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).

106

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

107

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

A
B
C
D
E

MS
MS
MS
MS
MS

Guar Gum 10 g/l

Plant Agar 3 g/l
Plant Agar 3 g/l
Isubgol 15 g/l
Isubgol 15 g/l

BAP 0.7 mg/l

BAP 0.7 mg/l
BAP 0.3 mg/l
BAP 0.7 mg/l
BAP 0.3 mg/l

Number of
initial inoculi
10
5
10
5
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

I.- MS+4 mg/l

kinetin

1
2
Average I
1
2
3
4
Average II

II - MS+8 mg/l
kinetin

Initial no. of
inoculi/vessel
5
5
5
5
5
5
5
5

No. of inoculi
resulted/vessel
54
30
42
93
162
116
118
122.25

Multiplication rate
10.8
6
8.4
18.6
32.4
23.2
23.6
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)

108

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

Vessel

I7g/l Gelcarin

1
2
3
4
Average I
1
2
3
4
Average II
1
2
3
4
Average III
1
2
3
4
Average IV

II
5g/l Gelcarin

III
4g/l Gelcarin

IV
2g/l Gelcarin

No. of
inoculi/vessel
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5

No. of inoculi
resulted/vessel
61
179
77
74
97.75
233
231
68
107
159.75
342
277
262
120
250.25
286
238
389
334
311.75

Multiplication rate
12.2
35.8
15.4
14.8
19.55
46.6
46.2
13.6
21.4
31.95
68.4
55.4
52.4
24
50.05
57.2
47.6
77.9
66.9
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

1
2
1
2
1
2
1
2
3

9 X 1-1.5 cm
9 X 1-1.5 cm
4 X 2.5 -3cm
4 X 2.5 -3cm
5 X 2.5 -3cm
5 X 2.5 -3cm
5 X 1-1.5 cm
5 X 1-1.5 cm
5 X 1-1.5 cm

II
III
IV

No. of inoculi
resulted
/vessel
574
645
321
343
462
634
696
394
515

Average no.
of inoculi
resulted/vessel
609.5
332
548
535

No. of inoculi
resulted/plantlet
63.777
71.666
80.25
85.75
92.4
126.8
139.2
78.8
103

Average
multiplication
rate
67.72
83.00
109.60
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.
109

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

Vessel

I
DKW+
0.5 mg/l BAP
II
DKW+
1 mg/l BAP

1
2
3
4
1
2
3
4

No. of inoculi
resulted/vessel
474
459
307
355
435
367
248
295

No. of inoculi
resulted/plant
94.8
91.8
61.4
71
87
73.4
49.6
59

Average no. of inoculi

resulted/vessel
398.75

Average
multiplication rate
79.75

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

1
2
3

No. of plantlets
resulted/vessel
8
7
6

No. of inoculi
resulted
/vessel
301
563
544

Multiplication
rate
37.625
80.428
90.666

Average no. of inoculi

resulted/vessel

Average
multiplication rate

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

110

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

A
B
C
D
E

Vessel

1
2
1
2
1
2
1
2
1
2

No. of initial
inoculi/vessel
10
10
5
5
10
10
5
5
10
10

No. of inoculi
resulted/vessel

Multiplication
rate/vessel

282
326
502
520
347
260
312
213
129
167

28.2
32.6
100.4
104
34.7
26
62.4
42.6
12.9
16.7

Average no. of
inoculi
resulted/vessel
304

Average
multiplication
rate
30.4

511

102.2

303.5

30.35

262.5

52.5

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 %).

111

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. 8. Bunches of plants rooted and acclimated

in float hydroponics

Fig. 7. Ex vitro rooting and acclimation in float

hydroponics

112

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.
REFERENCES
1. Clapa, D., Al. Fira and T. Rusu (2008). The use of Isubgol and Sequestrene 138 for the in
vitro propagation of the highbush blueberry (Vaccinium corymbosum L.), Journal of Food, Agriculture
& Environment JFAE, 6(1):132-134.
2. Bobrowski, V. L , P. C. Mello-Farias and J. A. Peters (1996). Micropropagation of
blackberries (Rubus sp.) cultivars, Rev. Bras. De Agrociencia, 2(1):17-20.
3. Erig, A. C and M. W. Schuch (2005). Tipo de luz na multiplicacao in vitro de framboeseira
(Rubus idaeus L.) Batum. Rev. Bras. Frutic., Jaboticabal 27(3): 488-490.

113

4. Fira, Al. and D. Clapa and C. Plopa (2009). Micropropagation of Blackberry Cultivar
Thornless Evergreen. Simpozionul Cercetarea pomicol o ans pentru agricultura romneasc, 12
nov. 2009, ICDP Pitesti Maracineni.
5. Fira, Al. and D. Clapa, (2009). Ex-Vitro Acclimation of some Horticultural Species in
Hydroculture. Bulletin UASVM, nr. 66 (1): 44-51.
6. Gajdosova, A., M. G. Ostrolucka, G. Libiakova, E. Odruskova and D. Simala (2006).
Microclonal Propagation of Vaccinium sp. And Rubus sp. and detection of genetic variability of
culture in vitro, Journal of Fruit and Ornamental Plant Research, 14 (1):103-119.
7. Jain, R. and S. B. Babbar (2005). Guar gum and isubgol as cost-effective alternative gelling
agents for in vitro multiplication of an orchid, Dendrobium chrysotoxum. Current Science, 88(2): 292295.
8. Minas, G. J. and D. Neocleous (2007). A Protocol for Rapid Clonal Propagation in vitro of
Primocane-Fruiting Red Raspberry Cultivars. Miscellaneous Reports 95, Agricultural Research
Institute, Lefkosia, Cyprus, May, issued by the Press and Information Office, Lefkosia
9. Murashige, T. and F. Skoog (1962). A revised medium for rapid growth and bioassays with
tabacco tissue culture. Physiol. Plant 15: 473-497.
10. Peek, D. R. and T. D. Reed (2008). Greenhouse Transplant Production. Burley Tobacco
Production Guide
11. Reed, T. D. (2009). Float Greenhouse Tobacco: Transplant Production Guide. Virginia
Cooperative Extension, publication 436-051.
12. Ross, D. S. and K. M. Teffeau (1995). Greenhouse Float Systems for Transplant
Production. Maryland Cooperative Extension, Fact Sheet 690;
13. Ruzic, D. and T. Lazic (2006). Micropropagation as Means of Rapid Multiplication of
Newly Developed Blackberry and Black Currant Cultivars. Agriculturae Conspectus Scientificus, 71
(4) :149-153
14. Ruzic, D. and T. Lazic (2007). Organogenesis In Vitro from the Leaf of Blackberry cv
aanska Bestrna. GENETIKA, 39(1): 69 -78.
15. Tyson, R. V., White J. M and King K. V. 1999. Outdoor Floating Hydroponic Systems for
Leafy Salad crop and Herb Production, Proc. Fla. State. Hort. Soc. 112:313-315.
16. Zawadska, M. and T. Orlikowska (2006). Factors modifying regeneration in vitro of
adventitious shoots in five red raspberry cultivars. Journal of Fruit and Ornamental Plant Research, 14:
5-5.

114