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In Vitro Micropropagation of Blackberry (Rubus fruticosus.)
Cultivar “JUMBO”
Şeyma ARIKAN, Muzaffer İPEK, Ahmet EŞİTKEN
University of Selçuk, Faculty of Agriculture, Horticulture Department-KONYA
Abstract
The objectives of the present study were to achieve improved techniques in in vitro propagation and to
determine a better substrate for blackberry. After sterilization, explants were placed into MS medium
supplemented with 1 mg/l GA3 in shoot initiation. 15 days after that, explants were cultured in Murashige
and Skoog (MS) medium supplemented with different concentrations and combinations of 6-Benzylamino
purine (BA), α-Napthalene acetic acid (NAA) and Indol acetic acid (IAA) in shoot proliferation stage. The
developed upon transfer to rooting on the MS medium containing Indole butyric acid (IBA) at 0.25, 0.50
and 1.0 mg/l. Results indicated that the best multiplication of shoots was obtained with 2.0 mg/l BA +
0.5mg/l IAA concentration. Rooting was successfully achieved with 100% rooted plants in 1.0 mg/l IBA.
Introduction
Blackberry Rubus fruticosus (Tourn.) L. subgenus Eubatus are found in temperate zones around the world
(McPheeters et al., 1988). Rubus species belong to the Rosaceae family. Blackberries are some of the most
consumed berries in the world. While a large amount of fruit is sold in the fresh market, most blackberries
are first processed into either individually quick frozen (IQF) fruit, puree, or juice that in turn are used as
ingredients in processed products such as preservers, jam, jelly, baked goods, dried products, ice cream and
yogurt (Hall, 1990). Blackberries are a rich source of anthocyanins and other polyphenolic antioxidants
(Siriwoharn et al., 2004).
Blackberry is commercially propagated by the classical methods of vegetative propagation, i.e. by
hardwood and softwood cuttings, by layering and bush division. However, successful application of these
methods of vegetative propagation is limited to certain extent. Although tissue-culturing blackberry is more
expensive than conventional vegetative propagation methods, problems with disease May be considered
more important than cost considerations (Caldwell, 1984). In vitro propagation through the development of
axillary buds eliminates the seasonal limitations encountered with these methods and needs a small quantity
of starting material, the in vitro plants propagated in this way in many species have proved to be healthy
and true to type (Shen et al., 1990). Micropropagation of blackberries 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 experiment were carried out to determine the best micropropagation media for then "Jumbo".
Material and Method
Blackberry shoots of “Jumbo” cultivar was utilized, coming from to shoot tip culture and kept in vitro.
Initial explants were with 1.0 cm length and contained axillary buds were placed in MS (Murashige and
Skoog, 1962) medium supplemented with 1 mg/l GA3 and 3% sucrose (w/v) and agar (0.7% ;w/v ). The pH
of the media was adjusted to 5.8 after the media were autoclaved at 121 ºC and 1.5 atm for 20 min.
Two weeks after initiation, the shoots were transferred to proliferation medium supplemented with different
combination of BA (0, 1.0 and 2.0 mg/l), NAA (0, 0.25 and 0.50 mg/l), IAA (0, 0.25 and 0.50 mg/l). Each
PGR combinations had 10 magenta boxes that has nine plantlets and contains 50ml solidified media, each
of magenta box regard as a replicates,
Cultures were maintained for 4 weeks (as long term salt stress) at the growth room (25±2°C) under a 16
hour photoperiod fluorescent light. Sub-culturing was done at regular intervals off our weeks up to 12
weeks (three subcultures). Number of shoots per explant after each subculture was noted. For rooting,
shoots (1,5- 2cm in height) were transferred to rooting media which has three concentrations of IBA (0,
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0.25, 0.50 and 1.0 mg/l). Then average number of shoot, rooting percentage, number of roots and length of
roots per rooted shoot were recorded in during micropropagation stages. Data were statistically analyzed
using the Duncan‟s multiple range test.
Results and Discussion
The table-1 shows that BA- IAA combinations has better results than only BA and BA- NAA combinations
on proliferation stages. The highest shoot proliferation for “Jumbo” cultivar was in the 2.0 mg/l BA and 0.5
mg/l IAA (10,9 shoot/explant). Similar results in blackberry and pear were obtained by Pasqual et al. (1991)
and Leite et al. (1993) respectively. Furthermore, Jafari Najaf-Abadi and Hamidoghli (2009) reported that
the greatest number of shoots was obtained MS medium containing 2.0 mg/1 BA and 0.5 or 1 mg/1 GA3. In
contrast to Jafari Najaf-Abadi and Hamidoghli (2009), Bobrowski et al. (1996) reported that GA3 was not
effective on multiplication rate. The study of Villa et al. (2005) shows that the highest numbers of shoot
were produced by WPM basal media containing 1.0 mg/1 BA.
Table 1. Effect of BA, NAA and IAA combinations on average number of shoot
Media
Avarage Number of Shoot
Control
1,59 h
1.0 mg/l BA
6,03 c
2.0 mg/l BA
4,40 e
1.0 mg/l BA+0.25 mg/l NAA 2,25 g
2.0 mg/l BA+0.25 mg/l NAA 1,18 i
1.0 mg/l BA+0.50 mg/l NAA 2,51 g
2.0 mg/l BA+0.50 mg/l NAA 3,85 f
1.0 mg/l BA+0.25 mg/l IAA
8,44 b
2.0 mg/l BA+0.25 mg/l IAA
5,44 d
1.0 mg/l BA+0.50 mg/l IAA
3,81 f
2.0 mg/l BA+0.50 mg/l IAA
10,90 a
There was a statistical difference among IBA levels (0.25, 0.50 and 1.0 mg/l). All IBA levels had
successfully rooting of shoots but a concentration of the best rooting was obtained from 1.0 mg/l IBA
(100%). The highest number of roots and length of roots was in the 1.0 mg/l IBA level (Table-2.). James et
al. (1980) reported that IBA stimulate rooting of red raspberry explants, just as study of Gonzalez et al.,
(2000) shows that medium containing IBA increased rooting percentage in blackberry. Also Bobrowski et
al., (1996) obtained 100% rooting in blackberry cultivars (cv. Ebano, Tupi and Guarany). However,
Fernandez et al. (1991), used a more complex medium (MS + 8.9 µM BA + 0.5 µM IBA + 0.29 µM GA 3)
for micropropagation of blackberry cultivar "Navaho” and obtained 90% rooting.
Table-2. Effect of different IBA levels on rooting
Media
Rooting rate (%)
Number
roots
of
Length
(cm)
Kontrol
16.66 d
2.33 c
1.33 c
0.25 mg/l IBA
43.33 c
3.67 bc
2.17 bc
0.50 mg/l IBA
76.66 b
4.67 ab
3.17 b
1.0 mg/l IBA
100.00 a
5.67 a
4.50 a
of
roots
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