Vaccinium myrtillus L. is a valuable wild fruit species of conservation concern in the Tuscan Apennines (Italy).
OBJECTIVE:
The development of a micropropagation protocol for the conservation of this species in in vitro-banks for the year-round mass production of specific selected superior bilberry genotypes, maintaining the genetic variability of natural populations, avoiding genetic erosion.
METHODS:
Nodal segments from seedlings were cultured on mMS medium with zeatin or 2iP for multiplication and elongation stage, afterwards shoots were rooting in vitro and in vivo. The RAPD analysis was applied to lines obtained and slow growth storage (4°C – darkness) was evaluated for the conservation.
RESULTS:
Zeatin concentration 9.1 μM was the most favorable for shoot proliferation. The activated charcoal was essential for in vitro rooting. Both in vitro and ex vitro methods were successfully used to root and acclimatize bilberry shoots. The RAPD analysis indicated a large genetic diversity among the micropropagated genotypes. Shoots were successfully in vitro preserved for up to 6 months with no apparent deterioration effect.
CONCLUSIONS:
In vitro propagation and conservation are fundamental tools for exploiting the whole potential of this wild crop species for breeding and genetic improvement purposes.
AncillottiC, CiofiL, PucciD, SagonaE, GiordaniE, BiricoltiS, et al.Polyphenolic profiles and antioxidant and antiradical activity of Italian berries from Vaccinium myrtillus L. and Vaccinium uliginosum L. subsgaultherioides (Bigelow) S.B. Young. Food Chem. 2016;204:176–184.
2.
GerdolR, SiffiC, IacuminP, GualminiM, TomaselliM.
. Advanced snowmelt affects vegetative growth and sexual reproduction of Vaccinium myrtillus in a sub-alpine heath. J Veg Sci. 2013;24(3):569–579.
3.
PatsiasK, BruhelheideH.
. Climate change – Bad news for montane forest herb layer species?Acta Oecol2013;50:10–19.
4.
NinS, PetrucciWA, DelBubba M, AncillottiC, GiordaniE.
. Effects of environmental factors on seed germination and seedling establishment in bilberry (Vaccinium myrtillus L.). Sci Hortic. 2017;226:241–249.
5.
DebnathSC, McRaeKB.
. In vitro culture of Lingonberry (Vaccinium vitis-idaea L.). Small Fruit Rev. b. 2001;1(3):3–19.
6.
NestbyR, PercivalD, MartinussenI, OpstadN, RoholfJ.
. The European Blueberry (Vaccinium myrtillus L.) and the Potential for Cultivation. A Review. Eur J Plant Sci Biotechnol. 2011;5(1):5–16.
7.
LambardiM, SharmaKK, ThorpeTA.
. Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.). In Vitro Cell Dev-Pl. 1993;29:189–199.
8.
GustavssonBA, StanysV.
. Field Performance of ‘Sanna’ Lingonberry derived by micropropagation vs stem cuttings. HortScience. 2000;35(4):742–744.
9.
OstroluckaMG, LibiakovaG, OndruskovaE, GajdosovaA.
. In vitro propagation of Vaccinium species. Acta Universitatis Latviensis Biol. 2004;676:207–212.
10.
DebnathSC, VyasP, GoyaliJC, IgamberdievAU.
. Morphological and molecular analyses in micropropagated berry plants acclimatized under ex vitro condition. Can J Plant Sci. 2012;92:1065–1073.
11.
GoyaliJC, IgamberdievAU, DebnathSC.
. Morphology, phenolic content and antioxidant capacity of lowbush blueberry (Vaccinium angustifolium Ait.) plants as affected by in vitro and ex vitro propagation methods. Can J Plant Sci. 2013;93:1001–1008.
12.
JaskowiakMA.
. Reviews of science for Science librarians: The conservation of endangered plants using micropropagation. Sci Tech Libr. 2014;33(1):58–70.
13.
ShibliRA, SmithMAL.
. Direct shoot regeneration from Vaccinium pahalae (Ohelo) and V. myrtillus (Bilberry) leaf explants. HortScience. 1996;31(7):1225–1228.
14.
ShibliRA, SmithMAL, NasiR.
. Iron source and cytokinin mitigate the incidence of chlorosis and hyperhydration in vitro. J Plant Nutr. 1997;20(6):773–781.
15.
JaakolaL, TolvanenA, LaineK, HohtolaA.
. Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots. Plant Cell Tiss Org. 2001;66:73–77.
16.
JaakolaL, RiihinenK, HaggmanHHohtolaA.
. Bilberry in vitro protocols and analyses of phenolic compounds.
In:Mohan JainSSaxenaPK
editors Methods in Molecular Biology, Protocols for in vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, Humana press; 2009; p.71–80.
17.
SgarbiE, SantoroE, SantoroS, GrimaudoM.
. Propagazione in vitro di mirtillo nero dell’Appennino settentrionale. Italus Hortus. 2009;16(2):151–153.
18.
DuanZ, LiJ, ZhaoY, WangH, HouL.
. The optimization of bilberry rapid micropropagation in vitro culture. J Nanling Forest Univ. (Natural Sciences Edition). 2010;34(3):168–170.
19.
CuceM, SokmenA.
. Micropropagation of Vaccinium myrtillus L. (bilberry) naturally growing in the Turkish flora. Turk J Biol. 2015;39:233–240.
20.
DebnathSC, McRaeKB.
. An efficient in vitro shoot propagation of cranberry (Vaccinium macrocarpon Ait.) by axillary bud proliferation. In Vitro Cell Dev-Pl. a. 2001;37:243–249.
21.
DebnathSC, McRaeKB.
. An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.). J Hortic Sci Biotech. 2002;77:744–752.
22.
PolashockJJ, VorsaN.
. Cranberry (Vaccinium macrocarpon Ait.). KhachatouriansG McHughen A ScorzaRNip WK Hui YH
editors. Transgenic plants and crops. New York: Marcel Dekker; 2003;383–396.
23.
DebnathSC.
. In vitro culture of lowbush blueberry (Vaccinium angustifolium Ait.). Small Fruit Rev. 2004;3:393–408.
24.
DebnathSC.
. Morphological development of Lingonberry as affected by in vitro and ex vitro propagation methods and source propagule. HortScience. 2005;40(3):760–763.
25.
DebnathSC.
. Zeatin-induced one-step in vitro cloning affects the vegetative growth of cranberry (Vaccinium macrocarpon Ait.) micropropagules over stem cuttings. Plant Cell Tiss Org. 2008;93(2):231–240.
26.
OstroluckaMG, GajdosovaA, LibiakovaG, HrubikovaK.
BezoM.
Protocol for micropropagation of selected Vaccinium spp.
In: SMJain HHaggman
editors. Protocols for micropropagation of woody trees and fruits. Berlin Heidelberg New York: Springer; 2007;445–455.
27.
OstroluckaMG, GajdosovaA, OndruskovaE, LibiakovaG.
. In vitro propagation of several Vaccinium corymbosum L. and Vaccinium vitis-idaea L. cultivars. Latv J Agron. 2009;12:75–80.
28.
McCownBH, ZeldinEL.
. Vaccinium. spp cranberry.
RELitz
editor. Biotechnology of fruit and nut crops. Biotechnology in agriculture series no. 29. Wallingford, UK: CAB International; 2005. p 247–261.
29.
PereiraMJ.
. Conservation of Vaccinium cylindraceum Smith (Ericaceae) by micropropagation using seedling nodal explants. In Vitro Cell Dev-Pl. 2006;42:65–68.
30.
GajdosovaA, OstroluckaMG, LibiakovaG, OndruskovaE, SimalaD.
. Microclonal propagation of Vaccinium sand Rubus sand detection of genetic variability in culture in vitro. J Fruit Ornam Plant Res. 2006;14(1):61–76.
31.
GajdoşováA, OstroluckáMG, LibiakováG, OndruşskováE.
. Protocol for micropropagation of Vaccinium vitis-idaea L.
Jain SM
H Haggman
In: , , editors. Protocols for micropropagation of Woody Trees and Fruits. Dordrecht: Springer; 2007. pp. 447–464.
32.
CuceM, SokmenA.
. In vitro production protocol of Vaccinium uliginosum L. (bog bilberry) growing in the Turkish flora. Turk J Agríc For. 2017;41:294–304.
33.
MeinersJ, SchwabM, SzankowskiI.
. Efficient in vitro regeneration systems for Vaccinium species. Plant Cell Tiss Org. 2007;89: 169–176.
34.
RuzicD, VujavicT, LibiakovaG, CerovicR.
. Micropropagation in vitro of highbush blueberry (Vaccinium corymbosum L.). J Berry Res. 2012;2:97–103.
35.
CuceM, BektasE, SokmenA.
. Micropropagation of Vaccinium arctostaphylos L. via lateral-bud culture. Turk J Agric For. 2013;37:40–44.
36.
PaprşteinF, SedlákJ.
. In vitro multiplication of lingonberry – Short Communication. Hort Sci. (Prague)2015;42(2):102–106.
37.
HungCD, HongC-H, KimS-K, LeeK-H, ParkJ-Y.
. , Dung CD, et al. In vitro proliferation and ex vitro rooting of microshoots of commercially important rabbiteye blueberry (Vaccinium ashei Reade) using spectral lights. Sci Hortic-Amsterdam2016;211:248–254.
38.
DebnathSC.
. A scale-up system for Lowbush Blueberry micropropagation using a bioreactor. HortScience2009;44(7):1962–1966.
39.
PanisB, LambardiM.
. Status of cryopreservation technologies in plants (crops and forest trees). The Role of Biotechnology, Villa Gualino, Turin, Italy; 5-7 March2005. p. 43–54.
40.
EngelmannF.
. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev-Pl. 2011;47(1):5–16.
41.
ReedBM.
. Design a micropropagation system: workshop presentation from the 1998 SIVB Congress on in vitro biology. In Vitro Cell Dev-Pl. 1999;35:275–284.
42.
KellerERJ, SenulaA, LeunufnaS, GrübeM.
. Slow growth storage and cryopreservation-tools to facilitate germplasm maintenance of vegetatively propagated crops in living plant collections. Int J Refrig. 2006;29(3):411–417.
43.
BenelliC, OzudogruEA, LambardiM, DradiG.
. In vitro conservation ornamental plants by slow growth storage. Acta Hortic. 2012;961:89–93.
44.
González-BenitoME, MartínC.
. In vitro preservation of Spanish biodiversity. In Vitro Cell Dev-Pl. 2011;47(1):46–54.
45.
RajasekharanPE.
. Gene Banking for ex situ conservation of plant genetic resources.
In: Bahadur ,BVenkatRajam, MSahijram,LKrishnamurthyK,
editors. Plant Biology and Biotechnology. New Delhi: Springer; 2015. pp. 445–459.
46.
EconomouAS, ReadPE.
. In vitro shoot proliferation of Minnesota deciduous Azaleas. HortScience1984;19:60–61.
47.
AlbertT, RaspéO, JaquemartAL.
. Influence of clonal growth on selfing rate in Vaccinium myrtillus L. Plant Biol. 2008;10(5):643–649.
48.
GattiE, SgarbiE, OzudogruEA, LambardiM.
. The effect of Plantform™ bioreactor on micropropagation of Quercus robur in comparison to a conventional in vitro culture system on gelled medium, and assessment of the microenvironment influence on leaf structure. Plant Biosystems2017;151:1129–1136.
49.
JaccardP.
. Nouvelles recherches sur la distribution florale. Bull Soc Vaud Sci Nat. 1908;44:223–270.
50.
DebnathSC.
. Propagation of Vaccinium in vitro. Int J Fruit Sci. 2007;6(2):47–71.
51.
HammerO, HarperDAT, RyanPD.
Paleontological Statistics Software: Package for Education and Data Analysis Palaeontologia Electronica. 2001.
52.
PeakallR, SmousePE.
. GenAlEx 6.5: genetic analysis in excel. Population genetic software for teaching and research - an update. Bioinformatics. 2012;28:2537–2539
53.
LewontinRC.
. Testing the theory of natural selection. Nature. 1972;236:181–182.
54.
GiordaniE, BiricoltiS, AncillottiC, PetrucciWA, GoriM, CalistriE, et al.Genetic diversity and changes in phenolic contents an antiradical activity of Vaccinium myrtillus berries from its southernmost growing area in Italy. Genet Resour Crop Ev. 2018;65:1173–1186.
55.
EtcherT, NoeN.
. Comparison between 2iP and zeatin in the micropropagation of highbush blueberry (Vaccinium corymbosum). Acta Hortic. 1989;241:185–190.
56.
ReedBM, Abdelnour-EsquivelA.
. The use of zeatin to initiate in vitro cultures of Vaccinium species and cultivars. HortScience1991;26(10):1320–1322.
57.
JaakolaL, TolvanenA, LaineK, HohtolaA.
. Micropropagation of Bilberry and Lingonberry. Acta Hortic. 2002;574:401–403.
58.
KaldmäeH, KarpK, StarastM, PaalT.
. Effect of donor plant physiological condition on in vitro establishment of Vaccinium angustifolium shoot explants. Acta Hortic. 2006;715:433–438.
59.
ChandlerCK, DraperAD.
. Effect of zeatin and 2iP on shoot proliferation of three highbush blueberry clones in vitro. HortScience1986;21:1065–1066.
60.
TetsumuraT, MatsumotoY, SatoM, HonshoC, YamashitaK, KomatsuH, et al.Evaluation of basal media for micropropagation of four highbush blueberry cultivars. Sci Hortic. 2008;119(1): 72–74.
61.
SerresRA, PanS, McCownBH, StangEJ.
. Micropropagation of several lingonberry cultivars. Fruit Varieties J. 1994;48:7–14.
62.
MarcotrigianoM, McGlewSP.
. A two-stage micropropagation system for cranberries. J Am Soc Hortic Sci. 1991;116(5):911–916.
63.
SmagulaJM, HarkerJ.
. Cranberry micropropagation using a lowbush blueberry medium. Acta Hortic. 1997;446:343–347.
64.
ThomasTD.
. The role of activated charcoal in plant tissue culture. Biotechnol Adv. 2008;26:618–631.
65.
FabbriA, MagnaniS.
. Nuova tecnica di radicazione di mora, lampone e mirtillo. Acta Italus Hortus2018;21:40–41.
66.
BrissetteL, TremblayL, LordD.
. Micropropagation of lowbush blueberry from mature field-grown plants. HortScience1990;25:349–351.
67.
FayMF.
. In what situations is in vitro culture appropriate to plant conservation?Biodivers Conserv. 1994;3:176–183.
ReedBM.
. Application of gas-permeable bags for in vitro cold storage of strawberry germplasm. Plant Cell Rep. 1991;10:431–434.
70.
ReedB.M.
. Cold storage of strawberries in vitro: a comparison of three storage systems. Fruit Varieties J. 1992;46:98–102.
71.
ReedBM.
. Improved survival of in vitro-stored Rubus germplasm. J Am Soc Hortic Sci. 1993;118:890–895.
72.
LisekA, OrlikowskaT.
. Factors influencing long-term storage of strawberry shoots in vitro. Acta Hortic. 2001;560:189–192.
73.
SuriyanC, KirdmaneeC.
. “Minimal growth in vitro culture for preservation of plant species.”Fruit Veg Cereal Sci Biotechnol. 2007;13–25.
74.
RoncasagliaR, BenelliC, De CarloA, DradiG.
OzudogruEA.
. 2009. Fattori che influiscono sulla conservazione in crescita rallentata di specie da frutto. Italus Hortus2009;16(2):234–238.
75.
OzudogruEA, PreviatiA.
LambardiM.
. In vitro conservation and cryopreservation of ornamental plants.In: Jain S, Ochatt S., editors.Protocols for In Vitro Propagation of Ornamental Plants. Methods in Molecular Biology (Methods and Protocols), Vol 589. Humana Press; 2010. p. 303–324.
76.
Cruz-CruzC, González-ArnaoM, EngelmannF.
. Biotechnology and conservation of plant biodiversity. Resour. 2013;2(2):73–95.
77.
OzudogruEA, BenelliC, DradiG, LambardiM.
. Effect of culture container and carbohydrate content on in vitro slow growth storage of the cherry rootstock 'Gisela® 5'. Acta Physiol Plant. 2017;39(4):94.
78.
ReedBM.
. Photoperiod improves long-term survival of in vitro-stored strawberry plantlets. HortScience2002;37(5):811–814.
79.
ReedBM.
. Cryopreservation of temperate berry crops. In:
ReedBM, editor. Plant Cryopreservation: A Practical Guide. New York: Springer; 2008, p. 333–364.
80.
PachecoG, GagliardiRF, VallsJFM, MansurE.
. Micropropagation and in vitro conservation of wild Arachis species. Plant Cell Tiss Organ Cult. 2009;99–239.
