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Bulletin UASVM Horticulture, 67(1)/2010 Print ISSN 1843-5254; Electronic ISSN 1843-5394

New Aspects Regarding the Micropropagation of Blackberry Cultivar Thornless evergreen

Alexandru FIRA1), Doina CLAPA1), Catita PLOPA2)

1) Fruit Research Station Cluj, 5 Horticultorilor Street, 400457 Cluj-Napoca, Cluj

Romania; www.scdpcluj.ro, doinaclapa@yahoo.com 2)

Research and Development Institute for Fruit Tree, Pitesti Maracineni, 402 Marului Street, 117450 Arges, Romania

Abstract. This paper presents results regarding the micropropagation of blackberry species Rubus laciniatus, cultivar Thornless evergreen. Several variants of nutritive media were tested, Murashige & Skoog 1962 (MS) and Driver & Kunyuki Walnut (DKW) were used as basal media, the growth regulators tested were kinetin and 6-benzylaminopurine (BAP) in various concentrations and, as gelling agents Plant Agar, Gelcarin GP-812, Isubgol and Guar gum were tested. Also, the optimization of number of inoculi/vessel and inoculum size in the multiplication phase was experimented. Ex vitro rooting and acclimation were carried out in several hydroculture experimental variants.

Keywords: multiplication, rooting, acclimation, float hydroponics

INTRODUCTION

Micropropagation of brambles was studied by many researchers (Bobrowski et al., 1996; Erig and Schuch 2005; Gajdosova et al. 2006, Minas and Neocleous 2007; Ruzic and Lazic, 2006). For multiplication, several variants of Murashige & Skoog (Murashige and Skoog, 1962) medium were used, supplemented with growth regulators (BAP , IBA and GA3) and, for rooting, variants of MS medium containing IBA. The ex-vitro rooting and acclimation of shoots excised directly from plantlets in the multiplication phase was also experimented. Another aspect to be studied was the in vitro regeneration of shoots from leaf explants, on media containing thidiazuron and auxins (Ruzic and Lazic, 2007; Zawadska and Orlikowska, 2006).

The technique of rooting ex-vitro in floating cell trays is based on the floatation method used in the USA for tomato and tobacco seedlings and for lettuce (Peek and Reed 2008; Reed 2009; Ross et al., 1999) and upon the fact that some plant species can be easily and efficiently rooted in water. Media gelled with Isubgol (Psyllium husk) and Guar gum ensured better growth and proliferation than media gelled with agar (Jain and Babbar, 2005) and Isubgol proved to be an effective gelling agent in the multiplication phase in blueberry cv. Blue Crop (Clapa et al., 2008)

At the Fruit Research Station of Cluj the micropropagation of blackberry cultivar Thornless evergreen was started in 2008 and good results were obtained regarding the initiation, multiplication, rooting and acclimation (Fira et al., 2009). In the present study, we tried to improve the micropropagation protocol for this cultivar, new culture media, growth regulators and gelling agents being tested. Also, new ex-vitro rooting and acclimation methods were successfully tested and viable, very vigorous plants were obtained, which could be planted into the field (Fira and Clapa, 2009).

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MATERIALS AND METHODS

The biologic material proceeded from in vitro cultures in the multiplication phase, in Murashige & Skoog 1962 (MS) media to which the following compounds were added: Myo inositol-100 mg/l, stock solutions of vitamin B1, B6 and nicotinic acid, the carbon source commercial crystal sugar, the iron source: FeNaEDTA, Plant Agar 6g/l, pH 5.8 and the growth regulator was BAP at 0.7 mg/l. In order to optimize the multiplication phase, several experimental series were done in which modified MS or DKW media were used as basal media; as growth regulators BAP was used at various concentrations (0.3, 0.5 and 0.7 mg/l), kinetin at 4 si 8 mg/l and various gelling agents were tested (Plant Agar, food grade agar, Gelcarin GP-812, Isubgol and Guar gum). Also, the optimization of the number of inoculi/vessel as well as the size of inoculi was pursued. In most of the experiments either 5 inoculi of 1-1.5 cm in length / Magenta vessel or 10 inoculi/820 ml jar were used and the cultures were incubated in the growth chamber for a period of 2 months. The exceptions regarding the number and dimensions of the inoculi as well as culture period are specified in the tables presented in the section Results and Discussion. The experimental series can be synthesized such as:

I. In order to test kinetin, Magenta GA7 vessels with MS medium were used and 2 experimental variants were set up: Variant I: 4 mg/l, Variant II: 8 mg/l. 5 inoculi/vessel were used.

II. For testing the Gelcarin GP-812 gelling agent, Magenta GA7 vessels were used containing MS medium + 0.7 mg/l BAP, with various concentrations of Gelcarin according to the following experimental variants: I: 7 g/l , II: 5 g/l, III: 4 g/l, 1V: 2 g/l.

III. For the experiment regarding the optimization of the number and size of inoculi Magenta vessels with MS+0.7 mg/l BAP were used.

IV. For the experiment of testing the DKW medium with various concentrations of BAP, Magenta vessels were used and 2 experimental variants: I: DKW+ 0.5 mg/l BAP, II: DKW+1 mg/l BAP. Also, some vessels from Variant I was kept for 11 weeks in order to establish the effect of prolonging the incubation period upon multiplication rate.

V. An in vitro inoculation experiment was done using only a pair of sterilized scissors for inoculation. The plant material was cut above the open culture vessels (one cut/vessel) and the shoots that fell onto the medium (MS+0.7 mg/l BAP) were pushed into the mass of medium only by means of the scissors.

VI. 820 ml jars were used, containing MS+0.5 mg/l BAP, 3 g/l Plant Agar, 100 ml medium/vessel. 10 inoculi of 2 cm in length were used in each vessel.

VII. An experiment was carried out in order to test various gelling agents in the nutritive medium. The experimental variants are presented in Table 1.

Ex vitro rooting and acclimation were done in the same phase, by using either cell trays immersed into plastic trays containing water (immersed trays) or cell trays containing polystyrene floats, set to float in improvised mini-basins that containing tap water with neutral pH (an adaptation of the 'float hydroponics' technique). The plant material consisted of abundantly branched plantlets cultured for 2 months on the multiplication medium. The plantlets were transferred ex vitro, the bases of the bushes were excised, resulting bunches of separate, individual shoots which were divided into smaller bunches and planted into the cells. The trays were kept for one month either in the growth chamber or in open air under a roof. The water in the trays was resupplied 2-3 times a week. A total of 88 shoots were used for indoor rooting and acclimation and 173 shoots were used for outdoor rooting and

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acclimation. The mini-basins were kept in the greenhouse and the liquid substrate was not resupplied during the entire rooting period.

Tab. 1 Gelling agents tested in the multiplication media for the blackberry

Variant Basal Medium Gelling agent Growth regulators Number of initial inoculi

A MS Guar Gum 10 g/l BAP 0.7 mg/l 10 B MS Plant Agar 3 g/l BAP 0.7 mg/l 5 C MS Plant Agar 3 g/l BAP 0.3 mg/l 10 D MS Isubgol 15 g/l BAP 0.7 mg/l 5 E MS Isubgol 15 g/l BAP 0.3 mg/l 10

RESULTS AND DISCUSSION

I. Kinetin at 8 mg/l ensured a reasonable multiplication rate which was, nevertheless, far lower as compared to the results obtained by using BAP as growth regulator (Tab. 2). The resulting plantlets were relatively vigorous.

Tab. 2 The influence of kinetin upon blackberry in vitro culture

Variant Vessel Initial no. of inoculi/vessel

No. of inoculi resulted/vessel

Multiplication rate

1 5 54 10.8 2 5 30 6

I.- MS+4 mg/l kinetin

Average I 5 42 8.4 1 5 93 18.6 2 5 162 32.4 3 5 116 23.2 4 5 118 23.6

II - MS+8 mg/l kinetin

Average II 5 122.25 24.45

II. The gelling agent Gelcarin GP-812 gave good results for the in vitro multiplication of the blackberry. The optimal concentration proved to be of 2 g/l. Increasing Gelcarin concentration lead to the decrease of multiplication rates (Fig. 1 and Tab. 3).

Fig. 1. Testing Gelcarin GP-812 in blackberry in vitro culture (2, 4, 5 and 7 g/l)

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Tab. 3 Testing Gelcarin GP-812 in blackberry in vitro culture

Variant Vessel No. of inoculi/vessel

No. of inoculi resulted/vessel

Multiplication rate

1 5 61 12.2 2 5 179 35.8 3 5 77 15.4 4 5 74 14.8

I- 7g/l Gelcarin

Average I 5 97.75 19.55 1 5 233 46.6 2 5 231 46.2 3 5 68 13.6 4 5 107 21.4

II 5g/l Gelcarin

Average II 5 159.75 31.95 1 5 342 68.4 2 5 277 55.4 3 5 262 52.4 4 5 120 24

III 4g/l Gelcarin

Average III 5 250.25 50.05 1 5 286 57.2 2 5 238 47.6 3 5 389 77.9 4 5 334 66.9

IV 2g/l Gelcarin

Average IV 5 311.75 62.4

III. The optimal number of inoculi/vessel regarding multiplicaton rate as well as regarding the number of inoculi resulted/vessel proved to be of 5. The use of large inoculi did not spectacularly increase multiplication rates (Tab. 4).

Tab. 4 The optimal no. of inoculi/vessel for blackberry multiplication

Variant Vessel No and type of initial inoculi

No. of inoculi resulted /vessel

Average no. of inoculi resulted/vessel

No. of inoculi resulted/plantlet

Average multiplication rate

1 9 X 1-1.5 cm 574 63.777 I 2 9 X 1-1.5 cm 645

609.5 71.666

67.72

1 4 X 2.5 -3cm 321 80.25 II 2 4 X 2.5 -3cm 343

332 85.75

83.00

1 5 X 2.5 -3cm 462 92.4 III 2 5 X 2.5 -3cm 634

548 126.8

109.60

1 5 X 1-1.5 cm 696 139.2 2 5 X 1-1.5 cm 394 78.8

IV

3 5 X 1-1.5 cm 515

535

103

107.00

IV. Doubling the concentration of BAP lead to the inhibition of the plantlets' growth in vitro, which lead to lower multiplication rates (Tab. 5 and Fig. 2). Also, increasing the incubation period for the cultures to 11 weeks instead of 2 months lead only to a small increase in multiplication rate due to the stagnation of the cultures; a multiplication rate of 89.6 was obtained.

V. The use of the inoculation method by means of the pair of scissors ensured reasonable multiplication rates. However, it is not to be recommended for the blackberry, as it leads to varying multiplication rates, non-uniform plantlet sizes and varying numbers of plantlets resulted/vessel.

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Tab. 5 The influence of BAP concentration upon multiplication rate in the blackberry

Variant Vessel No. of inoculi resulted/vessel

No. of inoculi resulted/plant

Average no. of inoculi resulted/vessel

Average multiplication rate

1 474 94.8 2 459 91.8 3 307 61.4

I DKW+ 0.5 mg/l BAP

4 355 71

398.75 79.75

1 435 87 2 367 73.4 3 248 49.6

II DKW+ 1 mg/l BAP

4 295 59

336.25 67.25

Fig. 2. The influence of BAP concentration upon multiplication rate in the blackberry (0.5 and 1 mg/l BAP)

Tab. 6. Blackberry multiplication rate by inoculation by the scissors

Vessel No. of plantlets resulted/vessel

No. of inoculi resulted /vessel

Multiplication rate

Average no. of inoculi resulted/vessel

Average multiplication rate

1 8 301 37.625 2 7 563 80.428 3 6 544 90.666

469.333 69.573

VI. The reduction of Plant agar concentration did not influence negatively the multiplicaton of this blackberry cultivar. Large plantlets resulted and the average multiplication rate was 95.7 (Fig. 3).

Fig. 3. Blackberry cv. 'Thornless Evergreen' multiplied on MS+3 g/l Plant Agar

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VII. All the gelling agents tested proved to be viable alternatives to agar for the in vitro multiplication of the blackberry.

Guar Gum (variant A) caused the reduction of the multiplication rate but the resulting plantlets were extremely vigorous and well-developed (Fig. 4).

Fig. 4. The use of Guar gum as gelling agent for blackberry cv. 'Thornless Evergreen'

The medium gelled with Plant Agar at the concentration of 3 g/l (Variant B) provided an average multiplication rate of 102.2. The reduction of BAP concentration to 0.3 mg/l (Variant C) ensured the reduction of multiplication rate but it increased shoot length. The resulting plantlets were non-uniformly developed, on the same bush there being long and extremely vigorous shoots alongside with thin and insufficiently developed shoots.

Isubgol caused the reduction of the multiplication rate in variants tested, respectively 52.5 in the variant with 0.7 mg/l BAP and 14.8 in the variant with 0.3 mg/l BAP.

The results are presented in Table 7. Tab. 7

Alternative gelling agents in blackberry in vitro culture

Variant Vessel No. of initial inoculi/vessel

No. of inoculi resulted/vessel

Multiplication rate/vessel

Average no. of inoculi

resulted/vessel

Average multiplication

rate 1 10 282 28.2 A 2 10 326 32.6

304 30.4

1 5 502 100.4 B 2 5 520 104

511 102.2

1 10 347 34.7 C 2 10 260 26

303.5 30.35

1 5 312 62.4 D 2 5 213 42.6

262.5 52.5

1 10 129 12.9 E 2 10 167 16.7

148 14.8

Ex-vitro acclimation and rooting were tested in the same phase, the following results being obtained:

The plantlets transferred ex vitro from the multiplication medium and planted into cell trays in plastic trays containing water (immersion hydroculture) presented the following rooting and acclimation percentages (Fig. 5 and Fig. 6). The rooting and acclimation percentages for both variants (protected space versus open air) are very similar (63 % and, respectively 64 %).

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a. b. Fig. 5. Rooting and acclimation percentages in protected space (a) and outdoors (b)

Fig. 6. Ex vitro rooting and acclimation in immersed cell trays

In the case of ex-vitro rooting in float hydroponics ex vitro rooting and acclimation in the cell trays was not satisfactory. From the total of 273 shoots in 10 cells (an average of 27.3 shoots/cell) only 176 generated viable, rooted plants (an average of 17.6/cell) The results of float hydroponics rooting and acclimation are presented in Figure 7, 8 and 9.

Fig. 7. Ex vitro rooting and acclimation in float hydroponics

Fig. 8. Bunches of plants rooted and acclimated in float hydroponics

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Fig. 9. Ex vitro rooting and acclimation in float hydroponics

CONCLUSIONS

The basal media tested, DKW and MS ensured good plantlet development and good multiplication rates in blackberry cv. 'Thornless Evergreen', the difference between the effects of the two media were insignificant. Kinetin at the concentrations of 4 and 8 mg/l is not recommended for the micropropagation of blackberry cv. 'Thornless Evergreen'. The plant growth regulator that ensures very good multiplication rates is BAP at 0.5-0.7 mg/l. The media gelled with Plant Agar ensure the highest multiplication rates. It is recommended to use 6 g/l of medium, but good results were also obtained by reducing the concentration of Plant Agar to 3 g/l. The gelling agent Gelcarin GP-812 gave good results in blackberry in vitro multiplication. The optimal concentration proved to be of 2 g/l, which ensured the highest multiplication rate, 62.4 times. Increasing gelcarin concentration resulted in the decrease of multiplication rates. Guar gum caused the reduction of multiplication rates, but the plantlets were very vigorous and uniformly developed. Isubgol at the concentration of 15 g/l can be an alternative gelling agent for this blackberry cultivar but it ensures lower multiplication rates. The optimal number of inoculi/Magenta vessel is 5, and the optimal length of the microcuttings is of 1-1.5 cm.

Acknowledgements. This work was supported by CNMP Romania, project number 52-165 PNII Program 4, Partnerships in high priority domains.

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