MIKROPROPAGÁCIA VYBRANÝCH DRUHOV MAGNÓLIÍ IN VITRO

MICROPROPAGATION OF SELECTED MAGNOLIA spp. IN VITRO

ING. AURÉLIA KAMENICKÁ, CSC., RNDr. MÁRIA LANÁKOVÁ, CSC., ING. JURAJ KUBA

Arborétum Mlynany SAV, Vieska nad Žitavou, 951 52 Slepcany e-mail: aurelia@nr.sanet.sk

Abstract A micropropagation method for three magnolia species was established using nodal segments of adult plants. The shoot cultures have grown under identical conditions on Standardi - Catalano medium supplemented with 2.22 µmol.dm-3 BAP and 1.07 µmol.dm -3 NAA. The growth and proliferation of M. liliiflora was less effected as compared with the species M. salicifolia and M. x soulangiana. Statistically significant were the differences in a total biomass production of shoots between tested species. The highest number of shoots per explant (<7) was obtained in M. salicifolia and M. x soulangiana, where as the greatest shoot length (<16) was obtained in M. salicifolia. The mean length of produced shoots was low in the species M. liliiflora. The method was proved to be convenient for propagation of magnolia species M. x soulangiana, M. liliiflora and M. salicifolia, respectively. Key words: Magnolia species, in vitro culture, regeneration List of abbreviations: 6-benzylaminopurine (BAP), a -naphthaleneacetic acid (NAA), 4-(3-indolyl) butyric acid (IBA) Súhrn

Práca popisuje postup mokropropagácie troch vybraných druhov magnólií (M. x soulangiana Soul.-Bod., M. liliiflora Desr., M. salicifolia L.) v podmienkach in vitro. Primaárne kultúry sme odvodili z nodálnych segmentov a pestovali na kultivacnom médiu STANDARDI a CATALANO (1985) s pridaním 2.22 µmol.dm-3 BAP a 1.07 µmol.dm-3 NAA.

Rastovú charakteristiku pletivových kultúr vybraných druhov udávame v tabulke 1. Najnižší pocet axilárnych výhonkov na explantát sme zistili pri M. liliiflora (3,0), ale výhonky boli dlhšie v porovnaní s M. x soulangiana a M. liliiflora. Výsledky potvrdili závislost multiplikácie výhonkov od genotypu. Pri vyhodnotení produkcie biomasy (tab. 2) vyšší prírastok cerstvej hmotnosti v prepocte na výhonok sme zistili pri M. salicifolia (199,7 mg). Rozdiely v pocte výhonkov v prepocte na kultivacnú nádobu medzi pletivovými kultúrami M. salicifolia a M . x soulangiana neboli štatisticky preukazné. Pri vyhodnotení sušiny výhonkov vyššie percento vody sme zistili pri pletivovej kultúre M. x soulangiana (94,6). Výhonky boli štavnaté, krehké s predlženými listami. Po oddelení tažšie zakorenovali.

Introduction

Magnolias are deciduous trees (Magnolia salicifolia L.), multi-stemmed large shrubs (M. x soulangiana Soul.-Bod.) and shrubs or small trees (M. liliiflora Desr.) belonging to the most glamorous woody plants. The propagation by cuttings is rather slow as compared with micropropagation which seems to be a quicker method of multiplication. In general, the genotype and source of primary explant are considered to be important factors in a micropropagation system (COLLIN and EDWARDS 1998). In magnolias, the shoot tips are usually used as primary explants (BIEDERMANN 1987, KAMENICKÁ, VÁLKA 1997, KAMENICKÁ, LANÁKOVÁ 2000). Magnolias are also amenable to propagation via somatic embryogenesis (MERKLE, WATSON-PAULEY 1994, MERKLE 1995). Axillary shoot proliferation in magnolias is difficult because of a high content of phenolic substances and the formation of vitrified shoots (MERKLE, WIECKO 1990, KAMENICKÁ, VALOVÁ 1994). The objective of this research was to study the growth, axillary branching of three Magnolia shoot cultures grown under identical in vitro conditions.

Material and Methods Plant material

Juvenile shoots (7-10 cm long) of Saucer magnolia (M. x soulangiana Soul.-Bod.) consisting of 3 to 4 internodes were collected in April from a 100-year old shrub. Apical shoots of Lily

flowered magnolia (M. liliiflora Desr., 5-3 cm long) with 2 or 3 internodes were collected from 40-year old shrub. Nodes from juvenile shoots of Japanese willow leaf magnolia (M. salicifolia Sieb. et Zucc.) Maxim.) were taken from 60-year old tree to start a shoot multiplication. All species were obtained from the gene pool of Arboretum Mlynany.

Micropropagation

After the removal of leaves the shoots were surface sterilised with 0.1% (w/v) mercuric chloride solution with a few drops of Tween 20 for five minutes. The shoots were soaked in 70% ethanol (20 sec.) and washed th ree times with sterile distilled water. Then they were cut on nodal segments (3-5 mm long) and cultured on S medium (STANDARDI, CATALANO 1985). The culture medium used for subculture of shoots was as follows: macrosalts and microsalts of S medium, NaFeEDTA 37.5 mg.l-1, sucrose 2% (w/v), meso-inositol 100 mg.l-1, pyridoxine 4.0 mg.l-1, BAP 2.22 µmol.dm-3 and NAA 1.07 µmol.dm-3 at pH 5.5 - 5.8 before autoclaving. As a gelling agent, 7.0 g.l-1 agar was added. Shoots of primary cultures were cultured in 100 ml culture vessels containing 25 ml of culture medium. Subculture of shoots was carried out in culture vessels containing 50 ml of medium. The culture vessels were closed with plastic light permeable closures with vent membranes (FÖRING-LOCK M). All chemicals were purchased from SIGMA (St. Louis, MO, USA). Shoot cultures were incubated in a growth chamber at 20-24 0C with a 16 h photoperiod by fluorescent tubes (light intensity 35-40 µmol.m-2.s -1). After the initiation phase, up to three months, the primary cultures started their development. At the end of subculture period, the good proliferating cultures were taken for multiplication.

Other parameters, the total number of shoots, the number of shoot x dry weight, fresh/dry weight ratio of shoots (FW/DW), and total capacity of proliferation (length x number of shoots) were recorded. For each genotype ten replicate culture vessels were used with four explants per vessel. All experiments were repeated at least three times. The data referring to number of shoots, their length, fresh and dry weights of axillary shoots were analysed by SAS/statistical program package for personal computers (SAS Institute Inc., USA, 1998).

Rooting of shoots and transfer to the soil

Shoots of approximately 40 mm in length were used. They were stimulated with 9.80 µmol.dm-3 of IBA in a reduced rooting medium according to STANDARDI, CATALANO (1985). During root induction and development the temperature was the same as at the multiplication stage. After 90 days of cultivation the number of rooted shoots were counted. Rooted plants were transferred to a greenhouse with the air temperature of 21-24 0C and the soil temperature of 24 0C. After acclimatisation (6-8 weeks), pots containing the plant were transferred to soil. These results have been archived before by KAMENICKÁ, VÁLKA (1997) and KAMENICKÁ (1998).

Results and Discussion Initiation of cultures from mature plants

The regeneration potential of the primary culture in tested magnolia species varied considerably. Table 1 summarises the results achieved during establishme nt of shoot cultures from three adult magnolia species. The mean number of shoots regenerated per explant varied significantly, depending on the species and ranged from 4.5 in M. x soulangiana to 3.0 in M. liliiflora . The greatest number of axillary shoots was produced by the primary cultures of M. x soulangiana and M. salicifolia (4.1-4.5/explant) as compared with the lowest number of axillary shoots in M. liliiflora culture (3/explant). At the beginning of in vitro cultivation, some species grew very quickly requiring a relatively long period of adaptation to the cultivation conditions (M. x soulangiana 3 months, M. salicifolia 5 months, M. liliiflora 6 months). After six or eight subcultures, the tested species were completely adapted to in vitro conditions.

Differences in multiplication response of different species have also been reported by VIEITEZ et al. (1983), COLEMAN, ERNST (1989). Also the rate of shoot formation varied between genotypes and provenances (PREECE et al. 1991). It is a generally accepted principle in

Komentár: nehodí sa mi tam slovo summarized

micropropagation that various clones that have to be cultured require a different "recipe". TUBESING (1998) has also reported that optimal conditions required by individual cultivars of magnolias are necessary to be modified. The production of axillary shoots per explant was affected by a species with number of shoots per explant (1-3) being greater in M. liliiflora than in other species tested. The average number of shoots reaching more than 6 shoots was characteristic for M . x soulangiana culture. The 17 % of M. salicifolia cultures and 20% of M. x soulangiana cultures produced average more than 7 axillary shoots per explant.

Table 1 Growth characteristic of three Magnolia shoot cultures

Growth parameters M. x soulangiana M. salicifolia M. liliiflora Number of shoots/explant 4.5 ± 0.13 4.1 ± 0.11 3.0 ± 0.11 Length of shoots (mm) 13.9 ± 0.21 15.3 ± 0.21 18.7 ± 0.43 Fresh weight (mg)/explant 753 ± 1.5 819 ± 3.8 712 ± 2.1 Dry weight (mg)/explant 41 ± 1.3 67 ± 2.5 74 ± 2.8 FW/DW 18.4 12.2 9.6 Total dry weight (%) 5.4 8.2 10.4 H2O (%) 94.6 91.8 89.6

The growth of shoot culture

The growth of shoots was influenced by a species. The length of axillary shoots was the greatest (18.7 mm) in the culture of M. liliiflora as compared with M. salicifolia and M. x suolangiana cultures (11-15 mm). These results are shown in Table 1. The results obtained so far suggest that the response of shoot culture to culture conditions is a species -dependent. At the end of proliferation period a positive effect of species on the fresh/dry ratio of shoot cultures was observed. The shoots of M. liliiflora species attained the lowest amount of biomass (FW/DW), whereas the corresponding values in the remaining species values were higher. A total shoot fresh weight was comparable in both M. liliiflora and M. x soulangiana. A higher fresh weight was recorded only M. salicifolia shoot culture. The species exhibited also a higher total proliferation capacity and a higher total number of shoots (Table 2). The differences in number of shoots per culture vessel for species M. salicifilia and M. x soulangiana were statistically significant (P< 0.05). These results suggest that each species has different physiological requirements for maximum shoot proliferation and for growth. Table 2 Biomass production of three Magnolia species

Genotypes Shoots/culture vessels

Fresh weight/per shoot (mg)

Total shoot/explant

Total capacity of proliferation/explant

M. x soulangiana 16.92* 167.3* 184 60.55 M. salicifolia 16.28* 199.7* 275* 62.73 M. liliiflora 10.08 96.2 222 56.10 ? significantly different distribution at P<0.05

Biomass increament

At the end of multiplication stage a positive effect of species on fresh weight of shoots was observed in M. liliiflora, but only in instances where the 62.5% proportion of shoots with more than 700 mg of fresh weight was observed. No such correlation has, however, been observed in M. salicifolia and M. x soulangiana (Fig. 1). As to the dry weight parameter, by its proportion of 86.1% of shoots with 70 mg dry weight M x. soulangiana has deviated significantly comparison with M. liliiflora and M. salicifolia (Fig. 2). The shoots of M. x soulangiana were usually succulent with a larger size of leaves than in M. liliiflora and M. salicifolia . Genotypic control of shoot regeneration has also been reported in Populus tremula, P.tremuloides (AHUJA 1983). WELANDER

Komentár: nerozumeiem tejto vete

and NYSTRÖM (1997) found that genotype of the donor plant was a decisive factor determining in vitro regeneration of Quercus robur. Significant clonal differences have also been revealed during the shoot tip proliferation of five Cotoneaster genotypes (MONIER, OCHATT 1995). DE ROGATIS and FABBRI (1997) have reported of significant interaction between the genotypes and culture media as well as between the genotypes and plant hormones during the regeneration of Prunus avium L. Considerable differences in number and size of new shoots were established among individual species or cultivars (VON ARNOLD, TILLBERG 1987). The authors have attributed them to the different level of endogenous hormones and to culture conditions. BIEDERMAN (1987) concluded that the best micropropagation of adult Magnolia genotypes was achieved by axillary shoot multiplication. In the light of presented results, it seems that the procedure is useful for the clonal propagation of the genotypes that we have attempted. Acknowledgement

This research was partially supported by the Grant Agency of the Slovak Academy of Sciences, project No. 51-51-1036/00-2000.

References AHUJA, M.R., 1983: Somatic cell differentiation and rapid clonal propagation of aspen. Silvae

Genetica, 32, s. 131-135. BIEDERMAN, I.E.G., 1987: Factors effecting establishment and development of Magnolia hybrids in

vitro . Acta Horticulturae, 212, s. 625-629. COLEMAN, G.D., ERNST , S.G., 1989: In vitro shoot regeneration of Populus deltoides , effect of

cytokinin and genotype. Plant Cell Rep., 8, s. 459-462. COLLIN, H.A., EDWARDS, S., 1998: Plant Cell Culture.Bios Scientific Publishers, Oxford, 157 s. DE ROGATIS, A., FABBRI, F., 1997: Improved regeneration of wild cherry (Prunus avium L.). In:

Ríordáin, F. (Ed.) Development of integrated systems for large-scale propagation of elite plants using in vitro techniques, COST 822, European Communities, Luxembourg, s. 52-53.

KAMENICKÁ, A., VALOVÁ, M., 1994: Effect of culture media on formation of axillary shoots of Magnolia x soulangiana Soul.-Bod. In vitro. Annali di Botanica, 52, s. 37-43.

KAMENICKÁ, A., VALKA, J., 1997: Cultivation and Propagation of Magnolias. Technical University Publishers, Zvolen, 99 s.

KAMENICKÁ, A., 1998: Influence of selected carbohydrates on rhizogenesis of shoots saucer magnolia in vitro . Acta Phyiol. Plantarum, 20, s. 425-429.

KAMENICKÁ, A., LANÁKOVÁ, M., 2000: Effect of medium composition and type of vessel closure on axillary shoot production of magnolia in vitro . Acta Phyiol. Plantarum, 22, s. 129 -134.

MERKLE, S.A., WIECKO,A.T., 1990: Somatic embryogenesis in three magnolia species. J.Amer. Soc. Hort. Sci., 115, s. 858-860.

MERKLE, S.A., WATSON-PAULEY, B.A., 1994: Ex vitro conversion of pyramid Magnolia somatic embryos. Hort Science, 29, s. 1186-1188.

MERKLE, S.A., 1995: Somatic embryogenesis in Magnoliaceae (Liliodendron and Magnolia). In: Bajaj, Y.P.S. (Ed.) Somatic embryogenesis and Synthetic seed I. Springer-Verlag Berlin Heidelberg, s. 388-403.

MONIER, C., OCHATT, S.J., 1995: Establishing micropropagation conditions for five Cotoneaster genotypes. Plant Cell Tissue and Organ Culture, 42, s. 275-281.

MURRAY, M. G., THOMPSON, W. F., 1980: Rapid isolation of high molecular weight DNA. Nucleic Acid Res., 8, s. 4321-4335.

PREECE, J.E., HUETTEMAN, C.A., ASHBY, W.C., ROTH, P.L., 1991: Micro- and cutting propagation of silver maple. II. Genotype and provenance affect performance. J.Amer. Soc. Hort. Sci. 116, s. 149-155.

STANDARDI, A., CATALANO, F., 1985: Tissue culture propagation of kiwifruit. Comb. Proc. Int. Plant. Prop. Soc., 34, s. 236-243.

TUBESING, CH.E., 1998: Magnolias with a future: Propagation and nursery culture. In : Hunt,D. (Ed.) Magnolias and their allies. Inter. Dendrology Society and The Magnolia Society, Sherborne, s. 199-200.

VIEITEZ, A.M., BALLESTER, A., VIEITEZ, M.L., VIEITEZ, E., 1983: In vitro plantet regeneration of mature chestnut. J.Hortic.Sci., 58, s. 457-463.

VON ARNOLD , S., TILLBERG, E., 1987: The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of Picea abies. Plant Cell Tiss. Org. Cult., 9, s. 253-261.

WELANDER, M., NYSTRÖM, J., 1997: The influence of explant type and ontogenetic age on shoot multiplication and rooting in vitro and acclimation and growth ex vitro of Quercus robur. In: Ríordáin, F. (Ed.) Development of integrated systems for large-scale propagation of elite plants using in vitro techniques, COST 822, European Communities, Luxembourg, s. 118 119.

Figure 1 Fresh weight of shoots per explant (%) of three magnolia species

Figure 2 Dry weight of shoots per explant (%) of three magnolia species

0

10

20

30

40

50

60

70

80

90

%

>70 71-100 <100

mg

M. salicifolia M. liliiflora M. soulangiana

0

10

20

30

40

50

60

70

%

>700 701-1000 <1000

mg

M. salicifolia M. liliiflora M. soulangiana