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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti
Short communication
a r t i c l e
i n f o
Article history:
Received 22 January 2013
Received in revised form 7 May 2013
Accepted 14 May 2013
Keywords:
Temporary immersion bioreactors
Photomixotrophic
Sucrose-reduced medium
a b s t r a c t
A novel protocol for raspberry (Rubus spp.) micropropagation in photomixotrophic conditions was optimized for the commercial genotypes Heritage, Meeker and Amity. Plant cultures were established in
Temporary Immersion Bioreactors (TIBs) under a controlled environment: 550 ppmv CO2 ; light intensity
80 M m2 s1 ; sucrose concentrations 15 gr/L and 30 gr/L. Results showed that both CO2 ux and chlorophyll uorescence were increased in plants cultured in TIBs with sucrose-reduced medium (15 gr/L) in
comparison with those plants micropropagated in TIBs + 30 gr/L sucrose, conrming a consistent photomixotrophic stage since the 5th day of culture. Raspberry plants multiplied in TIBs + 15 gr/L sucrose
demonstrated the highest values of plant size, total number of internodes, and percent of acclimatization
to greenhouse conditions. Studied variables could be related to an increase of the in vitro photosynthetic
activity which might prime plants for the ex vitro environment.
2013 Elsevier B.V. All rights reserved.
1. Introduction
The genus Rubus belongs to the family Rosaceae and contains cultivated raspberries, blackberries, hybrid berries, and others
species (Jennings, 1988). Raspberry fruits have been considered
nutraceuticals containing vitamins, bers, and phytochemical compounds that function as antioxidants and antimicrobial protective,
among other health-related properties (Poiana et al., 2010). The
wide diversity of Rubus species provides a potential source of novel
traits whereas breeding programs are actively working on releasing cultivars with excellent quality, high yields, greater adaptation
to adverse environmental conditions, and increased pest and disease resistance (Castillo et al., 2010). Rubus species and cultivars are
clonally propagated and maintained in greenhouses, screenhouses,
eld collections, and as tissue-cultured plants and cryopreserved
shoot tips (Wang et al., 2005; Gupta and Reed, 2006).
Clonal multiplication based on plant tissue culture is preferred
for the production of pathogen-free and genetic-delity commercial plants (Arencibia et al., 2011). Raspberries are susceptible to
numerous virus diseases, and sensitive cultivars may be killed or
severely weakened by virus infection; latent infections on tolerant
cultivars may shorten the planting life of a eld through reduced
yield and fruit quality. Incidence in Robus accessions of raspberry
50
(height), and in vitro shooting. The adaptability rate was determined after 30 days of planting (acclimatization). Data were
analyzed by ln(x). Variances were calculated for the micropropagation traits related to productivity and were compared between
treatments using Bartletts homogeneity of variances test STATISTICA V.6 software package (STATSOFT, Inc, 2003).
51
Fig. 1. Photosynthesis-related variables determined in raspberry plants (cv. Heritage) micropropagated in Temporary Immersion Bioreactors (TIBs). (ad) Signicant
differences (P < 0.01) determined by Duncan test at 5, 10, 15 and 20 days.
number of shoots was demonstrated to be higher in comparison with the agar-base procedure (control), evidencing the
enhancement effect of TIBs environment on plant multiplication.
In summary, the higher results for traits linked to plant micropropagation yields were demonstrated in raspberries multiplied
in CO2 -rich TIBs + 15 gr/L sucrose.
Fig. 2 illustrates the overall increase of micropropagation
efciency using the TIBs in comparison with the conventional
Table 1
Micropropagation traits evaluated during raspberry (Rubus spp.) micropropagation in CO2 -rich TIBs and agar-based procedures (control treatment).
Genotype
In vitro approacha
Adaptability (%)b
Heritage
Agar S 30 gr/l
5.78 2.41
(4.62)
5.97 3.14
(4.01)
9.21 3.67
(0.37)
4.98 2.59
(3.78)
6.73 3.21
(5.65)
10.67 5.35
(0.42)
5.65 3.12
(4.45)
6.41 2.71
(5.01)
12.07 5.54
(0.60)
6.52*
15.75 5.62
(12.77)
36.12 9.34
(21.44)
38.51 9.46
(28.52)
18.22 6.74
(9.22)
39.52 12.78
(30.45)
39.54 11.82
(27.83)
19.05 8.45
(10.75)
32.64 11.01
(28.32)
37.84 10.42
(32.74)
4.75*
74.65 21.10
(38.43)
186.40 32.97
(85.01)
345.52 40.35
(124.85)
84.32 28.43
(42.67)
198.43 41.55
(96.02)
360.21 61.64
(142.84)
74.65 21.10
(35.75)
186.40 32.97
(90.45)
345.52 40.35
(140.55)
5.62*
71.45 11.76
(35.76)
78.32 13.43
(10.54)
98.45 1.38
(7.31)
74.04 8.42
(42.56)
83.23 15.98
(23.78)
97.12 2.06
(16.51)
75.12 9.74
(40.65)
80.92 10.62
(19.43)
96.38 2.48
(11.83)
6.34*
TIBs S 30 gr/l
TIBs S 15 gr/l
Meeker
Agar S 30 gr/l
TIBs S 30 gr/l
TIBs S 15 gr/l
Amity
Agar S 30 gr/l
TIBs S 30 gr/l
TIBs S 15 gr/l
Bartletts test
a
b
*
52
Fig. 2. Micropropagation of raspberry (Rubus spp.) in Temporary Immersion Bioreactors (TIBs) under 550 ppmv CO2 and light intensity 80 M m2 s1 . (A and B) Plantlets (cv.
Heritage) of 15 days old growing in agar-base medium (control) and TIBs. (C) Raspberries (cv. Amity) of 25 days old multiplying in TIBs + 15 gr/L sucrose (L) and TIBs + 30 gr/L
sucrose (R). (D) Raspberries (cv. Heritage) after 3 weeks of transplanted to greenhouse.
Acknowledgments
To Anne Bliss PhD (University of Colorado, USA) and Patrick Matzler for the language revision and copyediting of the manuscript.
To the Regional Government of Maule (Chile) for nancial support.
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