Session IX
PROPAGATION AND IN VITRO MANIPULATION
Session IX
PROPAGATION AND IN VITRO MANIPULATION
Chairs
Luc Harvengt
Margarida Oliveira
Keynote Talks
SIX.1 SOME TRENDS AND PERSPECTIVE IN APPLICATION OF PLANT TISSUE
CULTURE FOR INDUSTRIAL FORESTRY
Luc Harvengt*, Jenny Aitken
Oral Communications
SIX.2 SOMATIC EMBRYOGENESIS OF SCOTS PINE: INITIATION OF CULTURES
FROM MATURE TREE EXPLANTS AND ENHANCEMENT OF CULTURE
SYSTEM
Tuija Aronen*, Leena Ryyanänen, Ravindra Malabadi
SIX.3 CLONAL PLANTATION OF EUCALYPTUS GLOBULUS IN WESTERN
AUSTRALIA
Akiyoshi Kawaoka*, Toshiaki Tanabe, Keiichi Shimizu, Yuji Fujii, Kazunori Hayashi,
Chris Schiller, Kunichika Murakami
SIX.4 TISSUE CULTURE IN CHERIMOYA: CURRENT STATUS
C.L. Encina*, E. Carmona Martin, I.M.G. Padilla, L. España Ramirez, E. Caro,
SIX.5 THE LONG WAY TO CLONAL VARIETIES FOR CHRISTMAS TREE
PRODUCTION: ACHIEVEMENTS AND PROBLEMS WITH SCALING UP IN
VITRO PROPAGATION USING SOMATIC EMBRYOGENESIS
Kurt Zoglauer*, Claudia Aurich, Markus Richter, Marion Zäpernick, Matthias Zander
SIX.6 GENETIC TRANSFORMATION OF EUCALYPTUS SALIGNA WITH P5CS GENE
Roberson Dibax, Marilia K.F. Campos, Cícero Deschamps, João C. Bespalhof F., Luiz G.
Vieira, Marguerite Quoirin*
SIX.7 BIOTECHNOLOGICAL APPROACHES TO CONSERVATION, PROPAGATION
AND BREEDING OF ENDEMIC TREE SPECIES FROM THE AZOREAN
ARCHIPELAGO
João Drumonde Neves*, Duarte Mendonça, Margit Laimer, Elena Popowich, Gerti
Steniczka, Veronika Hanzer, Artur da Câmara Machado
Poster Communications
SIX.8p
EFFECT OF DIPHENYLUREA DERIVATIVES ON ADVENTITIOUS ROOTING
AND SOMATIC EMBRYOGENESIS IN WOODY SPECIES
Enrico Rolli, Carmen Diaz-Sala, Angela Carra, Ada Ricci*
SIX.9p
TRANSFORMATION OF PINUS PINASTER FOR STUDYING NITROGEN
METABOLISM REGULATION
Ana Milhinhos*, Célia Miguel, M. Margarida Oliveira, Susana Tereso
SIX.10p IN VITRO CLONING OF CHERIMOYA (ANNONA CHERIMOLA MILL.)
C.L. Encina*, E. Carmona Martín, I.M.G. Padilla, N. Westendorp, E.Caro
SIX.11p CHERIMOYA (ANNONA CHERIMOLA MILL.): REGENERATION AND GENETIC
TRANSFORMATION
C.L. Encina*, I.M.G. Padilla, E. Carmona Martín, N. Westendorp, E.Caro
SIX.12p TOWRADS A BETTER UNDERSTANDING OF CONIFER SOMATIC EMBRYO
DEVELOPMENT VIA PROTEOMIC ANALYSIS
Caroline Teyssier*, Ludovic Bonhomme, Domenico Morabito, Michel Vallance,
Philippe Label, Marie-Anne Lelu-Walter
SIX.13p STUDIES OF BACTERIAL ENDOPHYTES AS A NECESSARY PRECONDITION
FOR SAFE MICROPROPAGATION AND GENE TRANSFER
Dietrich Ewald*, Kristina Ulrich
SIX.14p SOMATIC EMBRYOGENESIS IN ZYGOTIC EMBRYOS FROM HALF-SIB
FAMILIES OF SELECTED PINUS PINEA TREES
Elena Carneros*, Inmaculada Hernández, Jesús Jiménez, Dolores López-Vela, Jesús
Alegre, Mariano Toribio, Cristina Celestino
SIX.15p IN VITRO ESTABLISHMENT AND MULTIPLICATION OF THE HYBRID
EUCALYPTUS BENTHAMII MAIDEN & CAMBAGE X E. DUNNII MAIDEN
Gilvano Ebling Brondani, Leonardo Ferreira Dutra*, Fernando Grossi, Ivar Wendling,
Fabricio Augusto Hansel, Jefferson Hornig Azevedo
SIX.16p MINICUTTING OF LIQUIDAMBAR STYRACIFLUA L.: SUBSTRATUM EFFECT
AND TYPES OF PREPARATION
Gilvano Ebling Brondani, Ivar Wendling, Leonardo Ferreira Dutra*, Fabricio Augusto
Hansel
SIX.17p MICROPROPAGATION OF SELECTED ADULT PLANTS OF ARBUTUS UNEDO
L. (STRAWBERRY TREE)
Filomena Gomes*, Maria L. Lopes, Jorge Agrela, Jorge M. Canhoto
SIX.18p RELATIONSHIP BETWEEN EMBRYO DEVELOPMENTAL STAGE AND
SOMATIC EMBRYOGENESIS INDUCTION IN TWO SPANISH POPULATIONS
OF MARITIME PINE
Alicia Humanez, Jesús Muñoz-Bertomeu, Neftali Mesa, Carmen Brisa, Juan Segura,
Isabel Arrillaga*
SIX.19p MICROPROPAGATION OF BIGNONIACEAE GROWING IN SOUTH KOREA
Jae In Park
SIX.20p BAR GENETIC TRANSFORMATION OF MARITIME PINE
Jean-François Trontin*, Rémy Michel, Arnaud Germain, Hélène Pillet-Emanuel,
Francis Canlet, Luc Harvengt
SIX.21p EFFECT OF ACETOSYRINGONE ON AGROBACTERIUM-MEDIATED
TRANSFORMATION OF EUCALYPTUS CAMALDULENSIS
Regina Quisen, Yohana de O. U. Lima, Francine L. Cuquel, Marguerite Quoirin*
SIX.22p IMPROVING INITIATION OF SOMATIC EMBRYOGENESIS FROM
PORTUGUESE GENOTYPES OF MARITIME PINE (PINUS PINASTER AIT.)
Marta Simões*, Ana Milhinhos, Liliana Marum, Sónia Gonçalves, Pedro Simonini,
Célia Miguel
SIX.23p FASTER EVALUATION OF INDUCED FLORAL STERILITY IN TRANSGENIC
EARLY FLOWERING POPLAR
Hans Hönicka, Matthias Fladung*
SIX.24p TECHNOLOGIES FOR MASS PROPAGATION OF ELITE FOREST TREES: 30
YEARS ACTIVITIES AT THE CRA W.
R. Gruselle, J.P. Misson, J.M. Terzi, O. Arezki, Ph. Druart*
SIX.25p ECOLIRI: TO SUSTAIN TREE BIODIVERSITY OF THE RIVERSIDES
F. Lambot, R. Kipgen, R. A. Chandelier, P. Mertens, S. Adant, C. Husson, M. Jacques,
R. Gruselle, Ph. Druart*
SIX.26p PRE-FOREST: A NEW EUROPEAN TECHNOLOGY FOR COST EFFICIENT AND
ENVIRONMENTAL FRIENDLY PRODUCTION OF PRE-CULTIVATED FOREST
REGENERATION MATERIALS
Rosanna Bellarosa, Lorenzo Ciccarese, Olympia Dini-Papanastasi, Raquel Ferreira*,
Panagiota Kostopoulou, Anders Mattsson, Kalliopi Radoglou, Yannis Raftoyannis,
Bartolomeo Schirone, Marco C. Simeone, Gavril Spyroglou, Peter Johansson, Elisabetta
Margheriti, Nick Rammos
SIX.27p MATURE CORK OAK (QUERCUS SUBER L.) TRANSFORMATION WITH BAR
GENE
Ricardo Javier Ordás*, Rubén Álvarez, Jaime Humara, Mª Ángeles Revilla
SIX.28p SOME TISSUE CULTURE AND ANATOMICAL STUDIES OF SELECTED
TROPICAL TREE SPECIES
Rosna Mat Taha*, Noorma Wati Haron
SIX.29p ORNAMENTAL USE OF SILVER BIRCH WITH YELLOW-VEINED OR WHITEFLECKED LEAVES − MISSION IMPOSSIBLE?
Leena Ryynänen, Tuija Aronen*
SIX.30p MINICUTTING OF LIQUIDAMBAR STYRACIFLUA L.: SUBSTRATUM EFFECT
AND TYPES OF PREPARATION
Gilvano Ebling Brondani, Ivar Wendling, Leonardo Ferreira Dutra*, Fabricio Augusto
Hansel
SIX.31p SOMATIC EMBRYOGENESIS AND EFFICIENT PLANT REGENERATION
SYSTEM OF JAPANESE CEDAR (CRYPTOMERIA JAPONICA D. DON)
Tsuyoshi E. Maruyama*, Yoshihisa Hosoi
SIX.1 – Keynote talk
SOME TRENDS AND PERSPECTIVE IN APPLICATION
OF PLANT TISSUE CULTURE FOR INDUSTRIAL
FORESTRY
Luc HARVENGT1*, Jenny AITKEN2
1
AFOCEL, Biotechnology lab, Nangis, France, 2The Tree Lab P O Box 293, Rotorua,
New Zealand
[email protected]
[email protected]
Bibliographic review of tree biotechnology has been the subject of several good articles
and will not be developed in this presentation. GM technology is currently not applied at
all in commercial forestry outside China. Moreover, GM is the presently (but most likely
temporarily) both badly considered by the general audience and crowded by a high
number of patents on genes and methods. And current trends in regulatory matters as well
as biological constraints will most likely limit its extend to a few niche application.
Rather, a significant part of current GM research targets validation of candidate genes
(CG). CG to be used in marker assisted selection of trees to be mass-propagated through
seed, cuttings or tissue culture. A good clonal in vitro propagation system is an absolute
prerequisite for transgenesis and a very useful tool for genetic studies. Practical
significance for industrial forestry of in vitro propagation is limited to a handful of
species. Hardwoods are mainly poplar, eucalyptus and sweetgum propagated through
micropropagation. Softwoods are mainly pines and spruces propagated by somatic
embryogenesis. Other species x technics combinations are of interest only for specific
steps of breeding or "tissue conditioning" (eg micrografting for rejuvenilisation).
Universities and public research institutes have contributed significantly to initiate the
first achievement in somatic embryogenesis and to unravel basic mechanisms involved in
the phenomenon. But their studies much often involve a few or model lines/species and
run on short term. Private companies and research teams are confronted to the real world
and have to cope with a wealth of interaction effects as well as long term consequences of
tissue culture treatments. Big players like Arborgen and Cellfor dominate the world most
commercial achievements. Beside them, some private forest and industrial companies
achieve propagation of their own plant material. However, a few smaller teams can
reasonably contribute significantly to the practical application of plant tissue culture to
prepare future forests.
SIX.2
SOMATIC EMBRYOGENESIS OF SCOTS PINE:
INITIATION OF CULTURES FROM MATURE TREE
EXPLANTS AND ENHANCEMENT OF CULTURE
SYSTEM
Tuija ARONEN1*, Leena RYYNÄNEN1, Ravindra MALABADI1
1
Finnish Forest Research Institute, Punkaharju Research Unit, Finlandiantie 18,
FI-58450 Punkaharju, Finland
[email protected]
Scots pine (Pinus sylvestris L.) has been involved in forest tree breeding since its
beginning, but enhanced clonal testing and silviculture options have been unattainable
due to recalcitrance of the species in vegetative propagation. Somatic embryogenesis
(SE) of species, starting with immature zygotic embryos, suffers from low initiation rates
and difficulties in embryo germination, but is considered as the most potential method for
producing large amounts of clonal material.
The first objective of this study is regeneration of Scots pine plants via somatic
embryogenesis using vegetative shoot apices of mature trees as explants. This approach
would allow utilisation of selected adult trees to produce reforestation material having
superior growth and wood qualities. Cloning of mature trees has been successful e.g. in
Pinus patula, P. kesiya and P.roxburghii using methods developed by Malabadi et al. SEinitiations using shoot apex explants from 15-year-old Scots pines were done through
growing season 2006. During a period in spring the explants were responsive, and
embryogenic culture was raised from five of the 12 genotypes (42 %) tested. The SE
initiation depended not only on timing, but also on medium and incubation temperature.
According to microscopic observations, the proembryo formation in the initiated cultures
is abundant, and the embryos are well-formed with complete head and prolonged
suspensors. At the time of abstract submission, the SE cultures are under maturation, the
results being presented.
The second objective is to improve individual steps of somatic embryogenesis. In order to
accomplish this, different methods have been compared using material originating in
zygotic embryos, and the results will be presented. For initiation, induction with and
without subculturing was tested, the better initiation rate being achieved in the latter. For
proliferation of SE cultures, growth as calli or as cell masses on filter paper was
examined, the callus proliferation showing higher embryogenic capacity, while the
multiplication rate was better on filter papers. For somatic embryo maturation, the
methods used previously for embryo-driven SE-cultures of Scots pine and a modification
of the technique developed by Malabadi et al. for shoot apex -driven SE-cultures are
compared. For enhancing embryo germination, encapsulation of somatic embryos into
Na-alginate pearls and cold treatment are tested
.
SIX.3
CLONAL PLANTATION OF EUCALYPTUS GLOBULUS IN
WESTERN AUSTRALIA
Akiyoshi KAWAOKA1*, Toshiaki TANABE1, Keiichi SHIMIZU2, Yuji FUJII1,
Kazunori HAYASHI, Chris SCHILLER2, Kunichika MURAKAMI1
1
Forestry Science Research Laboratory, Nippon Paper Industries CO.,LTD, 5-21-1 Oji,
Kita-ku, Tokyo 114-0002, Japan, 2Collie Laboratory, Nippon Paper Industries 2CO.,LTD,
Skipworth Rd., Collie, WA 6225, Australia
[email protected]
Nippon Paper Industries has established clonal production system of E.globulus with a
scale in millions. Planted in our tree plantations for five years in Victoria and Western
Australia, they are maturing vigorously with 1.5 times faster growth than conventional
types, and with uniform size and shape. E.globulus is the profitable tree for pulp and
paper manufacturing. Due to its rapid growth, its harvesting cycle is quite short compared
to other species. This species is easily pulped, and the qualities of its fiber are one of the
best for paper production. Attempts at clonal production have been tried by several
organizations for years, in order to gain more profits from the best individuals (elite trees)
of E.globulus. However, E.globulus is well known as a “hard to vegetative propagate”
species, and a mass clonal production system for this species has not been reported by
others. Obtaining “elite trees” for propagating is another issue to overcome for our
project to succeed. We overcame these challenges using the following strategies:
1. Selecting superior E.globulus trees from our own tree plantation.
Elite candidates (plus trees) of more than 1.5 times of wood volume against the tallest
surrounding trees have been selected. And, the trees with the potential trait of drought
resistance from drought-damaged areas were also selected.
2. Mass propagating plus trees with our patented tissue culture system.
Using our tissue culture procedure, the elite candidates are propagating rapidly within a
short period. The unique photoautotrophic culture system enables E.globulus plants,
which usually have trouble forming roots, to improve rooting capacity at a commercial
level.
We will discuss the advantage of clonal propagation of E.globulus.
SIX.4
TISSUE CULTURE IN CHERIMOYA: CURRENT STATUS
C.L. ENCINA*, E. CARMONA MARTIN, I.M.G. PADILLA, L. ESPÃNA RAMIREZ,
E. CARO
Estación Experimental La Mayora (C.S.I.C.) 29750 Algarrobo-Costa. Málaga. Spain
[email protected]
Anonnaceae is an ancient family of plants, original from the Andean American area, and
seems to be natives to the Ecuador and Peru, including 50 genera whose expansion was
restricted by his agroclimatic requirements. The species cultivated are of the genus
Annona, as: A. squamosa, A. muricata, A. cherimola x A. squamosa and specially Annona
cherimola: the cherimoya, cultivated in Spain, Chile, California, and other countries.
The cherimoya is a species with a high degree of heterozygosis, and this advised against
his propagation by seeds. Also, the results of the useful of the traditional methods of
vegetative propagation were inefficient, due to the low morphogenetic potential of this
specie, especially at the rooting level. To solve these problems, we applied the in vitro
tissue culture techniques to propagate the cherimoya. In our works we use the Fino de
Jete variety, which is the main cultivar in Spain.
After developing the methodologies to micropropagate the juvenile material of cherimoya
(Encina et al., 1994), we optimized a protocol for the micropropagation of adult
cherimoya plants selected by his agronomic characteristics (Padilla and Encina, 2004).
In the present time, our research group develop a breeding program for the cherimoya,
developing several methodologies in vitro: a) Minigraphting, b) Somatic embryogenesis,
b) Adventitious organogenesis and cellular cultures, c) Ploidy manipulation of the
cherimoya, to obtain haploid, tetraploid and triploid plants (seedless), d) Genetic
transformation, for the genes introduction to control ripening processes and the genes
introduction to provide resistance to pathogen and insects.
References
Encina, C.L., Barceló-Muñoz, A., Herrero-Castaño, A. and Pliego-Alfaro, F. (1994) In
vitro morphogenesis of juvenile Annona cherimola Mill. bud explants. Journal of
Horticultural Science 69, 1053-1059.
Padilla, I.M.G. & Encina, C.L. (2004). Micropropagation of adult cherimoya (Annona
cherimola Mill). Cv. Fino de Jete. In vitro Cellular and Developmental Biology-Plant.
40: 210-214
SIX.5
THE LONG WAY TO CLONAL VARIETIES FOR
CHRISTMAS TREE PRODUCTION: ACHIEVEMENTS
AND PROBLEMS WITH SCALING UP IN VITRO
PROPAGATION USING SOMATIC EMBRYOGENESIS
Kurt ZOGLAUER1*, Claudia AURICH1, Markus RICHTER2, Marion ZÄPERNICK2,
Matthias ZANDER3
1
Humboldt University, Institute of Biology, Invalidenstr. 42, 10115 Berlin, Germany,
Horticultural Centre Münster-Wolbeck, Münsterstr. 62-68, 48167 Münster, Germany,
3
Humboldt University, Institute of Horticultural Science, Lentzeallee 55-57, 14195
Berlin, Germany
[email protected]
2
Abies species, first of all Abies nordmanniana, have an enormous commercial importance
for Christmas tree production in Germany and Europe. They are exclusively grown from
seeds harvested form natural population in the Caucasian mountains. Clonal varieties
would help to improve the quality of trees and the cultivation characteristics
considerably. Somatic embryogenesis based propagation methods are expected to become
a realistic possibility to solve these problems in the future. In Abies spp. somatic
embryogenesis has been reported for the first time 1989. In the recent years the
developmental patterns were described in detail and the protocols for initiation,
proliferation, maturation and germination were improved.
All steps of the general protocol for somatic embryogenesis were evaluated. Induction of
somatic embryogenesis was regularly achieved in 70% of the explants, i.e. isolated and
sterilized mature zygotic embryos. Approximately two third of the clones could be easily
propagated (on solid medium or in suspension) and maintained. Most of these clones
produced normally developed mature embryos, however, in less than 50% the embryo
yield was high enough (»100 mature embryos/g FM of plated embryogenic culture) for
large scale embryo production.
Conversion (esp. hypocotyl elongation and root growth) was improved by subculture to
fresh, sucrose-containing maturation medium at the end of the maturation period and
storage at 4°C for at least 4 weeks. Conversion occurred on a hormone-free, activated
charcoal containing medium for 14 days. With a new conversion procedure nearly 100%
of conversion (with low dependency on genotype only) was achieved. In large scale
acclimatization experiments with more than 50.000 plantlets substrates, potting systems,
physical conditions for acclimatization, plant protection and fertilisation were tested. The
results indicate that large scale propagation of Abies species by somatic embryogenesis is
possible. New achievements, problems and perspectives will be discussed.
SIX.6
GENETIC TRANSFORMATION OF EUCALYPTUS
SALIGNA WITH P5CS GENE
Roberson DIBAX1, Marilia K.F. CAMPOS2, Cícero DESCHAMPS1, João C.
BESPALHOK F., Luiz G. VIEIRA2, Marguerite QUOIRIN3*
1
Pós-graduação em Agronomia, Produção Vegetal, UFPR, rua dos Funcionários, S. n.,
Curitiba, PR, Brazil, 2Instituto Agronômico do Paraná, Rodovia Celso Garcia Cid, Km
375, Três Marcos, 86047-902, Londrina, PR, Brazil, 3Depto de Botânica, Universidade
Federal do Paraná, caixa postal 19031, 81531-990, Curitiba, PR, Brazil
[email protected]
The objective of this study was to obtain plants of Eucalyptus saligna expressing the
P5CSF129A gene of proline biosynthesis, by Agrobacterium tumefaciens-mediated
transformation and to measure their proline content. Young leaves from micropropagated
plants maintained in vitro were used as explants and inoculated with strain EHA105
containing a binary vector carrying P5CSF129A and gus gene both under the control of
the CaMV35S promoter, and nptII selection gene. The inoculation was done immerging
half leaves in a bacterial solution (OD600nm= 0.5) for 30 min, followed by a 5-daycoculture in the dark on modified MS culture medium (with half-strength KNO3 and
NH4NO3) supplemented with 0.1 µM NAA and 1 µM TDZ. The explants were then
transferred to the same medium containing 500 mg.L-1 cefotaxime (Cx) and 50 mg.L-1
kanamycin for two 28-day-periods and then the concentration of Cx was diluted at 250
mg.L-1. The calli that appeared on leaf explants were transferred on modified MS medium
containing 0.125 mg.L-1 NAA and 0.25 mg.L-1 BAP for more 28 days under light. Shoots
were first cultured on MS medium with 0.50 mg.L-1 GA3 for 14 days and finally on the
same medium containing 0.25 mg.L-1 BAP for shoot multiplication. The gus expression
was confirmed by histochemical test for ß-glucuronidase and by DNA amplification by
PCR. Transformation efficiency was 0.005%. One of the events was tested for proline
production and showed a four times enhanced production in comparison with not
transformed plant. The transformed plants will be used in physiological test in oder to
study their resistance to abiotic stresses.
SIX.7
BIOTECHNOLOGICAL APPROACHES TO
CONSERVATION, PROPAGATION AND BREEDING OF
ENDEMIC TREE SPECIES FROM THE AZOREAN
ARCHIPELAGO
João DRUMONDE NEVES1*, Duarte MENDONÇA1, Margit LAIMER2, Elena
POPOWICH2, Gerti STENICZKA2, Veronika HANZER2 , Artur DA CÂMARA
MACHADO1
1
Center of Biotechnology of the Azores, Departament of Agricultural Sciences,
University of the Azores, Terra-Chã, 9700-851, Angra do Heroísmo, Portugal, 2Plant
Biotechnology Unit, IAM, Department Biotechnology, BOKU, Muthgasse 18, 1190
Vienna, Austria
[email protected]
Conservation of non domesticated tree species needs to consider the high degree of
genetic variability among individuals, which in case of a small sized population requires
the establishment of several lines from individual plants. Treated as separate clones they
allow the conservation of as many alleles as possible. Their phytosanitary and
physiological status may vary considerably, thus influencing the successful establishment
in vitro. Efficient micropropagation protocols for three woody species, endemic to the
Azores archipelago, Picconia azorica (Tutin) Knobl., (Oleaceae), Prunus lusitanica L.
ssp. azorica (Rosaceae) and Vaccinium cylindraceum J.E.Sm. (Ericaceae) were
developed. Cultures of three Picconia azorica clones were initiated from nodal explants
of greenhouse-grown plants on Ruginis olive medium (ROM), supplemented with 5.0
mg/l 2iP. Different media were compared, and the best results were obtained on Driver &
Kuniyuki (DKV) medium supplemented with 5.0 mg/l 2iP and 0.4 mg/l indole-3-acetic
acid (IAA). For rooting, half strength DKV with different growth regulators was tested.
Cultures of seven Prunus lusitanica clones were initiated on woody plant medium
(WPM), supplemented with 1.5 mg/l 2iP, 0.4 mg/l 6-benzylaminopurine (BAP) and 0.05
mg/l indole-3-butyric acid (IBA). For shoot multiplication different media were
compared and the best results were obtained on DKV supplemented with 1.0 mg/l BAP
and 0.01 mg/l IBA. Half strength DKV with 0.5 mg/l IBA induced the highest rooting
rates. In vitro cultures of four Vaccinium cylindraceum clones were established. Shoot
cultures were maintained on WPM and Anderson’s medium (AM) either with zeatin, 2iP
riboside or 2iP. WPM supplemented with zeatin gave significantly higher multiplication
rates than AM. Root formation was induced in vitro on half-strength WPM with 1 mg/l
IAA.In this study the three studied Azorean species were rooted successfully and
acclimatized to greenhouse conditions with a survival rate above 80 %. Viable plant
material for replanting to the open field was obtained. Given the fact, that these trees and
shrubs have some ornamental potential, the most vigorous clones, beautifully changing
foliage coloration in autumn, will be used in an ornamental and forest plant breeding
programme.
SIX.8p
EFFECT OF DIPHENYLUREA DERIVATIVES ON
ADVENTITIOUS ROOTING AND SOMATIC
EMBRYOGENESIS IN WOODY SPECIES
Enrico ROLLI1, Carmen DIAZ-SALA2, Angela CARRA3, Ada RICCI1*
1
Dipartimento di Biologia Evolutiva e Funzionale, Viale G.P. Usberti 11/A, 43100,
Parma, Italy, 2Departamento Biologia Vegetal, 28871 Alcalá de Henares (Madrid), Spain,
3
Istituto di Genetica Vegetale CNR, Corso Calatafimi 414, 90129 Palermo, Italy
[email protected]
We report new insights on the biological activity of four symmetrically substituted
diphenylurea derivatives, the N,N’-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU), the
N,N’-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU), the 1,3-di(benzo[d]oxazol-5yl)urea (5-BDPU), the 1,3-di(benzo[d]oxazol-6-yl)urea (6-BDPU), in woody species.
Although these compounds are structurally similar to the urea-type cytokinins, none of
them show cytokinin-like activity. However, they all enhance adventitious root formation
in the presence of either endogenous auxin in the M26 apple rootstock microcutting test,
or exogenous auxin in the apple stem slice test. 2,3-MDPU and 3,4-MDPU also enhance
adventitious rooting in Pinus radiata hypocotyls in the presence of low auxin
concentration. In addition, both compounds induce somatic embryos from stigmas and
styles of Citrus madurensis Lour. and Citrus limon (L.), a higher embryogenic induction
was observed when compared with 6-benzylaminopurine (BAP), an adenine-type
cytokinin, and with N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU), an urea-type
cytokinin. The wide spectrum of action of diphenylurea derivatives, and the similar
responses obtained in Gimnosperms and Angiosperms, could indicate conserved
functions. More research is needed to understand their role at the biochemical and
molecular levels.
SIX.9p
TRANSFORMATION OF PINUS PINASTER FOR
STUDYING NITROGEN METABOLISM REGULATION
Ana MILHINHOS1*, Célia MIGUEL1, M. Margarida OLIVEIRA1,2 and Susana
TERESO1
1
Forest Biotech Lab, Instituto de Tecnologia Química e Biológica / Instituto de Biologia
Experimental e Tecnológica, Quinta do Marquês, 2784-505 Oeiras, Portugal,
2
Departamento de Biologia Vegetal, Faculdade de Ciências de Lisboa, Bloco C2, Piso 1,
Campo Grande, 1749-016 Lisboa, Portugal.
[email protected]
Tree improvement is one of the most important goals to achieve in order to face
increasing world demand for forest products. The controlled introduction and expression
of foreign or native genes in maritime pine may provide a powerful tool for the functional
analysis of genes related to plant development. These studies are important to understand
gene function and identify putative genes that, when introduced in elite plants may result
in increased yields. A positive correlation has been found between the higher expression
of glutamine synthetase (GS) gene and an increased growth in Populus. The present study
aims at understanding nitrogen metabolism regulation by glutamine synthetase and its
relation with carbon metabolism and plant development in pine. To accomplish this goal
we are analysing the expression and function of the genes GS1a and GS1b from Pinus
sylvestris in transformed Pinus pinaster. Embryogenic lines re-grown from
cryopreservation infected with Agrobacterium C58pMP90, carrying the GS1a (antisense)
or GS1b (sense or anti-sense) genes. The nptII gene, conferring kanamycin-resistance,
was used as selection marker. After infection, tissues were washed and sub-cultured on
medium containing timentin (400mg.l-1). Six out of ten embryogenic lines recovered from
cryopreservation were able to produce embryogenic tissues after infection. Kanamycin
selection of putative transformed tissues yielded 37 kanamycin-resistant lines, which
were all PCR-negative for virBG and PCR-positive for the nptII. From these lines, 17
were already confirmed as positive for the gene of interest. Maturation of transformed
embryogenic tissues into fully developed somatic embryos is currently taking place.
Presently, from more than 2000 mature somatic embryos obtained from the transformed
tissues, about 40% were able to elongate the hypocotyl and resulting plantlets are
germinating in Magenta flasks. The acclimatization of these potentially transformed
plantlets is underway. After molecular confirmation of the transgenic nature of plantlets
growing in the greenhouse, we aim to conduct biochemical studies to understand the
impact of manipulating glutamine synthetase expression on nitrogen metabolism and its
effects on plant growth.
Acknowledgements: Concepción Ávila and Francisco Cánovas for providing the gene
constructs. ST is supported by a Post-Doc grant (SFRH/BPD/14964/2004). This work is
supported by FCT project POCI/AGR/57157/2004.
SIX.10p
IN VITRO CLONING OF CHERIMOYA (ANNONA
CHERIMOLA MILL.)
C.L. ENCINA*, E. CARMONA, I.M.G. PADILLA, N. WESTENDORP, E. CARO
Estación Experimental La Mayora (C.S.I.C.) 29750 Algarrobo-Costa. Málaga. Spain
[email protected]
Cherimoya (Annona cherimola Mill.) is a difficult-to-propagate species using traditional
methods for vegetative propagation because of the low rooting capacity of adult
cherimoya material. This problem has been solved by the development of clonal
micropropagation protocols (Encina et al., 1994) after nodal explants of juvenile and
adult Annona cherimola cv ‘Fino de Jete’. After sprouting and multiplication of axillary
shoots on MS basal medium supplemented with different cytokinins (BAP and zeatin), a
95% rooting efficiency in juvenile material was achieved in three steps: 1) pretreatment
of in vitro shoots for 7 days on MS medium containing activated charcoal (0.1%); 2) root
induction on MS medium supplemented with IBA (500 µM), sucrose (58.4 mM) and
citric acid (200 mg l-1) for 10 days (7 days in the dark followed by three days light); and
3) elongation of roots in half strength MS medium. The 70% acclimatization rate then
recorded, has later been increased to 100% (Encina et al., 1999a). Using a similar method
adult explants of cherimoya have been micropropagated althought rooting and
acclimatization were about 30% less efficient (Padilla and Encina, 2004).
Other complementary methods such as inoculation of micropropagated plantlets with
arbuscular mycorrhizal fungi (Glomus spp.) improve vegetative growth and development
of plantlets in the glasshouse and substantially reduce acclimatization time and increase
plant recovery (Azcon-Aguilar et al., 1994b)
References
Azcón-Aguilar, C., Encina, C.L., Azcon, R. and Barea, J.M. (1994b) Effect of arbuscular
mycorrhiza on the growth and development of micropropagated Annona cherimola
plants. Agricultural Science in Finland 3, 281-287.
Encina, C.L., Barceló-Muñoz, A., Herrero-Castaño, A. and Pliego-Alfaro, F. (1994) In
vitro morphogenesis of juvenile Annona cherimola Mill. bud explants. Journal of
Horticultural Science 69, 1053-1059.
Padilla, I.M.G. & Encina, C.L. (2004). Micropropagation of adult cherimoya (Annona
cherimola Mill). Cv. Fino de Jete. In vitro Cellular and Developmental Biology-Plant.
40: 210-214
Encina, C.L., Padilla, I.M.G., Cazorla, J.M. and Caro, E. (1999a) Tissue culture in
cherimoya. Acta Horticulturae 497, 289-294.
SIX.11p
CHERIMOYA (ANNONA CHERIMOLA MILL.):
REGENERATION AND GENETIC TRANSFORMATION
C.L. ENCINA*, I.M.G. PADILLA, E. CARMONA MARTIN, N. WESTENDORP, E.
CARO
Estación Experimental La Mayora (C.S.I.C.) 29750 Algarrobo-Costa. Málaga. Spain
[email protected]
Regeneration of multiple adventitious shoots from hypocotyl has been reported in
Annona species: A. squamosa (Lemos and Blake, 1996a), A. muricata (Lemos and Blake,
1996b), A. cherimola x A. squamosa (Rasai et al., 1994) and A. cherimola ( Jordan, 1988;
Encina et al., 1999a).
Adventitious shoot development from leaf and internodal explants has been obtained in
A. muricata (Lemos and Baker, 1998) and in atemoya (Rasai et al., 1994). In cherimoya
protocols for adventitious organogenesis are still under development althought
regeneration at a low rate has already been obtained from juvenile and adult material.
Bud clusters from juvenile explants develop and root easily.
Transformation of cherimoya is also under development, and is focussed to the
introduction of genes (ACC synthase in antisense) in order to control fruit ripening ACC
synthase genes of cherimoya and atemoya have already been cloned.
Future progress on in vitro technologies combined with molecular genetic approaches
will support advances on genetic manipulation and improvement of Annonas.
References
Jordán, M. (1988) Multiple shoot formation and rhizogenesis from cherimola (Annona
cherimola L.) hypocotyls and petiole explants. Gartenbauwiss. 53, 234-237.
Jordán, M., Iturriaga, L. and Goreux, A. (1991). Promotion of Annona cherimola in vitro
shoot morphogenesis as influenced by antoxidants. Gartenbauwiss. 56, 224-227.
Lemos, E.E.P. and Blake, J. (1996a) Micropropagation of juvenile and mature Annona
squamosa L. Plant Cell Tiss.& Org. Cutl. 46, 77-79.
Lemos, E.E.P. and Blake, J. (1996b) Micropropagation of juvenile and mature Annona
muricata L. J. Hor. Sci. 71, 395-403.
Lemos, E.E.P. and Baker, D.A. (1998) Shoot regeneration in response to carbon source
on internodal explants of Annona muricata L. Plant Growth Regulation 25, 105-112.
Nair, S., Gupta, P.K., Shirgurkar, M.V. and Mascarenhas, A.F. (1984a) In vitro
organogenesis from leaf explants of Annona squamosa Linn. Plant Cell, Tiss. & Org.
Cult. 3, 29-40
Rasai, S., Kantharaj, A.S. and Dodd, W.A. (1994) The effect of growth regulators, source
of explants and irradiance on in vitro regeneration of atemoya. Australian J. Bot. 42, 333340.
SIX.12p
TOWARDS A BETTER UNDERSTANDING OF CONIFER
SOMATIC EMBRYO DEVELOPMENT VIA PROTEOMIC
ANALYSIS
Caroline TEYSSIER1*, Ludovic BONHOMME2, Domenico MORABITO2, Michel
VALLANCE1, Philippe LABEL1, Marie-Anne LELU-WALTER1
1
INRA, UR 588 Research Unit: Breeding, Genetic and Physiology of Forest Trees. 2163
Avenue de la Pomme de pin, BP 20 619 Ardon 45166 Olivet Cedex France, 2University
of Orléans, LBLGC, UPRES EA 1207. Rue de Chartres, BP 6759 F-45067 Orléans
Cedex 2, France
[email protected]
Development of clonal propagation method, such as somatic embryogenesis (SE) has
potentially numerous applications such as the production of a large number of genetically
improved plants. It is necessary to optimise somatic embryo development, which remains
difficult in pine species. Since the early protocols of SE were developed for spruce
species (Picea), it was assumed that they would be applicable to pines; however, it
became apparent that pines were less responsive. One limitation to large-scale
propagation of majority of commercial pine species through SE is the lack or low somatic
embryo maturation efficiency, which result in the inability to capture certain families.
This has caused great concerns among breeders and foresters of potential adverse
selection pressure.
Current maturation protocols lead to a development of mature somatic embryos that
morphologically resemble zygotic embryos. These somatic embryos are harvested after
arbitrarily chosen periods of time and are germinated and further grown in a greenhouse.
This procedure only gives an indication that the maturation was successful if produced
somatic embryos were able to convert to plants. Such an empirical approach does not
give any information of the quality of somatic embryos with respect to storage reserves
accumulation or water content, nor does it gives information on the optimal time for
harvesting to achieve maximal plant conversion rates. Therefore, there is a need to
develop markers that could be used for quality control of different batches of somatic
embryos that are matured, or when different maturation protocols are applied. Storage
protein accumulation and hydrous potential have been followed and the identity of the
proteins accumulated by the somatic embryos has been tested by 2D gel electrophoresis.
Final objective is to have a better understanding of the maturation of Pinus pinaster. The
description of SE would contribute to optimise the maturation process and the in vitro
production of plants.
SIX.13p
STUDIES OF BACTERIAL ENDOPHYTES AS A
NECESSARY PRECONDITION FOR SAFE
MICROPROPAGATION AND GENE TRANSFER
Dietrich EWALD*, Kristina ULRICH
Federal Research Centre for Forestry and Forest Products, Institute for Forest Genetics
and Forest Tree Breeding, Eberswalder Chaussee 3A, 15377 Waldsieversdorf , Germany
[email protected]
The occurrence of latent or covert bacteria is a serious problem in micropropagation
systems. Such endophytic bacteria could be detected in in vitro regeneration systems of
many woody plants. While endophytes are generally not harmful to plants, they may
affect culture growth under the modified conditions in vitro by acting as “vitropathogens”
or inducing hormone-mediated modifications. On the other hand, plant growth promoting
effects of bacterial endophytes have been reported in in vitro cultures. During
Agrobacterium-mediated transformation, the presence of endophytic bacteria results in a
potential risk of horizontal gene transfer of recombinant DNA from Agrobacteria to
endophytic bacteria.
The diversity of endophytic bacteria was investigated in in vitro cultures of poplar and
was compared to the bacterial communities found in poplars grown under field
conditions. In contrast to the high phylogenetic diversity of endophytic bacteria in poplars
from the field including Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes, in
most in vitro poplar clones isolates of the genus Paenibacillus dominated. This high
abundance of Paenibacillus was also found for other tree species, e.g. larch, spruce and
yew. Species or strains of this genus, known as plant growth promoting bacteria, seemed
to accumulate especially under in vitro conditions. The population density as well as the
community composition of endophytic bacteria in in vitro cultures were found to be
dependent on source, age and kind of culture. The use of isolated shoot meristems was
revealed as one possibility to establish plantlets free from endophytic bacteria. This in
vitro regeneration system should be taken into account as a measure preventing the
escape of Agrobacterium-mediated vector systems to the environment and providing
reproducible plant tissue culture systems.
SIX.14p
SOMATIC EMBRYOGENESIS IN ZYGOTIC EMBRYOS
FROM HALF-SIB FAMILIES OF SELECTED PINUS PINEA
TREES
Elena CARNEROS*, Inmaculada HERNÁNDEZ, Jesús JIMÉNEZ, Dolores LÓPEZVELA, Jesús ALEGRE, Mariano TORIBIO, Cristina CELESTINO
Instituto Madrileño de Investigación y Desarrollo Rural, Agrario y Alimentario
(IMIDRA). Apartado 127, 28800 Alcalá de Henares (Madrid).
[email protected]
Stone pine (Pinus pinea L.), a forest species widely distributed in the Mediterranean
region, is highly valued for its edible seeds. Due to its economic importance, breeding
programmes for Stone pine have been implemented in Spain. Somatic embryogenesis
may provide a propagation method with advantages in the improvement of forest species.
This work examines the capacity of zygotic embryos from several selected families for
the induction of somatic embryogenesis. The first objective was to determine the
developmental stage of zygotic embryos at each collection date by observing excised
megagametophytes under stereomicroscope. Secondly, was to assess the establishment of
embryogenic lines. This study focuses only on the initiation phase and reports the first
protocol to achieve somatic embryogenesis in Stone pine.
Open-pollinated cones from five selected trees were collected eight times at weekly
intervals during June and July 2006. The maternal trees are situated in a Stone pine clone
bank at the National Forest Breeding Centre 'Puerta de Hierro' (Madrid) of the Spanish
Ministry of Environment.
A modified Litvay’s medium supplemented with different concentrations of 2,4-D and
BAP was tested. Somatic embryogenic lines were obtained from immature zygotic
embryos originating from all of the five seed families assayed. On average, 5% of
explants responded to initiation condition (406/8301). However, only the 8.6 % (35/406)
of the responding explants could be maintained after 4 months of subculture. Proliferating
embryonal masses were obtained in all the collection dates. Three of the families gave the
best response when zygotic embryos were at the cleavage polyembryony stage and the
other two families when the dominant zygotic embryo was formed. Culture medium had
a significant effect on the initiation and establishment of embryogenic lines. The
interactions family x collection date and culture medium x collection date also had a
significant effect on the response.
In spite of the different stages of development and culture conditions, the embryogenic
response was relatively low. This shows that Stone pine is a recalcitrant species for
initiating somatic embryogenesis, and therefore further adjustments on culture conditions
should be done for increasing this response.
The collaboration in this work of Drs. Park, Bonga and Klimaszewska (Canadian Forest
Service) is highly appreciated. Funds were provided by National Project AGL200507585.
SIX.15p
IN VITRO ESTABLISHMENT AND MULTIPLICATION OF
THE HYBRID EUCALYPTUS BENTHAMII MAIDEN &
CAMBAGE X E. DUNNII MAIDEN
Gilvano Ebling BRONDANI1, Leonardo Ferreira DUTRA2*, Fernando GROSSI1, Ivar
WENDLING2, Fabricio Augusto HANSEL2, Jefferson Hornig AZEVEDO1
1
Universidade Federal do Paraná , Av. Lothário Meissner, 3400, Jardim Botânico,
2
80.210-170, Curitiba, Paraná, Brasil, Embrapa Florestas , Estrada da Ribeira, Km 111,
Caixa Postal 319, 83.411-000, Colombo, Paraná, Brasil
[email protected]
The establishment and multiplication are essential steps in the micropropagation of wood
species. The aim of this work was to test concentrations of sodium hypochlorite (NaOCl2)
during disinfestations, and combination of BA and NAA in the multiplication rate of the
spontaneous hybrid Eucalyptus benthamiii x E. dunnii. Young sprouts of ministumps of
three clones (H12, H19 and H20) produced by cuttings were the source of explants. The
ministumps were cultivated in semi-hydroponics system. Nodal segments with 2 axillary
shoots (1.5 cm in length) and without leaf were used as explants. Four concentrations of
NaOCl2 (0.5, 1.0, 1.5, and 2.0%) and twelve combinations of BA and NAA (0, 1, 2 and 3
µmol L-1 of BA with 0, 0.1 and 0.2 µmol L-1 de NAA, only at clone H12) were tested.
The explants were inoculated in MS and JADS mediums in the establishment and
multiplication steps respectively, both mediums were supplemented with 250 mg L-1 of
PVP40 and 30 g L-1 of sucrose, fixed in 6 g L-1 of agar at pH 5.8, and incubated at 25ºC
(±2ºC), photoperiod of 16 hours in light with luminosity of 84µmol m-2 s-1. Establishment
disinfestations: the explants were immersed in ethanol solution 70% for 15s, rinsed with
autoclaved water and then immersed in the treatments of NaOCl2 during 10 min. Then,
they were rinsed with autoclaved water for 3 times and inoculated in tubes (10 cm x 2
cm) with 10 mL of medium. Three typical responses of explants were evaluated weekly
(contamination, oxidation and healthy) and after 21 days the length of the shoot buds
were measured. All treatments were effective with no statistical difference. In general, the
rate of bacteria contamination and oxidation were very low. The fungi contamination was
about 41%. The healthy explants of clone H20 (66%) were bigger than clones H12 (45%)
and H19 (46%). The biggest length of shoots (0.9 cm) was estimated in the concentration
of 1.27% of NaOCl2. Multiplication steps: the shoots of clone H12 were inoculated in
tubes (10 cm x 10 cm) in 20 mL of medium. After 35 days of culture the number of
shoots and the mass of the explants were evaluated. There was an interaction between the
growth regulators used. The best combination was 2 µmol L-1 of BA with 0.2 µmol L-1 de
NAA, which yielded about 6 shoots per explant with a mass of explant around of 50.91
mg. In summary, the contamination of explants used in this work was not a problem, and
the multiplication of explant was viable with a rate of 6 shoots per explant in the best
treatment.
SIX.16p
MINICUTTING OF LIQUIDAMBAR STYRACIFLUA L.:
SUBSTRATUM EFFECT AND TYPES OF PREPARATION
Gilvano Ebling BRONDANI1, Ivar WENDLING2, Leonardo Ferreira DUTRA2*,
Fabricio Augusto HANSEL2
1
Universidade Federal do Paraná , Av. Lothário Meissner, 3400, Jardim Botânico,
2
80.210-170, Curitiba, Paraná, Brasil, Embrapa Florestas , Estrada da Ribeira, Km 111,
Caixa Postal 319, 83.411-000, Colombo, Paraná, Brasil
[email protected]
Minicutting is a clonal alternative for vegetative propagation of individuals with superior
genetic characteristics. Minicutting technique is widespread for Eucalyptus species, but
its application is not common seen to other wood species. The objectives of this work
were to evaluate the effect of different substratum compositions and the types of
preparation of the minicutting in the survival, rooting and vegetative vigor of sweetgum
minicuttings. Sprouts of ministumps produced by cuttings were collected. The
ministumps were cultivated in semi-hydroponics system. The minicuttings (5 cm in
length) had one pair of leaf with 50% of the foliar area. Then, minicuttings were
submitted to four different types of preparation: P1 = paraffin, P2 = paraffin + 8 g L-1 of
IBA, P3 = 8 g L-1 of IBA and P4 = control. The application of the IBA was realized with
the immersion of the basal region of minicuttings in a solution for a period of 10 seconds
and the paraffin application was done in the basal and apical portion of minicutting. The
minicuttings were planted in conic recipient (55 cm3) with three different substratum
combinations (v/v): S1 = rice rind + commercial substratum Plantmax® (1:1), S2 =
carbonized rice rind + commercial substratum Plantmax® (1:1) and S3 = rice rind +
commercial substratum Plantmax® + vermiculite (2:1:1). The minicuttings were kept
under controlled conditions of humidity and temperature in greenhouse; after 91 days of
rooting promotion they were transferred to shadow house for acclimatization during 15
days. Finally, the survival minicuttings were placed in an open garden for up to 35 days.
The survivals of minicuttings were evaluated in the end of each step (greenhouse, shadow
house and open garden). After 141 days of culture the shoots length, number of leaf,
biggest root length and the length of the root system were measured, as well as the visual
aspects of the plant and root formation were evaluated. The types of preparation of the
minicutting no presented statistical difference. However, there was a strong influence of
the substratum in the aerial and root formation. S2 substratum provided the biggest
percentage of rooting (69%), with no necrosis, presented statistical difference of S3
(45%) and S1 (12%). In resume, the rooting of minicuttings of L. styraciflua is
technically viable without the application of IBA and paraffin, with substratum
combination of carbonized rice rind + commercial substratum Plantmax® (1:1).
SIX.17p
MICROPROPAGATION OF SELECTED ADULT PLANTS
OF ARBUTUS UNEDO L. (STRAWBERRY TREE)
Filomena GOMES1*, Maria L. LOPES2, Jorge AGRELA1, Jorge M. CANHOTO2
1
CERNAS, Dep. Florestal, Escola Superior Agrária de Coimbra, Bencanta, 3040 – 316
Coimbra, Portugal, 2Instituto do Ambiente e Vida, Departamento de Botânica, Faculdade
de Ciências e Tecnologia, Universidade de Coimbra, 3001-455 Coimbra, Portugal
[email protected]
Arbutus unedo L. is a species characteristic of Mediterranean climates, widely
represented in southern Europe. The genus Arbutus (Ericaceae) includes about 20 species
from which Arbutus unedo, known as strawberry tree, is the most interesting from an
economic point of view. Several aspects contribute to the economic interest of this plant
including 1) fruit production (fresh or processed to make jellies); 2) production of a spirit
called “Medronheira”, that represents the main income for strawberry tree producers and
(3) the more recent utilization of shoots in the floral industry. Moreover, the plant is
highly resistant to forestry fires, as a result of its low resin content and its ability to
produce new shoots after burning. From an ecological perspective it should also be me
mentioned that A. unedo has the capacity to grow in poor as well as in water deficient
soils making the tree an ideal species to recover degraded lands and to prevent forestry
fires.
Adult plants of strawberry tree, growing in different regions of Portugal were selected for
its potential for fruit production. Branches (30 – 40 cm length) of these trees were
collected in the field and maintained in the greenhouse until epicormic shoots start to
develop. Following sterilisation, shoot tips (< 2 mm) and nodal segments (10-20 mm)
were then used to establish the in vitro cultures.
Best results (38.7 ± 9.8 %, survival rate) were obtained when shoot tips (< 2 mm) were
used. Optimum shoot proliferation was achieved on a basal De Fossard medium (FS; De
Fossard et al., 1974), containing Murashige and Skoog (1962) micro-nutrients, FS
organics, sucrose, and 9 µM benzyladenine (BA). Rooting of the formed shoots occurred
following auxin treatments. The highest root (93.3 %) rates were achieved when shoots
were inoculated in root induction medium, Knop (Gautheret, 1959), with 24.7 µM 3indolebutyric acid (IBA, during 6 days) or dipped on 9.8 x103 µM IBA (for 15 sec), and
followed by its subculture (5 weeks) on the same medium without growth regulators and
containing charcoal (1.5 %). Rooted plantlets were transferred to pots and 84.7 ± 4.6 % of
them acclimatized.
SIX.18p
RELATIONSHIP BETWEEN EMBRYO
DEVELOPMENTAL STAGE AND SOMATIC
EMBRYOGENESIS INDUCTION IN TWO SPANISH
POPULATIONS OF MARITIME PINE
Alicia HUMANEZ1, Jesús MUÑOZ-BERTOMEU1, Neftali MESA2, Carmen BRISA1,
Juan SEGURA1, Isabel ARRILLAGA1*
1
Plant Biology Dpt., University of Valencia, Vicent Andrés Estelles s/n, 46100 Burjassot,
Valencia-SPAIN; 2Dpt. Genética y Biotecnología Vegetal, University of Tolima, IbaguéCOLOMBIA
[email protected]
Pinus pinaster (Aiton), the most extensive conifer in the forests of the Iberian peninsula,
is geographically distributed in three major groups: the Atlantic, the Mediterranean, and
the North African (Curban & Petit, 2003, Mol Ecol). Somatic embryogenesis (SE) has
been applied to propagate selected maritime pine trees from Portugal and France. The
objective of this work is to apply SE to propagate maritime pine from two geographical
locations in Spain. Firstly the relationship between the developmental stage of zygotic
embryo and its capability to induce SE was tested.
Cones were collected weekly (June-July 2006) in Sierra Calderona mountains, Valencia ,
from wild growing trees belonging to the Mediterranean group (populations C1 and C2),
and a clone bank (Granja de San Ildefonso, Segovia) from 5 open pollinated plus clones
belonging to the Atlantic group (V1 toV5). Both mature and immature seeds were
removed from surface sterilized cones and cultured in the dark on two induction media: a
modified Litvay’s medium (Lelu et al. 2006, Plant Cell Rep) and a DCR medium (Miguel
et al. 2004, Plant Cell Tiss Org Cult). Percentage of embryogenic cultures was recorded
after 45 days and embryogenic calli subcultured onto the same medium. For histological
assays prefixed megagametophytes were sliced and stained with toluidine blue.
Embryogenic response (on average 20%) depended on genotype, sample date and culture
medium. Significant double interactions between the three factors were also observed.
Initiation of embryogenic calli occurred at all zygotic developmental stages, although it
was higher (up to 60-80% for V genotypes) when the dominant zygotic embryo started to
develop (early-middle July). Litvay medium was superior to DCR for SE induction but
there were not differences between media when the number of established SE lines was
recorded after 4 months in culture (153 vs. 155 proliferating SE lines, for Litvay´s and
DCR media respectively). Interestingly, some mother-derived SE lines were obtained
from megagametophytes of clones C2 and V3 (16.4 and 3.8%, respectively) collected at
the beginning of the experiment, before fecundation occurs. Maturation experiments are
in course.
Research supported by the Ministerio de Educación y Ciencia (Project AGL2005-0758502FOR). We thank the Generalitat Valenciana (Banco de semillas forestales & CIEF) and
the Ministerio de Medio Ambiente (DGB) for supply plant material.
SIX.19p
MICROPROPAGATION OF BIGNONIACEAE GROWING
IN SOUTH KOREA
Jae In PARK
Department of Forest Science CALES( College of Agriculture Life and Environment
Sciences ) Chungbuk National University Cheongju Chungbuk 361-763 Republic of
Korea)
[email protected]
In South Korea four woody plant species which are according to Bignoniaceae, grow in
open space. It consists of two genera(Catalpa and Campsis), each genus has two species
each. Genus Catalpa(Catalpa ovata and C. bignonioides) is tall straight tree, are planted
in the garden or along the street but Campsis(Campsis chinensis and C. radicans ),
creepers. Catalpa ovata. grows on the fence or on the trees, All of them are from foreign
countries( Catalpa ovata and Campsis chinensis are from China, and others from North
America. Catalpa species are known to have some pharmaceutical effects. The
pharmaceutical value of these species is still under investigation from different parts of
plant body for search of new materials. For propagation of these species
micropropagation technique was adopted. Three species( two Catalpas and Campsis
chinensis) were successful, other one is under progress. With juvenile materials, BAP(6Benzil aminopurine) was effective as additive to WPM solid media for production of
multiple shoots and microshoots was possible to be rooted on solid GD media with IBA.
Plantlets of all three species survived acclimatization.
SIX.20p
BAR GENETIC TRANSFORMATION OF MARITIME PINE
Jean-François TRONTIN*, Rémy MICHEL, Arnaud GERMAIN, Hélène PILLETEMANUEL, Francis CANLET, Luc HARVENGT
AFOCEL Biotechnology Lab ([email protected]), Domaine de l’Etançon, F-77370,
Nangis, France
[email protected]
The objective of this work was to transform our model genotype of maritime pine (Pinus
pinaster Ait.) by coculture of embryonal-suspensor masses with Agrobacterium
tumefaciens carrying 2 constructs with bar gene (phosphinothricin acetyltransferase,
Basta® tolerance) under the control of constitutive promoters P35S (from cauliflower
mosaic virus 35S RNA) or Pubi (from maize polyubiquitin gene). Both constructs were
cloned into binary vector pCambia1301 carrying hpt gene (hygromycin
phosphotransferase) and uidA reporter gene. After selection with hygromycin (H),
transformation yields obtained with Pubi-bar and P35S-bar were similar (59-65 resistant
H+ lines/g fresh weight). Using phosphinothricin (P) as selective agent, results were
slightly higher with Pubi-bar (97 resistant P+ lines/g) but significantly reduced with
P35S-bar (34 P+ lines/g) suggesting lower expression of bar when controlled by P35S.
Expression of uidA (GUS test) could be detected in most selected lines (73-93%). 30
independent H+/P+ lines per construct could be cryopreserved and 10 were subjected to
the maturation step. Despite low maturation yields related with line ageing during the
whole transformation process, P35S-bar lines were significantly more productive in
mature somatic embryos (SE) than Pubi-bar lines. In the latter case, a maturation
experiment with more juvenile material concluded that SE yield from hygromycinselected lines (9 SE/g ESM) is significantly reduced compared to phosphinothricinselected lines (20 SE/g) and untransformed control (18 SE/g). Transgenic somatic plants
could be regenerated for each construct and are currently growing in the greenhouse.
After 18 months, the growth of one pUbi-bar plant lot obtained from one P+ line
characterized by Southern blot (2-3 integrated bar copies) was found similar (collar
diameter) or even significantly higher (height) than non-transformed controls. Transgenes
were detected in needles of most plants tested using PCR (hpt, bar, 95%) and GUS
expression analyses (uidA, 89%). A non-destructive test performed on excised needles
showed that significant Basta® tolerance (at 5-fold the field dose) occurred in about half
tested plant (48%) compared to non-transformed controls. bar selection is currently used
to generate transgenic maritime pine expressing genes with direct, practical interest and
in functional genomic research studies.
SIX.21p
EFFECT OF ACETOSYRINGONE ON AGROBACTERIUMMEDIATED TRANSFORMATION OF EUCALYPTUS
CAMALDULENSIS
Regina QUISEN1, Yohana de O. U. LIMA2, Francine L. CUQUEL2, Marguerite
QUOIRIN3*
Embrapa Amazônia Ocidental, Manaus – AM, Brazil, 2 Pós-graduação em Agronomia,
Produção Vegetal, UFPR, rua dos Funcionários, S.N., Curitiba, PR, Brazil, 3Depto de
Botânica, Universidade Federal do Paraná, Caixa postal 19031, 81531-990 Curitiba, PR,
Brazil
[email protected]
1
The development and optimization of efficient protocols for production of transgenic
plants can offer great advantages to eucalyptus breeding programs. However, this process
is limited by the poor capacity of regeneration of recalcitrant species after inoculation
with disarmed Agrobacterium strain. Acetosyringone is a phenolic compound known to
improve the DNA transfer from the bacterium to the plant cell, but its effect on
organogenesis process has been little studied. In order to improve the efficiency of
Agrobacterium-mediated transformation of Eucalyptus camaldulensis, the effect of
acetosyringone added to the inoculation medium on explants transformation and
regeneration was studied. Cotyledons excised from 12-day-old etiolated seedlings were
inoculated with strain C58C1 carrying a binary vector containing pcgMT1gus where gus
reporter gene was under control of pcgMT1 promoter. After liquid culture, the bacterium
was resuspended in half-strength MS medium supplemented with 0, 20, 50 and 100 µM
acetosyringone and this solution used for explants inoculation. The expression of gus
gene was evaluated by counting the number of GUS-positive explants 5 days after a 3day-coculture with Agrobacterium tumefaciens. Shoot regeneration capacity was also
evaluated after 60 days of culture on medium containing selective agent and antibiotics.
A very high frequency (80%) of transient GUS expression level was obtained when 20
µM acetosyringone was added to the inoculation medium. However, the presence of
acetosyringone resulted in low regeneration rates, which ranged from 0.02 (100 µM) to
0.8 (20 µM) shoots/explant and 0.5 for control. Moreover, exogenous AS did not always
enhance satisfactorily the genetic transformation frequency, which depends on AS
concentration and probably on other factors.
SIX.22p
IMPROVING INITIATION OF SOMATIC
EMBRYOGENESIS FROM PORTUGUESE GENOTYPES
OF MARITIME PINE (PINUS PINASTER AIT.)
Marta SIMÕES*, Ana MILHINHOS, Liliana MARUM, Sónia GONÇALVES, Pedro
SIMONINI and Célia MIGUEL
Forest Biotech Lab, Instituto de Tecnologia Química e Biológica/Instituto de Biologia
Experimental e Tecnológica, Quinta do Marquês, 2784-505, Oeiras, PORTUGAL
[email protected]
Somatic embryogenesis (SE) is one of the most promising biotechnology tools for largescale multiplication of elite plant genotypes, especially for forest species due to their
economical value. One of the most problematic steps in SE process is the induction of
embryogenic cultures from selected trees. To improve initiation of SE in maritime pine
(Pinus pinaster Ait.) we studied the influence of basal culture medium, PGR’s and
mother tree genotype on the initiation rate of embryogenic cultures. Two basal
formulations were used, DCR and modified Litvay (LVm), containing several
combinations of the commonly used growth regulators 2,4-dichlorophenoxyacetic acid
(2,4-D), 6-benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA) and kinetin. Also
the brassinosteroid brassinolide and N-(2-chloro-4-pyridyl-N’-phenylurea (CPPU) were
tested for their ability to induce embryogenic tissue, as well as supplementation with
cobalt and nickel. In total, four different initiation media were used for this experiment.
One-hundred immature zygotic embryos from each of 3 open-pollinated (OP) families of
maritime pine were isolated and cultured on each initiation media. The highest SE
initiation rate was obtained on LVm basal medium with CPPU (Park et al. 2006). On the
other hand, DCR basal medium with 2,4-D and BAP (Miguel et al., 2004) and LVm basal
medium containing 2,4-D and BAP (Lelu-Walter et al., 2006) revealed the lowest
embryogenic tissue initiation capacity, with similar numbers of induced embryogenic
lines. The three OP families used showed a different behaviour amongst them. One of the
families showed very low initiation efficiency on all tested media when compared to the
other two. The obtained results confirm a strong influence of the mother tree (openpollinated family) on the initiation process. Regarding the initiation medium, we have
achieved significant improvement in global initiation rates when using LVm
supplemented with CPPU.
References:
Lelu-Walter MA, Bernier-Cardon M, Klimaszewska K (2006) Plant Cell Rep 25:767-776
Miguel C, Gonçalves S, Tereso S, Marum L, Maroco J, Oliveira MM (2004) Plant Cell Tiss Organ Cult
76:121-130
Park YS, Lelu-Walter MA, Harvengt L, Trontin JF, MacEacheron I, Klimaszewska K, Bonga JM (2006)
Plant Cell Tiss Organ Cult 86(1):87-101
Acknowledgements: Fundação para a Ciência e a Tecnologia is acknowledged for support through project
POCI/AGR/57902/2004 and grants SFRH/BD/32037/2006 (MS), SFRH/BD/1706/2004 (LM) and
SFRH/BPD/20657/2004 (SG)
SIX.23p
FASTER EVALUATION OF INDUCED FLORAL
STERILITY IN TRANSGENIC EARLY FLOWERING
POPLAR
Hans HÖNICKA, Matthias FLADUNG*
BFH, Institute for Forest Genetics and Forest Tree Breeding, Sieker Landstr. 2, D-22927
Grosshansdorf, Germany
[email protected]
A major concern over the use of transgenic trees is the potential for extensive transgene
dispersal through pollen and seeds. The incorporation of sterility genes into transgenic
lines of trees has been proposed to reduce or even avoid gene flow of transgenes into
non-transgenic relatives, which is one of the main ecological concerns with respect to
commercial use of transgenic plants. The evaluation of strategies for the induction of
sterility in transgenic forest tree species has been hindered by their long vegetative
periods. In this study early flowering 35S::Leafy poplar lines were used for the evaluation
of two different sterility constructs, TA29::Barnase and C-GPDHC::Vst1. The
combination of two transgenic approaches, one to induce early flowering and a second
for the induction of sterility, allowed evaluation of both sterility strategies two years after
transformation. This is a very short period of time considering the long vegetative period
of up to ten to twenty years common in forest tree species. This approach opens new
opportunities for the assessment of mechanisms for this plant group.
SIX.24p
TECHNOLOGIES FOR MASS PROPAGATION OF ELITE
FOREST TREES: 30 YEARS ACTIVITIES AT THE CRA W.
R. GRUSELLE, J.P. MISSON, J.M. TERZI, O. AREZKI. and Ph. DRUART*
CRA W Department Biotechnology, chaussée de Charleroi, 234, 5030 Gembloux,
Belgium
[email protected]
The need for elite forest trees is permanent for economical purposes. Compared to the
natural regeneration, cloning elite trees get advantage on seed regeneration before
planting; the plant material has been previously evaluated at the adult stage. However, a
technology of mass propagation is required at the lowest cost.
The “Plant propagation Unit” of the CRA W Department Biotechnology is involved in
the development of mass propagation technologies since 1978 when the first wild cherry
clones were established in vitro from meristem tips. From that time, several species have
been investigated with success, like alder, maple, ash, oak, cork-oak, whitebeam, poplar,
elm, walnut, horse chestnut, Eucalyptus, Acacia, Terminalia, Cypress and A.
nordmanniana.
The first approaches were designed on temperate fruit species as model plants: starting
from meristem culture and producing plants through axillary branching, shoot elongation
and in vitro rooting as successive steps. However, several adaptations were specifically
required at different stages of the processes making them mostly single or original
training technologies according to the species. Amongst the main adaptations, the
following are concerned:
the preparation of the initial explant from the stock plant before node culture,
involving forced shoot regeneration from roots/branches fragments, shoot flushing from
pre-harvested twigs, grafting, …
the regulation of the axillary branching and the in vitro rooting of walnut
(Gruselle et al., 1995, Dolcet-Sanjuan et al., 2004)
the investigation on single protocols involving meristematic aggregates for
Eucalyptus (Arezki et al., 2000) and somatic embryogenesis for A. nordmanniana
(Misson et al., 2006)
Among these species, elite wild cherries have been managed through the production of
thousands plants as a “polyclonal variety”. Using vitroplants, the performances of
classical methods for vegetative propagation have been improved as well.
References
- Gruselle, R., Nicaise, C. and Boxus, P. 1995. Bull. Rech. Agrom. Gembloux 30(1-2) : 47-53.
- Dolcet-Sanjuan, R., Claveria,E., Gruselle, R., Meier-Dinkel,A., Jay-Allemand, C. and Gaspar, Th. 2004.
Journal of American Society for Horticultural Science 129 (2): 198-203.
- Arezki, O., Boxus, Kevers, C. and Gaspar, T. 2000. In Vito Cell. Dev. Biol. 36:398-401.
-J.-P. Misson,P. Druart,B. Panis and B. Watillon (2006). Propagation of ornamental plants. 6, (1), 17-23.
http://www.cra.wallonie.be/research/themes
SIX.25p
ECOLIRI: TO SUSTAIN TREE BIODIVERSITY OF THE
RIVERSIDES
F. LAMBOT1, R. KIPGEN2, R.; A. CHANDELIER3, P. MERTENS4, S. ADANT1, C.
HUSSON5, M. JACQUES6, R. GRUSELLE7 and Ph. DRUART7*
1
Ministry of the Walloon Region, Water Division, DCENN, Namur,Belgium, 2Ministry of the
Interior, Water Management Agency, Grand Duchy of Luxemburg, 3CRA-W Department
Biological control and plant genetic resources, Gembloux, Belgium, 4Research Centre for Nature,
Forest and Wood, Gembloux, Belgium, 5INRA Nancy, UMR 1136, Pathology, Nancy, France,
6
Syndicat Intercommunal d’Aménagement de la Chiers, Nancy, France, 7CRA-W Department
Biotechnology “Plant propagation Unit”, chaussée de Charleroi,234, 5030 Gembloux, Belgium.
[email protected]
Within the framework of erosion and floods prevention, the public authorities decided to
stop with giving privilege to the civil engineering work techniques for the restoration of
the riparian zones. The most valuable contributions of a healthy riparian system to the
environment are (i) sediment filtering, (ii) bank stabilization, (iii) water storage and
release and (iv) aquifer recharge.
The supply of forest trees for the stabilization of river’s banks is generally inadequate in
quality. In absence of local origins, re-planted trees are actually collected from foreign
regions. Consequently, the genetic origin of these plants is unknown and their long- term
behavior towards pathogens and pedo-climatic conditions is not optimal
ECOLIRI project, financially supplied by FEDER, has the objective to establish an origin
secured production line of woody species from ecotypes of a trans border region
including the south of Wallonia (Belgium), the Grand-Duchy of Luxemburg and the
French Lorraine. These species are subject of collections as a function of the riversides
origin and of stock plants establishment for cuttings and seed orchards. Alder, willow and
ash are selected for complementary rooting systems (diffuse system for willow or alder
and tap-root system for ash) that must stabilize the banks durably.
After having prospected and fixed different origins in the 3 eligible regions, several
hundreds clones of alders are gathered. The re-implantation in their original sites is
foreseen after cuttings and/or in vitro propagation. Simultaneously, willows were
collected in Wallonia and a collection established. Planning and maintenance of stockplants to provide cuttings continuously will be realized. The discrimination of their
genetic variability is required using molecular tools for alder and aromatic profiles for
willows, because plant certification and sustainable biodiversity through seed production
are targeted.
The sanitation aspect is taken into account. A letal disease caused by a oomycetes called
Phytophthora alni now attacks black alder. So, the prospecting of alder clones in Lorraine
is made as a function of this parameter. Specific tests are developed in parallel to evaluate
disease tolerance. Single tolerant genotypes will be introduced directly in re-plantation
programs and in the design of seed orchards as insurances of banks stability and of
tolerance trait display respectively.
SIX.26p
PRE-FOREST: A NEW EUROPEAN TECHNOLOGY FOR
COST EFFICIENT AND ENVIRONMENTAL FRIENDLY
PRODUCTION OF PRE-CULTIVATED FOREST
REGENERATION MATERIALS
Rosanna BELLAROSA1, Lorenzo CICCARESE1, Olympia DINI-PAPANASTASI2, Raquel
FERREIRA1*, Panagiota KOSTOPOULOU2, Anders MATTSSON3, Kalliopi RADOGLOU2,
Yannis RAFTOYANNIS2, Bartolomeo SCHIRONE1, Marco C. SIMEONE1, Gavril
SPYROGLOU2, Peter JOHANSSON4, Elisabetta MARGHERITI5, Nick RAMMOS6
1
2
D.A.F. Department, University of Tuscia, via S. Camillo de Lellis, 01100 Viterbo, Italy, Forest Research
Institute, NAGREF, Vassilika 57006, Thessaloniki, GR, 3Dalarna University, Selma Lagerlöfsplatsen, S791 88 Falun, Sweden, 4QS Odlingssystem AB, Västbovägen 7, S-862 41 Njurunda, Sweden, 5Vivai
Torsanlorenzo, via Campo di Carne 51, 00040 Ardea (RM), Italy, 6Ditikomakedonika Fytoria, Grevena,
GR.
[email protected]
The project, funded by the European Commisison under the CRAFT 6th Framework
Research Programme, involves six partners, comprising both research institutions
(University of Tuscia, IT; Dalarna University, SE; National Agricultural Research
Foundation, GR) and private enterprises (Vivai Torsalorenzo, IT; QS Odlingssystem AB,
SE; Ditikomakedonika Fytoria, GR).
The main objectives of the project, short-handed Pre-Forest, are to introduce an
innovative, cost effcient and environmental friendly technology for cultivation of forest
reproductive material (FRM) in mini-plugs; develop a new technology for a grading and
transplanting robot adapted to mini-plugs; integrate these technologies into a functional,
flexible system for large scale production of pre-cultivated FRM adapted to mechanical
transplanting; and introduce this system at strategic locations in Europe.
The objectives will be achieved by basic technological, economical and biological studies
during the first year. Therefore, focus will be on the interplay between production
technology, mini-plug container design and rooting media, in order to develop a system
where high quality FRM can be produced. Growing manuals and protocols for the
cultural practices adapted to the new approach of pre-cultivation will be developed for the
species under study, a total of eight species, both broadleaves and conifers.
The system will be developed for year-around production and function in the same way
independently of climate variations in different parts of Europe.
The results will be validated by morphological, physiological, and performance tests as
well as genetic analysis by means of molecular markers. Genetic tests will be carried out
to evaluate the effect of the technique proposed on the conservation of genetic diversity.
The new technology will allow a large number of seedlings per square meter and hence
energy and space being efficiently used, leading to a very cost efficient production,
benefiting both nurserymen and end-users. On top of which it allows to meet the specific
requirements of the different tree species. Moreover, nurseries will be more competitive
on several aspects, namely at an economic level, a quality level, regarding environmental
protection and on addressing the market’s needs.
SIX.27p
MATURE CORK OAK (QUERCUS SUBER L.)
TRANSFORMATION WITH BAR GENE
Ricardo Javier, ORDÁS*, Rubén ÁLVAREZ, Jaime HUMARA, Mª Ángeles REVILLA
Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, C.\
Catedrático Rodrigo Uría s/n, Oviedo, Spain
[email protected]
The cork oak (Quercus suber L.) is one of the most characteristic species of the
Mediterranean ecosystem. Besides its great ecological value, it produces cork, a natural
renewable product of economic interest. Owing to the long generation time lag caused by
seed germination and flowering this species has been left unaltered in relevance to
genetic improvement or molecular breeding.
Herbicide resistance is a desirable trait not present within breeding population of Quercus
suber L. A genetic transformation protocol of mature cork oak has been reported (1),
which opens the possibility to insert this trait into desired genotypes.
Cork oak somatic embryos induced from a more than 100-hundred year old tree were
genetically transformed with AGL1 pBINUbiBAR. The binary vector pBINUbiBAR was
constructed in our laboratory, based on pBIN19, and carries the genes nptII (neomycin
phosphotransferase II) and bar (phosphinothricin acetyl transferase —PAT—). These
genes confer resistance to the antibiotic kanamycin and the herbicide phosphinothricin,
respectively.
The presence of both nptII and bar genes was assessed in the kanamycin-resistant lines by
PCR and Southern Blot. All lines carried both genes, and have three copies average of the
insert. The activity of PAT was checked culturing the lines with 20 mgL–1
phosphinothricin (commercial herbicide Finale®), and by chlorophenol red and enzimatic
assays. Only a 63 % of the lines showed PAT activity, and there were remarkable
differences in enzimatic activity between lines.
Hoping to be able to perform field analysis in a future, the transgenic embryogenic lines
were cryopreserved, following the protocol by Valladares et al. (2004), and the presence
and activity of the transgenes was confirmed in the recuperated lines.
Acknowledgements:
Authors thank Dr. M. Toribio for gently providing the cork oak embryogenic lines.
Rubén Álvarez was supported by a FICYT research fellowship funded by “Gobierno del
Principado de Asturias, Spain”.
References:
Álvarez, R, Alonso, P, Cortizo, M, Celestino, C, Hernández, I, Toribio, M & Ordás,
RJ(2004). Plant Cell Rep 23, 218-223.
Valladares, S, Toribio, M, Celestino, C & Vieitez, AM (2004). Cryo Lett 25, 177-186.
SIX.28p
SOME TISSUE CULTURE AND ANATOMICAL STUDIES
OF SELECTED TROPICAL TREE SPECIES
Rosna MAT TAHA* and Noorma Wati HARON
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
MALAYSIA.
[email protected]
Some tissue culture and anatomical studies were carried out on three important tropical
tree species. from Malaysia. The species investigated were Xylocarpus granatum (
Meliaceae), Psidium guajava L. and Eugenia cumini ( Linn) Druce ( Myrtaceae).
Complete plant regeneration was achieved from Psidium guajava and Eugenia cumini,
whilst regeneration of Xylocarpus granatum proved to be extremely difficult, only callus
formation was observed. Various explants ( leaf, petiole, stem and shoot tips) of Eugenia
cumini and Psidium guajava were successfully regenerated on MS ( Murashige &
Skoog,1962) medium supplemented with different concentrations of Naphthalene Acetic
acid ( NAA), Benzyl- amino purine ( BAP) and other hormones. The optimum medium
for regeneration of Psidium guajava was MS supplemented with 0.5 – 1.0 mg/l NAA
and 0.5- 1.0 mg/l BAP. The most responsive explant was shoot tip, however, leaf
explants could also be used for plant regeneration. Optimum regeneration medium for
Eugenia cumini was also MS supplemented with 1.0 mg/l NAA and 0.5 mg/l BAP.
Young stem was the most responsive explant for this species. Formation of callus was
observed from leaf explants of Xylocarpus granatum cultured on MS added with 20 mg/l
2,4,D ( Dichlorophenoxy acetic acid ). Anatomical studies were done on the leaves and
midribs of the two Myrtaceae species showed that similar characteristics of the cells were
found in both Psidium and Eugenia species in vivo and in vitro. However, some
differences in vascular bundles and shapes of cells were observed in Psidium grown in
vitro.
Xylocarpus species are considered important endangered mangrove species in Malaysia.
One of the economic importance of the species, particularly Xylocarpus granatum is for
wood carving, hence there is an urgent need to conserve the species. Anatomical study
was carried out on the midrib and lamina of the leaves of Xylocarpus granatum. The
results showed that the leaves have one to two layers of epidermal cells on the adaxial
and abaxial surfaces. On the midrib, the palisade cells are long and biseriate, vascular
bundles are arc-shaped surrounded with collenchyma cells. On the leaf lamina, thick
cuticles were present on both the adaxial and abaxial surfaces. The palisade and spongy
mesophyll cells were arranged in two layers. Tannin is observed as reddish-brown
patches in the midrib and lamina. Cross- section of the primary stem showed the presence
of a single layer epidermis. The cortex layer consists of parenchyma cells with
chloroplast while the pith contains parenchyma cells. Tannin is also observed in
parenchyma cells of the cortex.
Results from anatomical studies showed the presence of thick cuticle on both abaxial and
adaxial surfaces of the leaf. Tannin cells were also observed in the leaf lamina and
primary stem and this needs further investigation.
SIX.29p
ORNAMENTAL USE OF SILVER BIRCH WITH YELLOWVEINED OR WHITE-FLECKED LEAVES − MISSION
IMPOSSIBLE?
Leena RYYNÄNEN1, Tuija ARONEN1*
1
Finnish Forest Research Institute, Punkaharju Research Unit, Finlandiantie 18,
FI-58450 Punkaharju, Finland
[email protected]
There are few native deciduous tree species with colourful summer foliage among the
trees adapted to the harsh conditions in northern latitudes. The woody plants of more
southern origin imported for landscaping need a more temperate climate than that
prevailing e.g. in Finland, and they often die during the deep midwinter temperatures that
can fall to -40°C, or suffer from spring frosts. Nowadays it is possible to micropropagate
almost all the Nordic deciduous tree species, and several ornamental forms of native,
well-adapted species, mostly with dark red foliage, have become commercially available.
The aim of this study was to clone two new decorative forms of native birch (Betula
pendula Roth) having specific leaf colouration. The “golden-veined” (GV) and “whiteflecked” (WF) forms of birch were cloned by grafting and micropropagation, and
cryopreservability of the in vivo buds of the birches was also tested. All the methods were
applicable for both forms, but the WF birch was easier to propagate than GV birch.
Grafting of WF birch was more successful, and its regrowth percentages following
micropropagation and cryostorage were many times higher than those of GW birch,
which were only 6.3% and 6.9%, respectively. In the greenhouse and nursery, the 1- and
2-year-old micropropagated progenies of both leaf colour forms, with or without
cryopreservation, failed to express leaf colouration. The typical leaf colouration was,
however, observed in the leaves growing on the 2-year-old stems and branches of WF
birch grafts. The WF donor tree showed this same phenomenon where more pronounced
leaf colouration was found on leaves growing in the more mature parts of the tree.
Reference: Ryynänen, Leena & Aronen, Tuija, 2007. Phenotypic expression of leaf
variegation in two Betula pendula Roth genotypes following micropropagation,
cryopreservation and grafting. Propagation of Ornamental Plants 7(1):xx-xx. In press.
SIX.30p
MINICUTTING OF LIQUIDAMBAR STYRACIFLUA L.:
SUBSTRATUM EFFECT AND TYPES OF PREPARATION
Gilvano Ebling BRONDANI1, Ivar WENDLING2, Leonardo Ferreira DUTRA2*, Fabricio
Augusto HANSEL2
1
Universidade Federal do Paraná , Av. Lothário Meissner, 3400, Jardim Botânico,
2
80.210-170, Curitiba, Paraná, Brasil, Embrapa Florestas , Estrada da Ribeira, Km 111,
Caixa Postal 319, 83.411-000, Colombo, Paraná, Brasil
[email protected]
Minicutting is a clonal alternative for vegetative propagation of individuals with superior
genetic characteristics. Minicutting technique is widespread for Eucalyptus species, but
its application is not common seen to other wood species. The objectives of this work
was to evaluate the effect of different substratum compositions and the types of
preparation of the minicutting in the survival, rooting and vegetative vigor of sweetgum
minicuttings. Sprouts of ministumps produced by cuttings were collected. The
ministumps were cultivated in semi-hydroponics system. The minicuttings (5 cm in
length) had one pair of leaf with 50% of the foliar area. Then, minicuttings were
submitted to four different types of preparation: P1 = paraffin, P2 = paraffin + 8 g L-1 of
IBA, P3 = 8 g L-1 of IBA and P4 = control. The application of the IBA was realized with
the immersion of the basal region of minicuttings in a solution for a period of 10 seconds
and the paraffin application was done in the basal and apical portion of minicutting. The
minicuttings were planted in conic recipient (55 cm3) with three different substratum
combinations (v/v): S1 = rice rind + commercial substratum Plantmax® (1:1), S2 =
carbonized rice rind + commercial substratum Plantmax® (1:1) and S3 = rice rind +
commercial substratum Plantmax® + vermiculite (2:1:1). The minicuttings were kept
under controlled conditions of humidity and temperature in greenhouse; after 91 days of
rooting promotion they were transferred to shadow house for acclimatization during 15
days. Finally, the survival minicuttings were placed in an open garden for up to 35 days.
The survivals of minicuttings were evaluated in the end of each step (greenhouse, shadow
house and open garden). After 141 days of culture the shoots length, number of leaf,
biggest root length and the length of the root system were measured, as well as the visual
aspects of the plant and root formation were evaluated. The types of preparation of the
minicutting no presented statistical difference. However, there was a strong influence of
the substratum in the aerial and root formation. S2 substratum provided the biggest
percentage of rooting (69%), with no necrosis, presented statistical difference of S3
(45%) and S1 (12%). In resume, the rooting of minicuttings of L. styraciflua is
technically viable without the application of IBA and paraffin, with substratum
combination of carbonized rice rind + commercial substratum Plantmax® (1:1).
SIX.31p
SOMATIC EMBRYOGENESIS AND EFFICIENT PLANT
REGENERATION SYSTEM OF JAPANESE CEDAR
(CRYPTOMERIA JAPONICA D. DON)
Tsuyoshi E. MARUYAMA* and Yoshihisa HOSOI
Department of Molecular and Cell Biology, Forestry and Forest Products Research
Institute Matsunosato 1, Tsukuba 305-8687, Japan
[email protected]
Cryptomeria japonica is the most important conifers in Japan. However the pollinosis
from this species is one of the most serious allergic diseases in Japanese society. We are
interested in the application of biotechnologies for development of transgenic
Cryptomeria japonica that produces allergen-free pollen grains. Somatic embryogenesis
has become an important plant regeneration system for genetic engineering.
Agrobacterium-mediated gene transfer system, biolistic transformation approach, and
direct DNA transfer methods that targets embryogenic cells have been reported for a
number of tree species. This report describes an efficient plant regeneration system of
Cryptomeria japonica via somatic embryogenesis. Embryogenic cultures were initiated
from megagametophytes containing immature zygotic embryos. Embryogenic cultures
were maintained and proliferated by 2 to 3-week interval subcultures in medium
supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High
maturation frequencies of cotyledonary embryos were obtained on maturation media
containing maltose, polyethylene glycol, activated charcoal, and abscisic acid. Somatic
embryos germinated after transfer to plant growth regulator-free medium. Regenerated
somatic plants were acclimatized successfully and their growing has been monitored in
the field.