In vitro propagation of Gisela 5 (Prunus cerasus x P canescens)
Metka ŠIŠKO1
1 University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoče, Slovenia
The main purpose of the presented study was to investigate the techniques for in vitro propagation of Gisela 5 (a rootstock for cherry trees). Shoot tips and axillary buds were used as a source for explants. The buds were previously sterilized using two approaches. The first approach was based on dichloroisocyanuric acid and involved 23 buds, of which 95.5% survived. The second approach was based on sodium hypochlorite and involved 21 buds, of which 57.1% survived. Vital sprouts were subcultivated on the multiplication media G1 (based on MS medium) and G2 (based on woody plant medium). Out of 40 sprouts 119, sprouts were successfully regenerated on the G1 medium, and 169 on the G2 medium. The G2 culture medium has proved to be better, since the percentage of callused and failed sprouts was lower when compared with G1 culture medium. In vitro developed plants were transferred to four culture media for rooting, which differed in the content of auxines (IBA and NAA). In the culture medium with 0.5 mg/L IBA, the percentage of rooted plants was the highest (90%) compared with medium with 1 mg/L NAA which resulted in the lowest percentage (65%) of rooted plants. Rooted plants were transplanted into the growth substrate and acclimatised using two approaches. Acclimatization in the moist chamber was better when compared to the common growth chamber, since acclimatization in the moist chamber survived more plants (36.4%) compared with acclimatisation in the common growth chamber (11.5%).
Key words: Gisela 5, cherry rootstock, micropropagation, in vitro propagation
INTRODUCTION
Gisela 5 is known as a dwarfing rootstock for sweet cherry, developed from the cross between Prunus cerasus 'Schattenmorelle' x P. canescens. It is considered as very useful and economically important dwarfing rootstocks for intensive sweet cherry growing in temperate conditions. Production, marketing and trade rights for these rootstocks, which originate from a breeding program at the University of Giessen (Germany), are assigned to CDB (Consortium Deutscher Baumschulen), an association of German nurseries. There are very few data about micropropagation of Gisela 5 in the scientific literature.
The in vitro growth and development of a plant is determined by interaction of several complex factors: light, temperature, composition of the nutrient media (water, macro- and micro-elements, sugars, growth regulators, vitamins, etc.), gas exchange rate of the cultural vessels, etc (Pierik, 1997).
The aim of our study was to investigate different sterilization procedure for in vitro culture initiation of Gisela 5. Two nutrient media, one based on MS (Murashige and Skoog 1962) and another on the McCown woody plant medium (Loyd and McCown 1980), were compared in order to determine the medium with the best multiplication rate. Another aim of our study was to investigate different nutrient media for in vitro rooting of the Gisela 5 rootstock, which differed in the content of auxines (IBA and NAA) in order to determine the most suitable medium. One of the aims was also to compare two acclimatisation methods: in a common growth chamber (in a laboratory) and moist chamber (inside a greenhouse).
* Correspondence to: E-mail: metka.sisko@uni-mb.si
MATERIALS AND METHODS
Plant Material
Shoots of Gisela 5 were collected on June 20, 2008 from the experimental field Pohorski dvor, which belongs to the Faculty of Agriculture and Life Sciences, Hoce, Slovenia. In the laboratory for tissue culture, the shoot tips and axillary buds were dissected with a scalpel and used as a source for explants (Fig. 1).
Establishment of in vitro culture
Half of explants were sterilised with dichloroisocyanuric acid (DICA) (Aldrich, Germany). Explants were soaked for 15 min in DICA (16.6 g/L) to which 2-3 drops of Tween 20 (a wetting agent) were added and rinsed three times in sterile distilled water. The other half of explants were immersed in 70% ethanol for 40 sec, soaked in 1% sodium hypochlorite for 15 min and rinsed three times in sterile distilled water.
Sterilised explants were placed on the initiation medium which contained the following substances: MS mineral salts supplemented with 100.0 mg/L myo-Inositol, 0.1 mg/L thiamine-HCl, 0.5 mg/L pyridoxine-HCl, 0.5 mg/L nicotinic acid, 2.0 mg/L glycine, 0.1 mg/L IBA, 1.0 mg/L BAP, 0.1 mg/L GA3, 30 g/L sucrose and 8.0 g/L agar (Plant Agar, Duchefa). The pH was adjusted to 5.7, before autoclaving.
Fig. 1: Inoculated buds: shoot tips (A) and axillary buds (B) (Photo: Puster 2008)
Table 1: Number and percentage of contaminated, non vital and vital explants after sterilisation
Type of sterilisation	Number of inoculated explants	Number and percentage of contaminated explants after 12 days	Number and percentage of non vital explants	Number and percentage of sterile and vital shoots
DICA	23	2 (16.6%)	0 (0%)	21 (91.3%)
Sodium hypochlorite	21	0 (0%)	9 (42.9%)	12 (57.1%)
The cultures were maintained in growth chamber at 23°C with a 15 hours photoperiod (15.000 Lux). Every three days after inoculation, explants were checked for contamination (Tablel). Contaminated and non vital explants were discarded.
MULTIPLICATION
Vital and developed shoots were transferred on two different shoot proliferation media G1 and G2 (Table 2). Onto each medium 40 explants were transferred. The number of shoots after 56 days was counted and the multiplication rate was calculated for each medium (Table 3).
half of MS mineral salts supplemented with 100.0 mg/L myo-Inositol, 2.0 mg/L thiamine-HCl, 1.0 mg/L pyridoxine-HCl, 1 mg/L nicotinic acid, 2.0 mg/L glycine, 30 g/L sucrose and 8.0 g/L agar (Plant Agar, Duchefa). Four media (Rj, R2, R3, R4) differed in the content of auxines (IBA and NAA) as follows: Rj with 1.0 mg/L IBA, R2 0.5 mg/L NAA, R3 0.5 mg/L IBA and R4 1 mg/L NAA supplemented. The pH was adjusted to 5.7, before autoclaving.
On each of the four media mentioned before, 20 shoots were placed and maintained in a growth chamber at 23°C with a 15 hours photoperiod (15.000 Lux). The number of rooted plants was calculated after 14, 28, 42 and 56 days after cultivation.
Table 2: Shoot proliferation media G1 and G2
	G1	G2
MS	4.3 g/L	/
McCown	/	2.358 g/L
Sucrose	30 g/L	20 g/L
IBA	0.1 mg/L	/
NAA	/	0.005 mg/L
GA3	0.1 mg/L	/
BAP	1 mg/L	2 mg/L
Glycine	2 mg/L	2 mg/L
Myo-Inositol	100 mg/L	100 mg/L
Nicotinic acid	0.50 mg/L	1 mg/L
Thiamine	1 mg/L	2 mg/L
Pyridoxine-HCl	0.50 mg/L	1 mg/L
Agar	8 g/L	8 g/L
pH	5.7	5.7
Table 3: Multiplication rate on media G1 and G2
Medium	Shoots		Multiplication rate
	Inoculated	Proliferated	
Gi	40	119	2.98
G2	40	169	4.23
Rooting
For rooting, in vitro developed shoots (1.5-3 cm long) of Gisela were placed on four culture media for rooting (R1, R2, R3, R4), which differed in the content of auxines (IBA and NAA). All rooting media contained the following substances:
Acclimatisation
All plants with properly developed roots were transferred into the growth substrate (Bio-Potgrond, Klasmann). Before the transfer, roots were carefully washed under tap water to remove all remnants of agar. The transferred plants were acclimatised using two approaches. The first approach was based on acclimatisation in an ordinary growth chamber in the laboratory and the second approach was conducted in a moist chamber inside the greenhouse.
RESULTS AND DISCUSSION
With the sterilisation procedure DICA 91.3% of sterile plants was obtained and after sterilisation with sodium hypochlorite only 57.1% sterile and vital shoots were obtained (Table 1). After the hypochlorite sterilisation procedure, a lot of explants (42.9%) died without contamination, while with DICA sterilisation all explants that were not contaminated survived and produced sterile and vital shoots. Nacheva and Gercheva (2009) obtained from 85 to 100% sterile explants after sterilisation with calcium hypochlorite. Fidanci et al. (2008) report that the most appropriate time for taking explants from Gisela 5 is from the end of April to the beginning of June. Taking explants earlier caused a high level of contamination, whereas browning increased and growth and multiplication rates were reduced, for those collected later.
The results from the testing of two different nutrient media for multiplication show that higher number of shoots were obtained on G2 medium, based on Mc Cown woody plant medium, where the multiplication rate was 4.23
Fig. 2: Shoots obtained on G2 medium (A), acclimatised plants (B) and developed roots (C)
compared with G1 medium where the multiplication rate was 2.98. From 40 sprouts 119 sprouts,t were reproduce on the G1 medium, and 169 on the G2 medium. The G2 culture medium has proved to be better, since the percentage of callused (32.5%) and failed sprouts (0%) were lower than in the case of G1 culture medium with 60% of callused and 5% failed sprouts.
Table 4: Number and percentage of cultivated, rooted, non rooted and failed shoots on four rooting media
Medium	No. of shoots	Number of rooted plants after				No. and percentage of rooted plants		Number of non rooted plants		Number and percentage of failed shoots	
		14 days	28 days	42 days	56 days	No.	%	No.	%	No.	%
Ri	20	0	4	4	6	14	70	3	15	3	15
r2	20	2	10	2	2	16	80	1	5	3	15
Rs	20	0	4	5	9	18	90	0	0	2	10
R4	20	4	8	0	1	13	65	3	15	4	20
Nacheva and Gercheva (2009) investigated the effect of the type and the concentration of carbohydrates (sucrose and sorbitol) added to the nutrient medium with auxins (IBA, NAA and IAA) on the multiplication rate (number of shoots per plant). The best results (multiplication rate of 3.72 - 4.31 at 1.9 cm mean length of the shoots) were achieved in the variants containing 0.005 ^M IBA, 20 g/L of sucrose and 10 g/L of sorbitol.
The results from the testing of four different nutrient media for rooting show that in the culture medium R3 with 0.5 mg/L IBA, the percentage of rooted plants was the highest (90%) compared with medium R4 with 1 mg/L NAA, where the lowest percentage (65%) of rooted plants was obtained (Table 4). On the R3 medium, among 20 plants two were contaminated and 18 plants produced roots. On the media R1, R2 and R4, vital shoots without roots have been obtained.
ACKNOWLEDGMENTS
Thanks are due to Monika Puster for assistance with the experiments.
REFERENCES
1.	Fidanci A., Burak M., Erenoglu B., Akcay M.E. 2008. Determination of in vitro propagation techniques for some clonal cherry rootstocks. Acta Horticulturae. 795: 409-12.
2.	Lloyd G., McCown B. 1980. Commercially-feasible micropropagation of montain laurel, Kalmia latifolia, by use of shoot-tip culture. Int. Plant Prop. Soc. Proc. 30: 421-7.
3.	Murashige T., Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15(3): 473-97.
4.	Nacheva L., Gercheva P. 2009. Micropropagation of Gisela 5 (cherry dwarf rootstock): The effect of the type and the concentration of the carbohydrates in the nutrient medium. Acta Horticulturae. 825: 261-8.
5.	Pierik R. L. M. 1997. In Vitro Culture of Higher Plants, Kluwer Academic Publishers, Netherlands: 348 p.
Recived: February 21, 2010 Accepted in final form: May 11, 2011