Vegetative propagation of oak (Quercus robur and Q petraea) by cutting and tissue culture V Chalupa

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V Chalupa. Vegetative propagation of oak (Quercus robur and Q petraea) by cutting and tissue culture. Annales des sciences forestières, INRA/EDP Sciences, 1993, 50 (Suppl1), pp.295s-307s. ￿hal- 00882901￿

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Vegetative propagation of oak (Quercus robur and Q petraea) by cutting and tissue culture

V Chalupa

Faculty of Forestry, University of Agricultural Sciences, 165 21 Praha 6-Suchdol, Czech Republic

Summary —The potential of cuttings of Quercus robur and Q petraea to form adventitious roots de- creased rapidly with increasing plant age. The rooting ability of older plants was increased by hedg- ing. Hedging of stock plants offers an effective technique for the production of cuttings with high root- ing potential. Stock plant environment markedly affected rooting of leafy cuttings. A high percentage of cuttings collected from plants grown under continuous light rooted. Vigorous plants were pro- duced from cuttings which rooted quickly and were capable of rapid shoot growth immediately after rooting. Shoot growth of rooted cuttings was stimulated in suitable environmental conditions by suffi- cient mineral nutrition. Rooted cuttings which formed new long shoots and wintered in rooting medi- um in the same place in an unheated greenhouse exhibited high survival rates. For tissue culture propagation, 2 methods were used: micropropagation by axillary shoot multiplication and by somatic embryogenesis. Axillary shoot multiplication was stimulated on low salt media (BTM, or woody plant medium WPM) supplemented with a low concentration of benzylaminopurine (BAP) or N-benzyl -9- (2-tetrahydropyranyl) adenine (BPA) (0.2-0.6 mg·l-1). Rooting of microshoots was achieved in vitro and was also successful under non-sterile conditions in a rooting mixture of peat and perlite. The field growth of micropropagated trees was comparable to that of control seedlings. Embryogenic cul- tures were initiated from immature zygotic embryos of Q petraea cultured on modified Schenk and Hildebrandt (SH) medium supplemented with BAP (1mg·l-1). The majority of embryogenic cultures produced somatic embryos. The conversion of somatic embryos into plantlets was achieved after cold and desiccation treatment. Plantlets regenerated from somatic embryos were transplanted into potting mixture, where growth continued. vegetative propagation / Quercus spp / cutting / tissue culture / somatic embryogenesis

Résumé — Multiplication végétative des chênes par méthodes horticoles et culture de tissu. La potentialité des boutures de Quercus robur et Q petraea à former des racines décroît rapidement avec l’âge du pied mère. L’aptitude à l’enracinement d’arbres âgés est améliorée par une taille sévère du pied mère. Cette technique permet d’obtenir des boutures ayant une bonne aptitude à la rhizogenèse. Les conditions d’élevage des pieds mères ont une influence sur la production de ra- cines des boutures feuillées. Les boutures prélevées sur des arbres élevés en lumière continue s’enracinent plus facilement. Des plants vigoureux peuvent être produits à partir de boutures s’enracinant rapidement et capables de croître en hauteur immédiatement après s’être enracinées. La croissance en hauteur des boutures est améliorée par une nutrition minérale adaptée. Les bou- tures enracinées ayant développé de nouvelles pousses et maintenues durant d’hiver dans leur mi- lieu d’enracinement en serre non chauffée manifestent un taux de survie élevé. La multiplication végétative par culture in vitro implique deux techniques : la multiplication de pousses axillaires et l’embryogenèse somatique. La production de pousses axilliaires est améliorée sur des milieux faible- ment salins (BTM et WPM) et contenant de la BAP (ou BPA) en faible concentration (0,2-0,6 mg/l). L’enracinement de micropousses a été réalisé en conditions in vitro et en conditions non stériles sur des milieux constitués de tourbe et de perlite. La croissance au champ d’arbres issus de micropropa- gation est comparable à celle de semis. Les méthodes d’embryogenèse ont été réalisées à partir de culture d’embryons immatures de Q petraea faites en milieu SH additionné de BAP (1 mg/l). La ma- jorité des cultures produisirent des embryons somatiques. La conversion des embryons en plants s’est faite à l’aide de traitements par le froid et la dessication. Ces plants ont été transférés en pot pour leur développement ultérieur. multiplication végétative / Quercus sp / bouture / culture de tissu / embryogenèse somatique

INTRODUCTION for application of recombinant DNA tech- nology to improvement of oak trees. Plants of oak species used for reforesta- Experiments with vegetative propaga- tion are traditionally raised from seed. The tion of oak by cuttings were started a long vegetative propagation of oak was consid- time ago. The rooting of various oak spe- ered difficult and has not been successful cies proved to be difficult and the progress on a commercial scale. In many regions, in vegetative propagation of oak has been good acorn harvests are not frequent and slow. Propagation of juvenile cherrybark acorns are difficult to store. The vegetative oak (Q falcata) by cuttings was reported by propagation of oak may provide an ade- Farmer (1965) and later Cornu et al (1975, quate plant supply when there is a natural 1977), Kleinschmit et al (1975), Garbaye et shortage of seeds and could reduce the al (1977), Chalupa (1980, 1982, 1990a) demand for seed-grown planting stock, es- and Spethmann (1982, 1985, 1986) de- pecially during years following poor seed scribed the production of rooted cuttings of harvests. important European oak species (Q pe- The increasing interest in vegetative traea and Q robur). propagation of oak over the last decade Experiments with tissue culture propa- stimulated detailed studies, and new tech- gation of oak started after trials with niques have been developed which enable cuttings. Initially, efforts were focused on production of clonal plants either by a regeneration of plants from callus cultures. stem-cutting system or by in vitro meth- Callus formation was stimulated (Jacquiot, ods. Vegetative propagation is important 1952; Seckinger, et al 1979; Srivastava for oak tree improvement. The long repro- and Steinhauer, 1982), however, plant ductive cycle of oak is a serious obstacle propagation was not achieved. A system to effective tree improvement by conven- based on in vitro multiplication of shoots tional tree-breeding techniques. Vegeta- from axillary buds has been developed tive propagation is an important method (Chalupa, 1979, 1981, 1983, 1984; Bella- for preserving the unique characteristics of rosa, 1981; Pardos, 1981; Vieitez et al, some trees. In vitro propagation of oak 1985). Micropropagated plantlets were species can be used for the production of transplanted into soil and later were plant- plants with desirable genetic traits. Effec- ed in the field. The system of axillary-shoot tive plant regeneration from meristems multiplication was used for micropropaga- and embryogenic cultures is a prerequisite tion of various oak species: Q robur and Q petraea (Chalupa, 1979, 1981, 1983, 1984, ent ages (1-30-yr-old trees). For each treat- 1985, 1987b, 1988, 1990b; Vietez et al ment, 40-90 cuttings were used. Cuttings were All 1985; Pevalek-Kozlina and Jelaska 1986; collected between May 20 and July 20. cuttings were inserted into the rooting mixture Civinová and 1987; Favre and Sladky, 2-24 h after being taken from trees. Bases of San- Juncker, 1987; Meier-Dinkel, 1987; leafy cuttings (10-20 cm long) were soaked in a José et al 1988, 1990; Juncker and Favre, hormonal solution (20-24 h in indole-3-butyric 1989; Volkaert et al, 1990), Q suber (Bella- acid (IBA) 200 mg·1-1) or treated with a talc- rosa, 1981, 1989; Pardos, 1981; Manzane- based rooting powder (1% IBA + 10% benomyl or 0.5% IBA + 0.1% acetic acid ra and Pardos, 1990), Q Shumardii (Ben- naphthalene + 10% and inserted into nett and Davies, 1986), Q acutissima (Ide (NAA) benomyl, rooting mixture consisting of peat and perlite (1:1 or and Yamamoto, 1986; Sato et al, Q 1987), 1:1.5, v/v). Cuttings were rooted either under con- serrata (Ide and Yamamoto, 1987) and Q trolled environment (in growth cabinets equipped lobata (Johnson and Walker, 1990). with a fog system) or in a greenhouse under an intermittent After relative air Somatic embryogenesis has great po- fog system. rooting, and were re- tential to be used for mass clonal humidity temperature gradually propaga- duced, and rooted cuttings wintered in the rooting tion of somatic plants. Recently, embryo- mixture in the same place in the unheated green- genesis was induced in oak. Immature or house. Rooted cuttings were lifted the following mature embryos, anthers or seedling seg- spring (in early June, after formation of new ments were used as the initial explants for shoots) and were transplanted in the nursery. induction of somatic embryogenesis in Q robur and Q petraea (Chalupa, 1985, 1987a, 1990c; Jörgensen, 1988), Q suber Propagation by tissue culture (El Maataoui and Espagnac, 1987), Q acu- tissima Q rubra and Q (Sasaki et al, 1988), Plant material alba (Gingas and Lineberger, 1989), Q ilex (Féraud-Keller and Espagnac, 1989), Q For initiation of Q robur and Q petraea organ cerris (Ostrolucká and Pretová, 1991). cultures, explants were taken from shoots of Plant regeneration from oak somatic em- seedlings 3-6-months-old. As the source of ma- bryos proved to be difficult and the conver- terial from older trees, shoots or 6-year-old or of sion of embryos into plants was achieved hedged trees, stump sprouts (from stumps 40-yr-old trees) were used. After removing all only in some species and at a low frequen- leaves, the axis was cut into shoot-tip and nodal cy. segments 10-20 mm long, which were surface- In this report, results obtained in our ex- sterilized in 0.1% mercuric chloride solution for periments with vegetative propagation of Q 20-40 min. After 3 succesive rinses in sterile robur and Q petraea by cuttings and by tis- distilled water, the initial explants were placed on agar nutrient medium. sue culture are presented and discussed. For initiation of somatic embryogenesis, im- mature seeds collected from 5 open-pollinated trees were used for experiments. Fruits were MATERIALS AND METHODS collected weekly in July and August. Seeds were surface-sterilized in calcium hypochlorite solution (7.5%, w/v) for 20 min and then washed Propagation by cuttings twice with sterile distilled water. Immature em- bryos were excised from seeds and placed on Leafy softwood cuttings were used for rooting agar nutrient medium. Explants (immature em- experiments with Q robur and Q petraea. bryos, nodal segments) were cultured in 100 ml Cuttings were collected from 6-year-old hedged flasks containing 20 ml of nutrient medium. stock plants (hedged 4-10 cm above the Each treatment involved 30-60 explants and ground) and from seedlings and trees of differ- was repeated twice. Culture media and conditions Q robur and Q petraea the potential of cuttings to form adventitious roots de- Organ cultures creased rapidly with increasing plant age. taken from trees 1- and Explants were cultured on modified Gresshoff- Cuttings 3-year-old Doy (GD) medium (Gresshoff and Doy, 1972), rooted at high frequencies and produced BTM (Chalupa, 1984), or Woody plant medium well-developed root systems. Cuttings (WPM) (Lloyd and McCown, 1980). The basal from older trees (9-30-yr-old) rooted poorly media were supplemented with glutamine (table I). Difficulties associated with aging The media contained various (100 mg·l-1). make the direct use of from older concentrations of the cuttings (0.2-2.0 mg·l-1) cytokinin trees unsuitable for clonal (6-benzylaminopurine (BAP) or (N-benzyl-9-(2- rapid propaga- tetrahydropyranyl)adenine (BPA). For rooting, tion. The use of cuttings from young plants NAA and IBA were used in concentrations rang- is limited because the quantity of cutting ing from 0.2 to 1.0 mg·l-1. Difco Bacto agar material which is produced by young ortet (6 g·l-1) was used to solidify nutrient media and is low. sucrose (20 g·l-1) as a carbon source. The The of older oak trees can media were adjusted to pH 5.7 before steriliza- rooting ability tion by autoclaving at 121°C for 20 min. Cul- be increased by cutting down the trees and tures were grown at 25°C in light with a 16-h by hedging stock plants. In our experi- photoperiod under cool white fluorescent lamps ments, cutting down and hedging was ef- (60 uE·m-2 s-1). fective in Q robur and Q petraea. Rooting potential of cuttings harvested from Somatic embryogenesis hedged 6-year-old plants of Q robur was Explants were cultured on modified Murashige- high (table II). The stock plants were Skoog (MS) medium (Murashige and Skoog, hedged every year and elongated sprouts 1962), Schenk-Hildebrandt (SH) medium were used for rooting. Hedging of oak and Hildebrandt, and WPM (Schenk 1972), stock plants offers an effective technique (Lloyd and McCown, 1980), supplemented with for the production of cuttings with high glutamine (200 mg·l-1) or casein hydrolysate (500 mg·l-1). The media contained cytokinin BAP rooting potential and high survival. (0.2-2.0 mg·1-1), and auxin (IBA 0.0-1.0 mg·l-1, or 2,4-D 0.0-2.0 mg·l-1). Media were solidified with Difco Bacto agar (6 g·l-1). Sucrose was used as a carbon source (MS and SH medium 30 g·l-1 WPM: 20 g·l-1). Cultures were grown at 25°C either in the dark or in light (16-h photoperi- od or continuous light).

RESULTS

Vegetative propagation by cuttings

Rooting potential in relation to maturation and the effect of hedging

Vegetative propagation by cuttings is usu- ally restricted to young material because aging reduce the ability to root cuttings. In Cuttings harvested from hedged trees exhibited significantly higher frequencies of formation of new shoots than cuttings col- lected from intact control trees (table II). Shoot growth of rooted cuttings were also stimulated by mineral nutrition. Regular watering (every 2nd d) of rooted cuttings with diluted WPM (1/10 strength of macro- elements) or incorporation of slow-release fertilizers into rooting mixture enhanced root quality and stimulated shoot growth. Supplemental nutrition with diluted WPM had a favorable influence on shoot elonga- tion. The formation of new shoots was also stimulated by supplemental lighting. Cuttings grown under continuous light (cool white fluorescent lamps) formed new shoots Effect of physiological condition at higher frequency (87%) than cuttings of stock plant on rooting potential grown under a natural photoperiod. Rooted cuttings, which formed new Stock plant environment markedly affected shoots and reached a total length of 30-50 rooting of harvested leafy cuttings. Irradi- cm in the autumn, wintered in the rooting ance, photoperiod and their interactions with mixture in the same place in an unheated nutrients had a marked effect on the rooting greenhouse and suffered only small loss- potential of leafy cuttings. In our studies, a es. The following spring, rooted cuttings long photoperiod (continuous light) im- were lifted (in early June) and transplanted proved rooting of Q petraea cuttings. in the nursery, where the growth continue. Cuttings from seedlings grown under contin- Their survival rate was high (78-94%) and uous light rooted in significantly higher per- vigorous plants were produced during the centages (92%) than those from seelings growing season. grown under natural daylength (76%).

Stimulation of shoot Vegegative propagation growth tissue culture after rooting of cuttings by

For successful vegetative propagation of At present, two methods can be used for oak, it is important not only to achieve root- tissue culture propagation of oak: axillary ing of cuttings, but to produce plants with shoot multiplication and somatic embryo- low mortality and rapid growth. In our ex- genesis. periments with Q robur, cuttings which, af- ter rooting, formed new shoots and had an Micropropagation by axillary shoot active metabolic exchange between root multiplication system and stem, exhibited high survival rates. Vigorous plants were produced from To establish cultures, we used actively cuttings which rooted quickly and were ca- growing shoots collected after bud flush- pable of rapid shoot growth immediately af- ing. Sterile nodal segments and shoot-tips ter rooting. of juvenile origin were placed on nutrient medium and started to grow within 1-2 BAP (2 mg·l-1). The multiplication rate weeks. Among the media tested, the high- (number of segments usable for the next est multiplication rate was obtained on low multiplication cycle) achieved on WPM salt media (BTM, WPM) supplemented supplemented with BAP was high (3-8, de- with a low concentration of cytokinin (BAP pending upon the clone). 0.2-0.6 Within 4-5 weeks, shoots mg·l-1). A new cytokinin, BPA effectively stimulat- elongated considerably and leaves devel- ed the formation of axillary buds and shoot on salt media oped. Explants grown high proliferation. Tested clones of Q petraea (MS, SH) produced short shoots. produced more shoots on media containing The number of new shoots that were BPA than on media supplemented with formed during the multiplication stage was BAP. Many shoots were produced on WPM moderated by cytokinin. Cytokinins BAP containing 0.6 mg·l-1 BPA (table III, fig 1). and BPA were the best stimulators of Tissue culture propagation of adult trees shoot proliferation of Q petraea and Q ro- was more difficult than propagation of bur. The of shoots was growth axillary Shoots initiated at the base of stimulated on WPM with a seedlings. supplemented the trunk retain characteristics and low concentration of BAP (0.2 mg·l-1). juvenile were used as the initial for the es- Higher concentration of BAP (0.4-0.6 explants tablishment of adult tree cultures mg·l-1) induced shoot proliferation and the (stump of 12 trees were number of produced shoots increased (ta- sprouts 40-yr-old used). The explants of adult trees were grown on ble III). Shorter shoots were produced on the same media as cultures. Ex- medium containing a high concentration of seedling plants from 7 trees produced multiplying cultures. The mean multiplication rate of cultures of adult origin was lower (by about 28%) than the rate of juvenile cultures, however, two genotypes exhibited the same proliferation rate as cultures of seed- ling origin. Rooting of microshoots was achieved in vitro and was also successful under non- sterile conditions in rooting mixture. Agar media used for in vitro rooting contained no cytokinin and had a lower level of min- eral salts. Cytokinins are strong inhibitors of adventitious rooting, and high-salt media had indirect inhibitory effects. GD agar me- dia and WPM (half- or full-strength) con- taining a low concentration of auxin (IBA or NAA 0.2-1.0 mg·l-1) stimulated root induc- tion. Within 2-3 weeks, 68-92% of micro- shoots of juvenile origin (depending upon the clone) produced roots. Rooting per- centages of microshoots initiated from adult trees were lower (by 24-78%, de- pending upon the clone), than those of mi- croshoots of seedling origin. High rooting percentages of juvenile shoot quality was very important in ex vitro microshoots were also obtained by direct rooting. Small microshoots (10-15 mm rooting in potting mixture. After auxin treat- long) exhibited higher mortality rates. Fully ment (a quick dip of the microshoot base developed leaves of microshoots were into liquid IBA, 1.0 g·l-1, for 1 min), micro- metabolically beneficial to rooting. Stem el- shoots were inserted into potting mixture ongation and formation of new leaves stim- (peat and perlite, 1:1, v/v) and kept under ulated adventitious root formation. The a plastic sheet in a humid atmosphere. treatment of microshoots with rooting hor- Mean rooting percentages of juvenile mi- mone was useful for increasing the speed croshoots ranged from 54 to 80% (depend- and uniformity of rooting and the number ing upon the clone). Ex vitro rooting was of adventitious roots. For ex vitro rooting, less laborious than in vitro rooting. Micro- humidity control was important. Shortly af- ter adventitious root formation, active from immature seeds collected in July and shoot growth resumed and the size of the early August produced embryogenic tissue plantlets increased substantially. The new- most frequently; 48-76% of cultured imma- ly formed leaves were much less suscepti- ture zygotic embryos produced embryo- ble to desiccation. Plantlets were grown genic cultures (table IV). Embryogenic cul- under high humidity for 5-8 weeks, then tures were initiated on modified SH and humidity was gradually reduced to normal MS media and WPM (containing 500 mg·l-1 levels. Plantlets grown under continuous of casein hydrolysate), supplemented with light maintained shoot growth after root BAP (1 mg·l-1) or BAP (1 mg·l-1) plus IBA formation and exhibited higher survival (1 mg·l-1). The immature zygotic embryos rates. cultured on these media produced embryo- tissue within 7-9 weeks The After plantlets formed new adapted genic (fig 2). was maintained leaves on elongated shoots and reached embryogenic competence tissue subculture. Em- the height of 10-20 cm, they were trans- by embryogenic tissues cultured on modified SH ferred outdoors and grown in partial shade bryogenic for 2-3 months. Most rooted plantlets of medium containing cytokinin kept their em- for more than 3 juvenile origin survived (76-94%) and con- bryogenic potential years. somatic were often tinued to grow. After hardening off, the Developing embryos attached to tissue. Secon- plants were planted in the field, usually in loosely parent somatic was early summer. Planted trees attained a dary embryogenesis frequent. Adventitious height of 20-30 cm at the end of the sec- embryos developed gradually into mature somatic ond growing season. In the following embryos. years, the growth of micropropagated Somatic embryos conversion was trees continued. Indeed there was no sig- achieved after alternations of physical con- nificant difference in growth between the ditions and medium changes. The conver- micropropagated plants and control seed- sion of somatic embryos into plantlets was lings. At the end of the 8th growing sea- son, the micropropagated trees were more than 230-290 cm high. The trees exhibited normal growth and appearance.

Plant regeneration by somatic embryogenesis

Somatic embryogenesis is a promising method of clonal oak multiplication. Our experiments showed the feasibility of us- ing immature zygotic embryos for initiation of highly embryogenic tissue and forma- tion of oak somatic embryos. In our experiments with somatic em- bryogenesis in Q petraea embryogenic cultures were initiated from immature zy- gotic embryos cultured on modified SH and MS media and on WPM supplement- ed with cytokinin. Zygotic embryos excised stimulated by exposure to cold (2-3 °C for 3-4 wk) and desiccation (dehydration of somatic embryos inside sterile sealed dishes for 2-3 wk). After desiccation, so- matic embryos were transferred into WPM containing a low concentration of cytokinin (BAP 0.1 mg·l-1) and were cultured under continuous light to induce conversion; 12- 18% of embryogenic cultures produced germinating somatic embryos. Some so- matic embryos produced only roots, some embryos produced shoots and roots (fig 3). The plantlets with growing shoots and roots were subcultured individually on WPM without cytokinin. More than 90 plantlets of Q petraea regenerated from somatic embryos were transplanted into potting mixture. Plantlets were grown un- der high air humidity and continuous light. After acclimatization, 62 plants of Q pe- traea regenerated from somatic embryos were planted in the nursery. DISCUSSION The importance of tissue culture as a propagation method of oak continues to grow. A system based on offers the micropropaga- Vegetative propagation opportun- tion by axillary shoots has been developed to use valuable in ity genotypes commer- (Chalupa, 1979, 1981, 1983, 1984; Bella- cial is an forestry. Vegetative propagation rosa, 1981; Pardos, 1981; Vieitez et al, alternative to a based on breeding system 1985) and proved to be effective. Recently seed orchards. It seems that seed or- the system has been refined (Bennett and chards are difficult to use in breeding oaks Davies, 1986; Meier-Dinkel, 1987; Chalu- due to their and long reproductive cycle pa, 1988, 1990b; San-José et al, 1988, low acorn production. 1990) and used for production of plants for The problem of aging plays an impor- field testing. Experiments indicate that tis- tant role in vegetative propagation (Bonga, sue culture propagation of oak will become 1982, 1987; Durzan, 1984, 1990). The a useful tool for the clonal multiplication of idea to propagate mature-plus oak trees is selected plants. Plants produced from tis- not easily applicable. For successful clonal sue cultures are as vigorous as plants pro- oak propagation, juvenile tissue is essen- duced by conventional methods. Field tial as the initial explant. Shoots originating growth of micropropagated oak trees of from juvenile zones of the tree exhibit juve- juvenile origin was comparable to that of nile characteristics (Schaffalitzky de Muck- control seedlings. It is anticipated that the adell, 1954, 1959). Experiments with vari- axillary-shoot multiplication method will ous tree species (Bonga, 1982, 1987; continue to be the main tissue culture Hartmann and Kester, 1983; Franclet et al, method for oak propagation. 1987) and our experiments with oaks indi- Development of somatic embryogenesis cate that cuttings made from stump as a propagation method continues and and from sprouts hedged stock plants cut new information on initiation of embryogen- back every year are juvenile explants ic culture and oak regeneration has been which root easily. Experiments show that published (Chalupa, 1987a, 1990c; Sasaki cutting down and hedging of oak trees is et al, 1988; Gingas and Lineberger, 1989). an efficient method to obtain juvenile ma- Experiments showed the feasibility of us- terial from older trees. ing immature embryos for initiation of high- For possible use of cuttings in commer- ly embryogenic tissue and for formation of cial forestry, rooted cuttings with high sur- oak somatic embryos. In vitro induced em- vival rates and good growth and morpholo- bryogenesis often depended upon the gy must be produced. The physiological presence of growth regulators in the nutri- status of stock plants had great influence ent medium, however, their role is not on rooting potential and mortality of rooted clear. Some species required the presence cuttings. Correct timing of cutting collec- of auxin in medium for the induction of em- tion, sufficient mineral nutrition, a reliable bryogenesis, for other species this sub- fog system and effective irradiance during stance was not essential. The main prob- lem is the of the rooting process favored the production low frequency conversion of of rooted cuttings with high survival rates. oak somatic embryos into plantlets. Before somatic is used as a Rooting cuttings, which formed new embryogenesis prop- shoots shortly after rooting and wintered in agation method, many problems must be solved. an unheated greenhouse, exhibited high survival and rapid shoot growth during the Currently available results and knowl- following growing season. edge indicate that a stem-cutting system and micropropagation by tissue culture are Chalupa V (1984) In vitro propagation of oak promising methods for clonal oak propaga- (Quercus robur L) and linden (Tilia cordata tion. Close association of micropropaga- Mill). 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