor and health are major factors influencing both rooting and grafting processes Hartmann et al 2002 The objective of this work was to comprehensively investigate 1 the genetic variation in rooting ability of A chilensis considering the influence of the season of cuttings collection the ontogenetic stage of the donor plant and the exogenous auxin supply and 2 the grafting capacity and compatibility of different genetic origins of A chilensis under the influence of season and type of vegetative material We report basic information to support future research works and to develop practical methodologies for conservatio
25. ncy was observed in relation to the season of cutting collection In the winter experiment with juvenile ortets only one cutting rooted on a total of 230 0 43 while in mid summer experiment seven juvenile cuttings rooted on a total of 150 4 66 Moreover three of the four rooted adult cuttings were ob tained in the late spring experiment These results could be indicating that although A chilensis would be a hard to root species even in juvenile stages the activity period would be more favorable for adventitious root formation In difficult to root woody species the ease of adventi tious root formation tends to decline with the physiological age of the stock plants due to several progressive changes in morphological anatomical and biochemical traits Hartmann et al 2002 The mentioned behavior of P12 progeny 33 of rooting ability composed of 10 open pollinated five year old seedlings resulted contrasting to all the other treatments probably indicating that ontogeny results crucial in determining the rooting capacity of A chilensis In relation to the supply of exogenous auxins the fact that none of the control cuttings 0 on 289 in any of the experiments produced roots and that the 10 rooted cuttings within the IBA plots corresponded to doses between 4 200 and 7 000 mg L could be indicating that higher levels of exogenous auxins are needed to promote rooting In general the more lignified the vegetative ma terial
26. ntity treatment Each plot corresponded to one replicate of each treatment One different level of indole 3 butyric acid IBA alcoholic solution 1 400 2 800 4 200 5 600 and 7 000 mg L was randomly assigned to each plot corresponding to the adult genotypes in all the four experi ments and to the juvenile treatments in experiment 4 An additional 5 000 mg L IBA plot was included for all the treatments in experiment 4 and for the adult genotypes and P12 juvenile progeny in experiment 2 Quick dip method Mes n 1998 was used to apply the auxin alcoholic solu tions with a five seconds immersion time Additionally two commercial products with o naphtalene acetic acid NAA 1 000 mg L concentration gel and powder were applied to all the treatments in experiment 2 and to P12 juvenile progeny in experiment 4 A control plot 0 mg L auxin level was included for each assayed genetic entity in all the four experiments Our experimental designs allow analyzing the response variable percentage of rooting using the linear random coefficient regression model Littell et al 2006 with the auxin level IBA level as the covariate BOSQUE 30 1 18 26 2009 Vegetative propagation patagonian cypress Table 1 Cuttings donor trees used in the rooting experiments Genotipos utilizados como dadores de estaquillas en los ensayos de enraizado Denomination Provenance Ontogeny Sex Type of stem Experiment SR1 San Ram n Adult Female
27. on fera vulnerable del bosque subant rtico de Sudam rica de gran importancia ecol gica y potencial econ mico Su diversidad gen tica se encuentra seriamente amenazada En este trabajo se examin la capacidad de diferentes genotipos de poblaciones x ricas m sicas y h medas de A chilensis para propagarse asexualmente por enraizado de estacas y por injertos Se analiz adem s la influencia de auxinas ex genas de la estaci n de colecta del estadio ontog nico de las plantas donantes de la estaci n de injerto y del tipo de p a La capacidad de enraizado fue muy pobre tanto para ortets adultos promedio 0 27 como para juveniles promedio 2 10 S lo una progenie juvenil de procedencia x rica mostr resultados remarcables con 33 de enraizado Los injertos mostraron buenos resultados a comienzos de primavera usando esquejes vigorosos y semile osos con un 80 95 de prendimiento La compatibilidad entre genotipos de diferentes procedencias usados como p a y portainjertos fue amplia Estos resultados indican que aunque A chilensis es una especie de dif cil enraizado su propagaci n por injertos es factible utilizando tecnolog a de bajo costo Palabras clave Austrocedrus Cupressaceae enraizado de tallos injertos INTRODUCTION in forest research most of the efforts were traditionally focused on propagation of timber species a scenario of Vegetative propagation is an irreplaceable tool for rapid climate changes IPCC 2008
28. ots 0 mg L IBA level Grafting Early spring was found to be the most adequate season for apical cleft grafting in A chilensis After one year 80 95 34 on 42 of the practiced grafts succeeded in experiment 5 Grafting success was significantly different according to scion type yx 10 11 P 0 0064 type 1 scions hardwood had only 37 50 of success while types 2 semi hardwood and 3 semi herbaceous showed 92 85 and 90 00 of successful grafts respectively Success was independent of rootstock and scion provenances indicating a wide compatibility among genotypes from the different origins Only genotype SR8 did not produce any successful graft in this experiment All the practiced grafts showed callus formation in early spring experiment In experiment 6 mid spring percentage of graft success was very low with an average of 6 52 6 on a total of 92 Positive responses corresponded to genotype SR12 1 on 18 genotype FIAV 1 on 11 and F2LM 4 on 42 Genotype SR8 did not produce any successful graft In opposition to early spring results no positive re sults were obtained in experiment 7 suggesting that late summer is not favorable for A chilensis propagation by apical cleft grafting Callus formation was not observed in this experiment DISCUSSION Genetic variation in rooting ability Typically the capac ity of producing adventitious roots from stems is related to several natural adaptations of species i e
29. ple geographic distribution its ecological and multi purpose functions and its potential timber value It ranges through a wide variation in eco logical conditions regarding latitude altitude soils and pluviometric regime Gallo et al 2004 which suggests a large phenotypic plasticity Its adaptation to extreme xeric conditions makes it a suitable species for commercial forestry in arid environments Its wood has been widely used in the region during all the last century due to its good technological quality and beautiful design Even without forest management and genetic improvement A chilensis wood is the most valuable among the conifers in Argentina SAGPyA 2008 The remaining total cover area of A chilensis has been measured in 142 000 ha Bran et al 2002 from an estimated original of about two million Schmaltz 1992 being the species categorized as vulnerable Conifer Specialist Group 1998 The total of the wood of A chilensis consumed in the region still comes from natu ral stands and an increasing demand exists for seedlings for commercial plantations and restoration purposes For these reasons a domestication and conservation program has been recently initiated at the Instituto Nacional de Tecnologia Agropecuaria Bariloche In Argentina the distribution of A chilensis corre sponds to a Mediterranean like climate with a tendency to a moderate to severe aridity One remarkable environ mental gradient given by the prec
30. strocedrus chilensis is a hard to root species Genetic variation for rooting ability as an adaptive trait deserves more research Probably the most genetically variable and endangered xeric marginal populations have relatively higher frequencies of genotypes with natural rooting ability which would be an advantage for conservation and breed ing objectives once the environmental factors affecting the rooting process are optimized It is likely that acceptable rooting percentages can be achieved for certain genotypes when using juvenile or rejuvenated material and higher exogenous auxin doses Grafting capacity of A chilensis was high and compatibility among genotypes was wide enough to assure good results at low costs The optimal season for apical cleft grafting was early spring in north Patagonia Several objectives of present and future research programs could be enhanced using vegetative propagation of A chilensis by scion grafting Rescue of genotypes from the extreme marginal populations the most genetically diverse and endangered should be a priority for conserva tion of the genetic resources of the species as well as for other rare genotypes such as the fastigiated variety that counts with less than 10 known individuals in the wild Grafting could also be useful to study the transference and the genetic resistance of a widespread disease locally known as cypress decay ACKNOWLEDGEMENTS This work was developed with the
31. with increasing land tree domestication and breeding and its advantages and degradation and genetic diversity loss makes it necessary to implications have been widely treated in literature Wright focus also on species that are important for other functions 1976 Zobel and Talbert 1984 Park et al 1989 Although such as support of ecosystems and supply of non timber 18 products or have a remarkable potential for quality wood production Programs involving indigenous species and im poverished communities have become important in the last decades Leakey et al 2005 and the development of low cost vegetative propagation technologies is one of its most relevant aspects e g Tchoundjeu et al 2004 Atangana et al 2006 Despite the advances in tissue culture for many conservation domestication and breeding programs lower cost macro propagation methods continue to be the most convenient approaches even when human and financial resources were not scarce e g Wollemia nobilis J H et Allen program Wollemi Pine International 2004 Only a few tree species native to the subantarctic forests of South America are included in domestication and conservation programs being several species of Nothofagus spp among the most relevant Donoso and Gallo 2004 In the Argentinean Patagonian region patagonian cypress Austrocedrus chilensis D Don Pic Ser et Bizzarri Cupressaceae is one of the most important native tree species because of its am
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