Báo cáo khoa học: "Embryogenesis Jin Quercus petraea"

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Original article in Quercus petraea Embryogenesis J Jörgensen Lower Saxony Forest Research Institute, Department of Forest Tree Breeding, 3513 Staufenberg-Escherode, Germany Summary — Embryogenesis in Quercus petraea was achieved by culture of zygotic embryos and anthers on modified woody plant medium with benzylaminopurine and 2,4 dichlorophenoxyacetic acid. After counting chromosomes and isoenzyme analysis the embryos from anther culture were subdivided into haploid and somatic clones. Protoplasts that had been isolated from haploid and dip- loid tissues were regenerated to embryos. embryogenesis / androgenesis / protoplast culture / Quercus petraea somatic Résumé — Embryogenèse chez Quercus petraea. L’embryogenèse de Quercus petraea a été réalisée par culture d’embryons zygotiques et d’anthères sur du milieu WP contenant de la BAP et du 2,4 D. L’analyse des isoenzymes et le dénombrement des chromosomes ont montré que les em- bryons issus de culture d’anthères étaient subdivisés en clones haploïdes et somatiques. Les proto- plastes isolés à partir de tissus haploïdes et diploides ont été régénérés en embryons. embryogenèse somatique / andfogenése/ culture de protoplastes / Quercus petraea hardly be achieved; 3) the fo- INTRODUCTION quality can constantly supplied with rests cannot be oak seeds or plants because oak seeds In contrast to the breeding of agricultural cannot be stored for more than 3-4 years. and horticultural plants, the breeding of This situation is quite unsatisfactory oak faces special difficulties: 1) inbred when confronting simultaneous increases lines, which help to combine attributes ex- of forest decline and wood demand. actly, cannot be produced due to the long generation period of oak; 2) the rate of Although at the beginning of this pro- gram only shoot cultures had been de- rooting in cuttings of older mother trees is quite low (Spethmann, 1986) so that a suf- scribed for this species (Pevalek-Kozlina ficient supply of clones of high genetic and Jelaska, 1986; Meier-Dinkel, 1987),
Calli that had developed in anther culture tried to find a combination of in vitro we induced to embryogenesis on modified were techniques and conventional methods, for WPM with 2.5 μM BAP at 2000-3000 lux and which it would also be easier or even nec- 25°C±1. essary to use physiologically young mate- rial, to offer possibilities in tree breeding, comparable to those of traditional breeding Culture of androgenic of early flowering plants. and somatic embryos Somatic embryos, which are genotypi- similar to the mother tree, are ideal cally Androgenetic and somatic embryos were cloned for the development of artificial seed, modified WPM with 2.5 μM BAP. The em- on whereas gametophytic embryos are ideal bryos developed plants on the same basic medi- um without hormones. as a basic material for protoplast culture; when the latter are hybridized which shortens the time of breeding and for get- Protoplast culture ting them independent of flowering, they can be used as somatic embryos. Both can be stored in liquid nitrogen types In order to isolate protoplasts, haploid and dip- (Jörgensen, 1990) thereby achieving inde- loid oak embryos were cut into small pieces with a razor blade and suspended in an enzyme so- pendence of the unsteady seed produc- lution consisting of 3% Rohamend PC, 5 mM tion. (2+) Ca and 0.5 M mannitol and cultivated at 800- 1000 lux at 25 °C ± 1 for 8-15 h. Afterwards, the isolated protoplasts were concentrated accord- MATERIALS AND METHODS ing to a method described by Binding and Koll- mann (1985) and Binding et al (1988), then washed and embedded at a concentration of about 10 protoplasts/ml in VKM medium 4 -10 3 Androgenesis (Binding and Nehls, 1977) solidified with aga- and somatic embryogenesis rose. VKM-medium (1 ml) was added and the Petri dish was closed and sealed with parafilm. The cultivation was done at 800-1000 lux and Anthers with pollen in the single nucleus or tet- 25 °C ± 1. The liquid VKM medium was re- rade stage of 3 mature and immature zygotic newed every 2-3 days. After the development of embryos of 5 mother trees of Quercus petraea 1-2 mm microcalli, the agarose drops with the were prepared under sterile conditions, laid on a regenerates were laid on modified WPM with woody plant medium (WPM) (Lloyd and 2.5 μM BAP and further cultivated under the McCown, 1981) with MS (Murashige and conditions. same Skoog, 1962) microelements: 1000 mg/l gluta- mine, 50 mg/l glycine and 50 mg/l serine (modi- fied WPM) with combinations of benzylaminopu- rine (BAP) and 2,4-dichlorophenoxyacetic acid Genetic investigations (2,4-D) each at one of the following 6 different of the androgenic embryos concentrations: 0.0, 0.5, 1.0, 2.5, 5.0 and 10.0 μM. Sugar, agar, macro- and microelements After fixing the material in a mixture of 4 parts without Fe-ethylenediaminetetraacetic acid (FeEDTA) and K were autoclaved and ethanol and 3 parts acetic acid, the tissues were 4 HPO 2 smashed and stained in carminoacetic acid, and FeEDTA, K amino acids, vitamins and , 4 HPO 2 the chromosomes were counted. Isoenzyme in- hormones were sterilized by filtration and added vestigations were carried out by Dr Müller- afterwards. The pH was adjusted to 5.8. The tis- Starck (Müller-Starck and Jörgensen, 1991) at sues were cultivated at 2000-3000 lux and the Forest Genetic Institute of the University of 25 °C ± 1 (zygotic embryos) or 30 °C ± 1 (an- Göttingen. thers).
RESULTS and from pollen being in the single-nucleus stage for the 1 st day (3 clones) proved to be haploid, whereas embryos from pollen Androgenic and somatic embryogenesis being in the single-nucleus stage for the 2nd day were somatic (3 clones) (see be- Two to 3 months after cultivation of the an- low). We could not assign the clones from thers on a modified WPM with 0.5 to 10 this year to haploid or somatic, because to μM BAP and 0.5 to 10 μM 2,4 D, the walls date there has not been enough material of 1-3 of 50 anthers, from 2 trees in which to evaluate. On modified WPM without hor- the pollen was in the tetrade or for the 1 st mones, embryos of the last years devel- 2 days in the single-nucleus stage, were oped plants (fig 2) that were transferred to ruptured by the growing calli. It is not yet greenhouse conditions. possible to give a statistical analysis, be- Zygotic embryos of 4 mother trees culti- cause the reactions of the anthers differed vated on modified WPM with 0.5-5 μM from clone to clone ranging from 0 to 6%. BAP and 2.5-10 μM 2,4 D developed so- On the other hand no organgenesis oc- matic embryos in the lower 3rd of the coty- curred in 2 of 6 years, whereas in other ledon and on the hypocotyl. Here too, the years up to 6% of the cultivated anthers of regeneration rate on the different media 2 of the 3 tested mother trees showed or- varied from clone to clone between 1 and ganogenesis. This difference in reaction 4 of 5 acorns in a year during which the re- seems to depend upon the environmental generation rate was very high, whereas, in conditions of the mother trees. These calli other years, the regeneration rates of the were totally transferred to embryos on same mother trees were between 0 and 3 modified WPM with 2.5 μM BAP. Secon- of 5 acorns, placed in one Petri dish. The dary embryos developed on the surface of first plants were regenerated on modified the primary embryos on the same medium WPM without hormones. (fig 1).Embryos resulting from tetrades
velopment of the first embryos (Jörgensen, Protoplast culture in press). days after the beginning of the Three to 5 protoplast culture, the first divisions were Genetic investigations observed. Altogether, 0.5-3% of the proto- of the androgenic embryos plasts divided. About 70% of these cells developed through sustained divisions to As somatic cells, ie, anther walls were gen- 1-to-2-mm microcalli within 3-4 weeks. Af- erative cells, and pollen were cultivated to- ter the transfer of the agarose drops with gether in the framework of the anther cul- the microcalli to modified WPM with 5% ture, the origin of the regenerates had to coconut milk and 2.5 μM BAP, between 5 be examined. The haploid clones were and 24% of the microcalli grew to calli of identified as pollen regenerates by count- about 4-5 mm diameter within 2-3 weeks ing the chromosomes, whereas the diploid (fig 3); development of embryos occurred clones proved to be regenerates of the an- after 11 weeks. The evolution of the 4 rep- ther wall. Isoenzyme analysis carried out etitions was very similar. The same experi- by Dr Müller-Starck (Müller-Starck and ments in beech showed that a period of at Jörgensen, 1991) showed that a duplica- least 2 months was necessary for the de-
tion of the haploid chromosome set could useful for the practice of oak breeding. The be excluded (fig 4). fact that the experiments to produce andro- genic embryos can be done only a few days per year and only in years in which DISCUSSION the trees are flowering seems to be the biggest obstacle to generating enough haploid clones for plant breeding in vitro. The results described above represent an important step forward in the breeding of In addition to androgenesis, it is impor- Quercus petraea in a shorter time, but the multiply valuable adult trees. To do tant to number of different clones obtained within so, somatic embryogenesis is a useful the short period of this project were not method, because it reactivates attributes sufficient to realize crossing experiments which are typical of young material, like
A, Zuba N, Mordhorst G (1988) Protoplast rooting ability, growth etc. In Quercus acu- culture in agarose media with particular em- tissima (Sasaki et al, 1988), Q petraea phasis to streaky culture lenses. Bot Acta (Jörgensen, 1990), Q rubra (Gingas and 101, 133-139 Lineberger, 1989) and Q robur (Chalupa, Chalupa V (1990) Plant regeneration by somatic 1990), somatic embryos were observed, embryogenesis from cultured immature em- but they are only of scientific interest, be- bryos of oak (Quercus robur L) and linden cause they were induced on zygotic em- (Tilia cordata Mill). Plant Cell Rep 9, 398-401 bryos, which means on genetically indefi- Feraud-Keller C, Espagnac H (1989) Conditions nite material. Only Feraud-Keller and d’apparition d’une embryogenèse somatique Espagnac (1989) with Q ilex and les cals issus de culture de tissus foli- sur aires de chêne vert (Quercus ilex L). Can J Jörgensen (1988, 1990) with Q petraea Bot 67, 1066-1070 could induce somatic embryogenesis with Gingas VM, Lineberger RD (1989) Asexual em- tissues of adult trees. bryogenesis and plant regeneration in Quer- Protoplasts from embryogenic material Plant Cell Tissue Cult 17, 191- Organ cus. be regenerated to embryos and, con- can 203 sequently, to plants in Quercus petraea in Quercus J (1988) Embryogenesis Jörgensen and Fagus sylvatica (Jörgensen, in press), petraea and Fagus sylvatica. J Plant Physiol so that the hybridization of these species 132, 638-640 should be possible, whereas, as shown J (1990) Somatic embryogenesis in Jörgensen only in Fagus sylvatica, the use of meso- Quercus petraea, Fagus sylvatica, Aesculus hippocastanum and Pyrus communis. In: Ab- phyll protoplasts leads only to callus for- stracts of the IUFRO Working Party S 2-04- mation (Lang and Kohlenbach, 1988). 07. 15-19 Oct 1989, Placerville, CA (Ahuja RM, ed) (poster) Plenum Press, New York (1991) ACKNOWLEDGMENTS Jörgensen J (1993) Application of in vitro tech- niques for conservation and breeding of oak, beach and horse-chesnut. In: Conservation I wish to thank Dr Müller-Starck for the isoen- and manipulation of genetic resources in fo- zyme analysis and Manuela Hoffmann, August restry (Kim ZS, Hattemer, eds). Proc joint Ko- Kapelle and Margit Günter for technical assis- rean/German symp on Forest Genetics, Ko- tance. The project was financed by the Ministe- rea Univ, Sept 10-15 1991, Kwagmungag rium für Ernährung Landwirtschaft und Forsten, Publ. Co Seoul, Korea (in press) Nordrhein-Wesffalen, for about 3 years, and is Kohlenbach HW (1988) Callus forma- now funded by the Bundesministerium für For- Lang H, tion from mesophyll protoplasts of Fagus syl- schung und Technologie. vatica L. Plant Cell Rep 7, 485-488 Lloyd G, McCown BH (1981) Commercially fea- sible micropropagation of moutain laurel (Kal- REFERENCES mia latifolia) by use of shoot tip culture. Proc Int Plant Propag Soc 30, 421-427 Binding H, Kollmann R (1985) Regeneration of Meier-Dinkel A (1987) Propagation of Prunus protoplasts. In: In vitro Techniques - Propa- and Quercus by tissue culture. Proceedings gation and Long-Term Storage (Schäfer- of the European Seminar on Wood Produc- Menhur A, ed) Comm Eur Community Nij- tion and Harvesting. Vol 2,1.1: Selection and hoff-Junk Publ, Dordrecht, 93-99 improvement of forest reproductive material, Binding H, Nehls R (1977) Regeneration of iso- Bologna, 2, 76-83 lated protoplasts to plants in Solanum dulca- Müller-Starch G, Jörgensen J (1991) Enzyme mara L. Z Pflanzenphysiol 85, 279-280 gene markers as indicators of the initial Binding H, Görschen E, Jörgensen J, Krumbie- ploidy in anther cultures of trees. Can J For gel-Schroeren G, Ling HQ, Rudnick J, Sauer Res 21, 1141-1144
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