SK84493A3 - Method of multiplying in vitro of magnolia soulangiana soul.bod. - Google Patents

Abstract

Solution is concerning the method of multiplying of Magnolia soulangiana Soul.bod. by method in vitro in aseptic conditions from explanates removable from grown-up wood species. Primary explanates (buds, apexys, nodal segments a.s.o.) are after sterilization cultivated on modificated cultivating mediums consolidated by agar and is with additing of growth regulators and active coal.

Description

The present invention relates to a method for the propagation of a magnolia soulangiana Soul (Point.) By an in vitro method, under aseptic conditions, from explants taken from adult woody plants. The primary explants (buds, apexes, nodal segments, etc.) are cultured on modified agar-reinforced culture media with the addition of growth regulators and activated carbon after sterilization.

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SMonium Magnolia Magnification Method

BACKGROUND OF THE INVENTION The present invention relates to a method for propagation of Magnolia soulangeane (Magnolia x soulangeane Sou) by the in vitro method, τ of mature woody plants.

D o t e r i j i s t. and in. technique

Magnolias are among the decorative woods of great interest. To date, M. soulange's reproduction has been largely ensured by vegetative methods (cuttings). [Hieke, K .: Magnolia Cutting, Zahradnickie Listy 5, 125-154 (1967); Seneta, W .: Magnolia Magnification. Ogrodnictwo 6, 180-181 (1973); Bojarczuk, K .; Spreading of magnolia from seedlings zielnycíi z zastosowaníem various czynników stymulu yacych ukorzenianie, Arboretum Kornickie XXVII, 170-185 (1982); Bärtels, A .: Reproduction of woody plants, SZN Praha 1988, 451). This method of magnolias propagation is associated with considerable difficulties such as long rooting time (1 -? Years), low percentage of rooted cuttings, limited production of cuttings and the like. The cuttings are very difficult to root vegetatively, and without the application of growth stimulators, the root formation% is very low. Collection of cuttings. I c the most advantageous leri in a period of intensive growth, which limits not only the harvest period but also the amount of cuts taken. The roots of the cuttings are also negatively affected by the age of the mother tree and the place of collection of explanatory tatas.

At present, therefore, there is an interest in the possibility of their reproduction under controlled conditions (even in vitro), which will ensure qualitatively and quantitatively higher efficiency of seedling production. In vitro magnolia propagation experiments generally employ juvenile material.

The essence of the]] _; zu

The inadequacy of the above methods of reproduction is overcome by the in vitro multiplication of the magnolia soulangea (Magnolia x soulangeana Onul.) Method according to the invention. The essence of the invention lies in t.om. that primary explants (buds, apexes, nodal segmnets) are taken from adult species (parent plant), sterilized and cultured after sterilization under aseptic conditions on agar-reinforced, modified culture media containing growth regulators (axillary growth) up to 12 weeks). The formed axillary shoots are separated from the primary culture and further cultured on a modified rooting medium until the root is formed (typically 4 to 8 weeks).

It was found. that it is advantageous to add growth regulators, in particular growth regulators of the cytokinin family, e.g. benzylaminopurine (BAP) in an amount of 0.2 to 0.3 mg.l ¹ and from the group of auxins, e.g. α-naphthylacetic acid (NAA) at a concentration of 0.01 to 0.1 mg.l ^-1 . Sucrose is added as a carbon source in an amount of up to 20,000 mg.l ¹ ) and cultivation is performed under aseptic conditions, at a temperature of 20 to 24 ° C, with an illumination intensity of 2.0 to 2.5 klx for 14 to 16 hours per day.

It was also found that it is preferred that the rooting medium contains an acid (1-ylbutyric indole (IBA) in 1.0 to 6.0 mg.] ^'1 and activated carbon in a concentration of about 0.3%.

The in vitro method of magnolization according to the invention brings a number of advantages, namely the possibility of producing the material throughout the year regardless of the season, faster propagation in saving space, since it is possible to grow more regenerants in a smaller space compared to conventional propagation, multiplication coefficient compared to traditional methods (1 st primary explanatory 5 to 10 mm long produces 4 to 9 shoots) and the possibility of repeating the propagation yoke 2-3 times during the year, shortening the time to root the shoots (4 to S weeks) as opposed to 1 to 2 years in the case of trimmings), the production capacity of the regeneration method according to the invention is at least 1000 per plantation per explant per year.

EXAMPLES OF EMBODIMENTS AND INVENTION

A. Production of shoots

Example 1

They were used as primary explants. magnolia buds, taken in spring (March) from a 40-year-old tree (M. soulanaeova Soul. growing in Arboretum Mlyňany). After collection, the buds were sterilized with 0.1% mercuric chloride with Tween (0.05%). After sterilization, the buds were repeatedly washed in distilled and subsequently in redistilled water and stored on a culture medium with a modified proportion of growth regulators (vicf tab.1). The resulting shoots in the laminar box were divided into segments with a length of 3-5 mm, distinguishing between apex and nodal segments.

Cultivation of these segments was done in 370 ml sealed glass containers containing 50 ml of 6.5 gI- ¹ agar-reinforced culture medium and 20 g.I- ¹ sucrose. Culture agar media was adjusted to pH 5.6-5.8 by addition of IM NaOH, to which were added growth regulators from the group of cytokinins (benzylaminopurine 0.3 mg.l · ¹ ) and auxins (α-naphthyl acetic acid).

0.1 mg.l ^-1 ) and autoclaved at tef? 1 ° C for 20 minutes. The explants (apexes and nodal segments) were cultured at temperatures ranging from 20 to 24 ° C. in an air-conditioned room on shelves under lighting panels with an illumination intensity of 2.0 to 2.5 klx, at 14-16h fluorescent lighting (8-10h without light), for 10 weeks. The production effect was 7 to 9 shoots per segment, with an average shoot length of 16 mm.

T i b u i k no. 1

Composition of culture media

¾ 1 o / en i e Staridai di a Catalano (1965) [mg.1 ¹ 1 Medium of Cown and Lloyd (1981) [mg.1 ¹ ] Knog 1800 - Ca (H ¢ 13) 2 - 4H? 0 1200 5 56 NH4NO3 400 400 rtgSOzí. 7H? 0 360 370 K2SO4 - 990 KH2PO4 270 170 CaC12-2H20 - 96 FeS04 27.85 27.85 NaEOTA 37.25 37.25 ZnS04 10 B 6 H3BO3 10 6.2 ΜΠ3Ο4.H20 18 22.3 CuS0 ₄ .5H2O 0,025 0.25 N32MO04.2Η2Ο 0.25 0.25 ΚΙ - - K '/ s. nicotinic 5 0.5 G1ycín 2 2 pyridoxine 0.5 0.5 Tiamíη 0.5 1.0 K y s. Folic 0.5 - biotin 0.65 - 61utam i n - 2 Myoinozi tol L00 100 agar 7 000 7 000 sucrose 20 000 20 000 PH 5.4-5,6 5.4-5.6 Standardi, A. Catalano, F .: Tissue culture propagation 0

kiwifruit, Combined Proceedings I n t. Plánt Propag. Soc., 34, 234-243 (1985): Mg Cowri, 8H, Lloyd. G .: Woody plant medium (WPM) and mineral forces icrit lormulation For mi cror.ulture of woody plant species, Sci Sci ·? Π <: · = · lf ·. 453 (1931).

He advanced on the as in Example 1 with the difference that in kul t.i vačnom medium the following components have been modified: ZnS04.7H2O (8.6 mg.1 “ ¹ ) H3BO3 (6.2 mg.I ^-1 ) MnS04.H2O (1.0 mg.1 ' ¹ 1 KI (0.08 m g. 1 “ ¹ ) C0CI2 (0.025 mg.I- ¹ ) thiamine HCl (4.0 mg.I ' ¹ ), excluded the following components: nicotinic acid, glycine, pyridoxine HCl, folic acid, biotin. growth regulators - BAP and NAA unchanged

concentration.

The production effect of the explant culture was 5 to 7 shoots per segment, with a shoot length of 12-16 mm.

Example 3

The procedure was as in Example 1 except that modified McCown and Lloyd medium were used (Table 1). BAP concentration: 0.2 mg.I ^-1 , NAA concentration: 0.1 mg.I ^-1 . The production effect was 4-5 shoots per segment, with a shoot length of 14-16 mm.

Example 4

The procedure was as in Example 1 except that the spring apical portions of actively growing shoots from adult trees were used as the primary explants. Leaves were removed from shoots containing 2-3 internodes and leafless shoots were perfectly sterilized (Example 1). The explants thus prepared were divided into sterile conditions into apex and 2-3 nodal segments. The production effect of the explant culture was 4 to 8 shoots per segment, average length 17 to 14 mm.

B. Rooting of shoots

Example 1 3. d 5

Vi cli f er eric -xxildr not bulbs. who reached a length of 2 to 4 cm, were separated from the primary culture and rooted in conditioned Standard and Catalano medium. The content of growth regulators from the group of auxins - β-indolylbutyric acid (IBA) was 6.0 mg.I ^-1 , with the addition of 0.3% activated carbon, the proportion of macros oel einen products was adjusted as follows:

K NO? (900 mg.l ¹ ) Ca (NO3) 2.4H2O (600 mg.l ^-1 ) NH4NO3 (200 mg.l- ¹ ) MgSO4.7H2O (180 mg. ¹ L) KH2PO4 (135 mg.l- ¹ ) Culture medium was agar-reinforced (6.5 gl ¹ ) and replenished sucrose (20 000 mg.I ^-1 ). Shoots rooted in

370 ml glass culture flasks containing 50 ml medium. 1-2 shoots grown in the culture conditions of Example 1 were placed in the culture vessels thus prepared.

The best rooted shoots were 2 to 3 cm long (2 to 3 internodes). without leaf reduction. Shoots rooted in a period of 4 to 6 weeks. The production effect of the roots was 4 to 8 roots per 1 shoot, with an average length of 21 to 26 mm. The rooting percentage ranged from 97 to 99.

Example 1 <> d 6

The procedure was as in Example 5 except that the culture medium contained 1.0 mg.l ^-1 IBA and 0.3% activated carbon. per-

cento rooting ranged from 80 to 83, production effect average length 28 to 30 was 1 mm. to 2 kor en e per 1 sprout, i ch At KLA il 7 The procedure sa like in case 1-i d 5 -i : · Team difference that cultivation medium obs- h oval 0 3,0 m g. .1 ¹ IBA a 0.3 activated carbon.

The rooting percentage ranged from 60 to 65. the number of roots per shoot was 0.9 to 1.0, the average length was 31 to 33 mm.

Example 8

The procedure was as in Example 5 except that the culture medium did not contain any growth regulators and 0.3 µl of activated carbon was added. The rooting percentage was from 40 to 45. The number of roots per explant was 0.7 to 1.0, the average length being 30 to 32 mm.

P Γ. i e m y. s e 1 n. y tein tein se

IN

The invention can be used in ornamental horticulture to produce Magnolia soulange seoul seedlings, to build genobanks and enrich the gene pool of botanical gardens and arboretums, to create and protect the environment, to save and reproduce the gene pool in areas affected by industrial pollution, in breeding programs.

Claims (7)

An in vitro method of propagation of a magnolia of soulange (Magnolia x soulangiana Soul.), Characterized in that from explants, the artificial culture regulators are grown to adult trees, harvested primarily, and after sterilization on agar and growth media until axillary. shoots. the formed axillary shoots are separated from the primary culture and grown on modified rooting media until the root system is formed. 2. The method of claim 1, wherein the plant growth regulators in the development of axillary shoots and the Rollover a 6-benzyladenine (BA) at 0.2 _f to 0.3 mg.l ^-1. Method according to claims 1 and 2, characterized in that alpha-naphthylacetic acid (NAA) is added at a concentration of 0.01 to 2 as the growth regulator of the multi-peak explant culture in the prolongation of axillary shoots 0.1 mg.I ^-1 . The method according to claim 1, characterized in that the culture medium for multiplication and rooting and shoots contains 20 g. I ^-1 sucrose. _T Method according to claim 1, characterized in that the explants are grown under aseptic conditions at a temperature of 20 to 24 ° C, a light intensity of 35-40 µmol.s and a photoperiod length of 14 hours. Method according to claim 1, characterized in that the rooting medium contains beta-indolylbutyric acid in a concentration of 1.0 to 6.0 mg.I ^-1 . Method according to claim 1, characterized in that the rooting medium contains activated carbon in a 0.3X concentration.