THE ROOTING OF CHRYSANTHEMUM [CHRYSANTHEMUM × GRANDIFLORUM (RAMAT.) KITAM] IN IN VITRO AND IN VIVO CONDITIONS

Alicja Tymoszuk¹, Natalia Miler², Małgorzata Zalewska¹, Małgorzata Borawska^3
1 Department of Ornamental Plants and Vegetable Crops, University of Technology and Life Sciences in Bydgoszcz, Poland
² Department of Ornamental Plants and Vegetable Crops, Faculty of Agriculture and Biotechnology, University of Science and Technology, Bydgoszcz, Poland
³ Department of Ornamental and Vegetable Crops, University of Technology and Life Sciences, Bydgoszcz, Poland

ABSTRACT

The purpose of the present paper is a comparison of the quality of chrysanthemum plants of three cultivars: 'Bislet', 'Euro' and 'Reddy', which were acclimatized after having been rooted in vitro, with the quality of plants acclimatized and rooted at the same time in vivo in selected substrates. In in vitro culture the plants were rooted on MS medium with an addition of 2mg·dm^-3 IAA, and next acclimatized in a mixture of peat with perlite in the volume proportion of 2:1 (v:v). Peat, perlite and a mixture of peat with perlite in the volume proportion of 2:1 (v:v) were used for direct rooting in vivo taking place simultaneously with acclimatization. A positive effect of in vivo rooting in perlite on the increase of the length and weight of the root system was found out. Plants rooted in vitro had numerous short roots of high weight. The largest fresh weight of the roots was observed in 'Bislet' and 'Euro' chrysanthemums. 'Bislet' produced the fewest roots, but they were the longest. In 'Reddy' cv. it was observed that the roots had the smallest fresh weight and they were the shortest. The highest increase of the shoot length was observed in the plants previously rooted in vitro and in the plants rooted in vivo and directly acclimatized in perlite. The most of new leaves were formed by microcuttings rooted in vitro.

Key words: acclimatization, in vitro rooting, in vivo rooting, Chrysanthemum × grandiflorum (Ramat.) Kitam., substrates.

INTRODUCTION

In the commonly used systems of micropropagation of plants, the following stages can be distinguished: initial stadium, stadium of intensive propagation, rooting and, following them, acclimatization. Adventitious roots formed in in vitro culture have no root hairs or they are poorly formed, and the vascular bundles are not fully developed, which is why these roots function worse than those formed in the substrate [5]. In vitro rooting requires considerable work expenditures and it constitutes from 35 to 75% of total costs of micropropagation [4]. Specific conditions of in vitro culture cause that in comparison to plants growing in vivo, stomata do not function in microcuttings. Their leaves are covered with a thin layer of cuticle and they have weakly developed palisade cells with big inter-cellular spaces [15, 16]. That is the reason why acclimatization is a critical stage of each plant production using in vitro techniques and it is often decisive of it success. After microcuttings are transferred from the in vitro culture, they must have the proper conditions allowing them to increase the photosynthetic activity, excessive transpiration should be prevented and a supply of nutrients should be enabled [10]. Hardening and successful rooting of microcuttings are to a large extent determined by the proper choice of the subsoil where the plants are transferred from the in vitro culture [3]. Planted shoots are placed under small foil tunnels, frequently sprinkled and moderately aired. After a week, this cover can be removed for a longer and longer period until the plants are rooted. To make it easier for the microcuttings to adapt to the new conditions, it is recommended that before planting they should be lit with the light of higher intensity together with gradually increased temperature and decreased humidity [19]. In chrysanthemums, a typical proceeding after in vitro propagation of plants is to root them on a medium with an addition of auxin, which lasts about two weeks, after which acclimatization to in vivo conditions takes place [9].

Replacing in vitro rooting with direct in vivo rooting, taking place simultaneously with acclimatization, considerably reduces the production costs, enables to place the shoots in the substrate [5] and allows a simplified process of micropropagation [1]. Besides, the period when the microcuttings reach the value of the planting material is shortened. Direct in vivo rooting is successfully used, for example, in plants from the families of Araceae, Marantaceae, Zingiberaceae, fern, kalanchoe, miniature roses [15].

The purpose of the present paper was to compare the quality of chrysanthemum plants of three cultivars which were acclimatized after having been in vitro rooted with the quality of plants simultaneously acclimatized and rooted in vivo in selected substrate.

MATERIAL AND METHODS

The experiment was conducted between February and March, 2008 at the Laboratory of Biotechnology and the glasshouse of the Department of Ornamental Plants and Vegetable Crops of University of Technology and Life Sciences in Bydgoszcz. The experimental material were three cultivars of chrysanthemum [Chrysanthemum × grandiflorum (Ramat.) Kitam.], namely 'Bislet', 'Euro' and 'Reddy'. The plants were propagated in vitro using single-node explants on a modified medium according to Murashige and Skoog (MS) [12], with the content of calcium and iron increased by half. The medium, containing 30 g·dm^-3 saccharose, was solidified with 8 g·dm^-3 agar, and its pH was established before autoclaving at the level of 5.8.

Microcuttings prepared for acclimatization directly after the stage of in vitro propagation, were taken out from the medium and planted into plastic cuvettes at the spacing of 5×5 into three kinds of substrate: a mixture of peat with perlite in the volume proportion 2:1 (v:v), peat and perlite, which had been earlier treated with a 0.2% solution of fungicidal Benlate 50 WP. High de-acidified peat with pH 5.5-6.5 was used. Microcuttings were covered with a perforated film and they were regularly sprinkled and aired. Acclimatization lasted 2 weeks.

After the stage of propagation, the shoots intended for rooting in vitro were subcultured onto the MS medium, prepared as above, with an addition of 2 g·dm^-3 of indole-3-acetic acid (IAA). After two weeks, the rooted microcuttings were taken out from the medium, the remains of the medium were washed off the roots and the seedlings were planted in plastic cuvetted at the spacing of 5×5 into a mixture of peat with perlite in the volume proportion of 2:1 (v:v). The procedure was the same as above throughout the two weeks of acclimatization.

Propagation and in vitro rooting took place in a sterile growth room. Acclimatization to in vivo conditions proceeded in an unsterile growth room. The conditions in both rooms were strictly controlled. Temperature of 24 ± 2°C, a 16-hours' photoperiod and Phillips fluorescent lamps TDL 36 W/54 emitting daylight were used. The intensity of quanthum flux density was established at the level of 52 ± 4 ľmol·m^-2.s^-1.

Two weeks after acclimatization, the proportion of rooted and acclimatized plants as well as their number, length and fresh root weight were estimated. On the days when acclimatization began and after it was finished, the shoot length from the substrate surface was measured with the aim of calculating their increase. Microcuttings intended for acclimatization were, on average, 2.5 cm in height and had from 4 to 6 leaves. On the day acclimatization began, the first fully developed leaf under the apical bud was marked, which made it possible to calculate the new leaves during acclimatization.

Results were statistically analyzed using the analysis of variance for a two-factor experiment, and the object means were estimated with Tukey's test at the level of significance α = 0.05. The experiment studied 12 combinations: 3 cultivars × 4 manner of rooting in 5 replications, 5 plants in each replication.

RESULTS AND DISCUSSION

Due to the specific physiology and anatomy, ex vitro plants in the course of acclimatization are vulnerable to a strong water, light and temperature stress. A lot of them are not able to get adjusted to the changing conditions and they die [8]. Special treatment of acclimatized plants and the proper choice of the substrate is of special importance. An important physical property of the substrate for the cultivation of ex vitro microcuttings is porosity, which determines the access of the adequate amount of air to the roots [11]. The subsoil must also be clean, it must have big water capacity and a permanent structure. These requirements are met by mineral substrates, e.g. perlite [2]. The subsoil of peat and perlite is characterized by good airiness and permeability and, like peat, it has a low capacity weight [17], due to which it is very often used for acclimatization. In the present experiment, acclimatization was successfully completed in all the applied substrate, both in plants rooted in vitro and acclimatized directly after the propagation stage. A lower percentage of rooted and acclimatized plants (80%) was observed only after two weeks of in vivo rooting in 'Bislet' transferred to a mixture of peat with perlite and in 'Reddy' placed in peat. Microcuttings of glasshouse carnation, after having been planted into different garden substrate, got acclimatized the best in the mixture of perlite with sand in the volume proportion of 1:1 (90%), in sand (86.7%) and in perlite (83.3%) [2].

In the present experiment, the plants rooted in vitro before acclimatization and plants directly acclimatized in perlite had the most of roots with the highest fresh weight (Table 1 and 2). Dąbski [2] observed the highest fresh weight of roots of glasshouse carnation 8 weeks after planting the plants from in vitro cultures into the substrate composed of perlite and sand in the proportion of 1:1, and the largest number of the main roots in pure perlite. In the experiment by Borkowska [1], who compared in vitro rooting of strawberry on a medium supplemented with 0.1 mg·dm^-3 IBA and in vivo in multiplates filled with mineral wool, the fresh weight of in vivo regenerated roots was higher than that of the roots regenerated in vitro. The root system produced in vitro consisted only of the main roots, which were breakable while the plants being pulled out from the medium. The most distinctly marked difference between the plants of strawberry rooted in vitro and in vivo was the relation of the dry weight of shoots to the roots. This parameter was smaller in the shoots rooted in vivo, thus indicating better development of the root system.

Table 1. Number of roots on chrysanthemum microcuttings depending on the rooting conditions and the cultivar

Rooting conditions (B)		Cultivar(A)			Means
in vitro rooting	Substrate for acclimatization	Bislet	Euro	Reddy
Yes	peat + perlite	18.2	21.04	20.84	20.03
No	peat + perlite	6.24	10.76	7.88	8.29
	peat	6.76	9.20	7.44	7.8
	perlite	8.24	12.52	20.20	13.65
Means		9.86	13.38	14.09	–
NIR0.05; LSD0.05		A = 1.94, B = 2.47, B/A = 4.28, A/B = 3.89

Table 2. Fresh root weight (mg) of chrysanthemum microcuttings depending on the rooting conditions and the cultivar

Rooting conditions (B)		Cultivar (A)			Means
in vitro rooting	Substrate for acclimatization	Bislet	Euro	Reddy
Yes	peat + perlite	140.31	113.52	126.92	126.92
No	peat + perlite	121.45	120.96	43.29	95.23
	peat	92.51	112.78	29.97	78.42
	perlite	91.35	182.31	128.86	134.17
Means		111.41	132.39	82,26	–
NIR0.05; LSD0.05		A = 26.55, B = 33.79, B/A = 58.53, A/B = 53.1

The stimulating effect of perlite on the formation of numerous roots of high weight can be explained by the fact that perlite as an artificial substrate is characterized by good oozing, it is uniform, thanks to which, the root system of each plant develops in the same conditions [13]. In the experiments by Gabryszewska and Warabieda [6], the dry weight of roots of common lilac shoots rooted in vitro on the substrate with auxins was significantly smaller than the root dry weight of plants rooted in vivo. As a result of in vivo rooting in a mixture of peat and perlite (2:1) as compared to in vitro rooting, better developed root system and higher evaluation in bonitation scale were also obtained.

The roots regenerated on chrysanthemum plants rooted in in vitro conditions and next acclimatized were shorter than those that were regenerated during direct acclimatization in all three kinds of substrate (Table 3). The fact that a higher number of shorter roots was obtained was most probably caused by the addition of auxin to the medium at the stage of rooting. Auxins stimulate the initiation of adventitious roots, but they may inhibit their elongation [14]. In the experiment by Świstowska and Kozak [18], the microcuttings rooted in vitro had numerous, short and compact roots.

Table 3. Root length (cm) of chrysanthemum microcuttings depending on the rooting conditions and the cultivar

Rooting conditions (B)		Cultivar (A)			Means
in vitro rooting	Substrate for acclimatization	Bislet	Euro	Reddy
Yes	peat+ perlite	2.03	1.56	1.06	1.54
No	peat + perlite	3.75	2.66	1.04	2.48
	peat	3.84	3.33	1.08	2.75
	perlite	3.33	2.83	2.39	2.85
Means		3.24	2.59	1.39	–
NIR0.05; LSD0.05		A = 0.52, B = 0.66, B/A = 1.14, A/B = 1.04

Differences were also found in root regeneration between the cultivars (Phot. 1, 2, 3). The biggest fresh weight of roots was observed in chrysanthemums 'Bislet' and 'Euro'. 'Bislet' produced the fewest roots, but they were the longest. On the other hand, the roots of 'Reddy' had the smallest fresh weight and they were the shortest. Similar differences in the ability of rhizogenesis between the cultivars were observed in Benjamin's fig [10] and in carnation [7].

Photo 1. Microcuttings of 'Bislet' chrysanthemum (from the left): rooted in vitro and next acclimatized in a mixture of peat and perlite (2:1), acclimatized directly in a mixture of peat and perlite (2:1), in peat and perlite

Photo 2. Microcuttings of 'Euro' chrysanthemum (from the left): rooted in vitro and next acclimatized in a mixture of peat and perlite (2:1), acclimatized directly in a mixture of peat and perlite (2:1), in peat and perlite

Photo 3. Microcuttings of 'Reddy' chrysanthemum (from the left): rooted in vitro and next acclimatized in a mixture of peat and perlite (2:1), acclimatized directly in a mixture of peat and perlite (2:1), in peat and perlite

Table 4. Increase of chrysanthemum microcuttings in length (cm) during acclimatization depending on the rooting conditions and the cultivar

Rooting conditions (B)		Cultivar (A)			Means
in vitro rooting	Substrate for acclimatization	Bislet	Euro	Reddy
Yes	peat + perlite	2.04	1.04	2.22	1.77
No	peat + perlite	1.06	0.63	0.44	0.71
	peat	0.57	0.75	0.99	0.77
	perlite	1.32	1.03	1.41	1.25
Means		1.25	0.86	1.27	–
NIR0.05; LSD0.05		A = n.s., B = 0.64, B/A = n.s., A/B = n.s.

Table 5. Number of new leaves produced during acclimatization on chrysanthemum microcuttings depending on the rooting conditions and the cultivar

Rooting conditions (B)		Cultivar (A)			Means
in vitro rooting	Substrate for acclimatization	Bislet	Euro	Reddy
Yes	peat + perlite	4.32	4.24	4.32	4.29
No	peat + perlite	2.84	3.08	3.2	3.04
	peat	3.00	2.72	2.66	2.79
	perlite	2.48	3.28	3.56	3.11
Means		3.16	3.33	3.44	–
NIR0.05; LSD0.05		A = n.s., B = 0.35, B/A = 0.61, A/B = 0.55

After the plants were transferred into in vivo conditions, the greatest increase of the shoot in length was observed in the plants rooted in vitro and directly acclimatized in perlite (Table 4). The biggest number of new leaves was produced by microcuttings rooted in vitro (Table 5). Like in the case of lilac, it was found out that the shoots rooted in vitro were longer and they had more nodes [6]. On the other hand, strawberry rooted directly in vivo during acclimatization produced a bigger number of new leaves as compared to the plants previously rooted in vitro [1].

CONCLUSIONS

1. In the studied cultivars of chrysanthemum, in vivo rooting taking place simultaneously with acclimatization can successfully replace in vitro rooting and acclimatization following it and thus the period of obtaining new ex vitro seedlings can be shortened and the costs of micro-propagation can be reduced.

2. The best substrate for direct acclimatization of chrysanthemum microcuttings is perlite.

REFERENCES

1. Borkowska B., 2001. Morphological and physiological characteristics of micropropagated strawberry plants rooted in vitro or ex vitro. Sci. Hort. 89, 195–206.

2. Dąbski M., 1996. Wpływ podłoży ogrodniczych na początkowy wzrost goździka szklarniowego (Dianthus caryophyllus L.) po wysadzeniu z kultur in vitro [Influence of horticultural beds on initial growth of carnation (Dianthus catyophyllus L.) after planting from culture in vitro]. Zesz. Probl. Post. Nauk Rol. 429, 77–79 [in Polish].

3. Dąbski M., 2002. Badania nad ukorzenianiem mikrosadzonek wybranych gatunków roślin ozdobnych [Studies on the rooting of micro-seedlings of selected species of ornamental plants]. Rozprawy naukowe AR w Lublinie. 262, 21 [in Polish].

4. Debergh P. C., Maene L. J., 1981. A scheme for commercial propagation of ornamental plants by tissue culture. Sci. Hort. 14, 335–345.

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7. Garrido G., Cano E.A., Arano M. B., Acosta M., Sánchez-Bravo J., 1996. Influence of cold storage period and auxin treatment on the subsequent rooting of carnation cuttings. Sci. Hort. 65, 73–84.

8. Hazarika B. N., 2003. Acclimatization of tissue-cultured plants. Current Science 85(12), 1704–1712.

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10. Łukaszewska A., 2001. Wpływ światła na ukorzenianie sadzonek figowca Benjamina [The effect of light on the rooting on the cuttings of Benjamin's fig]. Ogrodnictwo 5, 23–24 [in Polish].

11. Matysiak B., Nowak J., 1997. Czynniki wpływające na aklimatyzację roślin ozdobnych rozmnażanych in vitro [Factors influencing acclimatization of ornamental plants reproduced in vitro]. Mat. Konf. "Rozmnażanie roślin in vitro". Skierniewice, 19 marca 1997, 21–26 [in Polish].

12. Murashige T., Skoog F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15, 473–497.

13. Oświecimski W., 1996. Aktualne tendencje w wykorzystaniu podłoży nieorganicznych w uprawach pod osłonami [Actual trends in practical use of artificial media in greenhouse production]. Zesz. Probl. Post. Nauk Rol. 429, 9–13 [in Polish].

14. Pierik R.L.M, 1987. In vitro culture of higher plants. Martinus Nijhoff Publishers, Dordrecht, Netherlands.

15. Podwyszyńska M., 1991. Możliwości podniesienia efektywności mikrorozmnażania roślin doniczkowych w Polsce [Possibilities of raising the effect of micropropagation of pot plants in Poland]. Mat. Konf. Nauk. "Rośliny doniczkowe". Łódz, 15–16 listopada 1991, 43–56 [in Polish].

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17. Startek L., Wojcieszczuk T., 1996. Wpływ właściwości podłoża i regulatora wzrostu na ukorzenianie się niecierpka nowogwinejskiego [Influence of substrate and growth regulator properties on rooting of New Guinea impatiens]. Zesz. Probl. Post. Nauk Rol. 429, 287–292 [in Polish].

18. Świstowska A., Kozak D., 2004. Wpływ auksyn na ukorzenianie mikrosadzonek i adaptację roślin Columnea hirta Klotzsch et Hanst. Cz. I. W kulturze in vitro [The influence of auxins on the rooting of microcuttings and acclimatization of plants of Columnea hirta Klotzsch et Hanst. Part I. In in vitro culture]. Acta Sci. Pol., Hortorum Cultus 3(2), 229–238, www.acta.media.pl [in Polish].

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Scientific work co-financed by the European Social Fund and the State Budget as part of  Integrated Operational Programme of Regional Development, Actions 2.6 "Regional Innovation Strategies and Knowledge Transfer" of own project of Kujawsko-Pomorskie voivodeship "Scholarships for PhD students 2008/2009 – IOPRD"

 

Accepted for print: 8.07.2009

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Alicja Tymoszuk
Department of Ornamental Plants and Vegetable Crops,
University of Technology and Life Sciences in Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 22
email: alicjaskowronek@wp.pl

Natalia Miler
Department of Ornamental Plants and Vegetable Crops, Faculty of Agriculture and Biotechnology, University of Science and Technology, Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 22
email: nmiler@utp.edu.pl

Małgorzata Zalewska
Department of Ornamental Plants and Vegetable Crops,
University of Technology and Life Sciences in Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 36
email: zalewska@utp.edu.pl

Małgorzata Borawska
Department of Ornamental and Vegetable Crops,
University of Technology and Life Sciences, Bydgoszcz, Poland
Bernardyńska 6, 85-029 Bydgoszcz, Poland
Phone: (+48) 52 374 95 22

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