In the present study, it is aimed to investigate embryo and protoplast
isolation from orchid seeds, because of know-how deficiency. As material Barlia robertiana seeds were subjected enzymatic
process. Globular embryos with suspensor were fully removed by enzymatic
maceration for between 100-180 min of incubation. The
highest obtained embryo yields were found inRapidase
EX Color and Rapidase Vino Super, with degree of 89.2-90.3 ×103·g-1 MF and 86.4-91.3 ×103·g-1 MF, respectively.
According to multicomponent maceration tests, a treatment comprising 150 min of
the enzymatic reaction with 0.18% (E/S) at 50℃ was the most
optimal for obtaining high embryo yields. Regarding protoplast isolation tests,
enzyme combinations C (1% Cellulase “Onozuca” R-10 + 0.2% Macerozyme R-10 and
0.1% Driselase) and F (1% Cellulase “Onozuka” + 1% Macerozyme + 0.5% Driselase)
after 15 hours produced the best results for protoplast isolation and were significantly
different compared to other enzyme combinations. Greater protoplast yields were
obtained using a rotatory system with protoplasts incubated in the dark. Also
the present findings were discussed from the point of view of in vitro plant manipulation and orchid
cultivation in cultural media and then their importance emphasized in molecular
Cite this paper
M. Aybeke, "Embryo and Protoplast Isolation from Barlia robertiana Seeds (Orchidaceae)," American Journal of Plant Sciences, Vol. 4 No. 6, 2013, pp. 1-8. doi: 10.4236/ajps.2013.46A001.
 B. G. Keel, “Assisted Migration as a conservation Strategy for Rapid Climate Change: Investigating Extended Photoperiod and Mycobiont Distributions for Habenaria Repens Nuttal (Orchidaceae) as a Case Study,” Ph.D. Thesis, Antioch University, Antioch, 2007.
 H. Koopowitz, P. S. Lavarack and K. W. Dixion, “The Nature of Threats to Orchid Conservation,” In: K. W. Dixon, et al., Eds., Orchid Conservation, Natural History Publications, Kota Kinabalu, 2003, pp. 25-42.
 M. M. Hossain, “Asymbiotic Seed Germination and in Vitro Seedling Development of Epidendrum Ibaguense Kunth. (Orchidaceae),” African Journal of Biotechnology, Vol. 7, No. 20, 2008, pp. 3614-3619.
 CITES, “Appendies I and II,” 2010.
 J. Nadarajan, S. Wood, T. R. Marks, P. T. Seaton and H. W. Pritchard, “Nutritional Requirements for in Vitro Seed Germination of 12 Terrestrial, Lithophytic and Epiphytic Orchids,” Journal of Tropical Forest Science, Vol. 23, No. 2, 2011, pp. 204-212.
 M. Aybeke, “The Pollen and Seed Morphology of Some Ophrys L. (Orchidaceae) Taxa,” Journal of Plant Biology, Vol. 50, No. 4, 2007, pp. 387-395.
 P. S. Huehnea and K. Bhinijaa, “Application of Cryoprotectants to Improve Low Temperature Storage Survival of Orchid Seeds,” Scientia Horticulturae, Vol. 135, 2011, pp. pp. 186-193. doi:10.1016/j.scienta.2011.11.026
 S. Nontachaiyapooma, S. Sasiratb and L. Manochc, “Symbiotic seed Germination of Grammatophyllum speciosum Blume and Dendrobium draconis Rchb. F., Native Orchids of Thailand,” Scientia Horticulturae, Vol. 130, No. 1, 2011, pp. 303-308.
 F. R. Hay, D. J. Merritt, J. A. Soanes and K. W. Dixon, “Comparative Longevity of Australian Orchid (Orchidaceae) Seeds under Experimental and Low Temperature Storage Conditions,” Botanical Journal of the Linnean Society, Vol. 164, No. 1, 2010, pp. 126-141.
 A. R. Roy, R. S. Patel, V. V. Patel, S. Sajeev and B. C. Deka, “Asymbiotic Seed Germination, Mass Propagation and Seedling Development of Vanda coerulea Griff ex. Lindl. (Blue Vanda): An in Vitro Protocol for an Endangered Orchid,” Scientia Horticulturae, Vol. 128, No. 3, 2011, pp. 325-331.
 D. K. Das and A. Rahman., “Expression of a Rice Chitinase Gene Enhances Antifungal Response in Transgenic Litchi (cv. Bedana),” Plant Cell Tissue and Organ Culture, Vol. 109, No. 2, 2012, pp. 315-325.
 S. T. Hosomi, C. C. Custodio, P. T. Seaton, T. R. Marks and N. B. Machado-Neto, “Improved Assessment of Viability and Germination of Cattleya (Orchidaceae) Seeds Following Storage,” In Vitro Cellular & Developmental Biology—Plant, Vol. 48, No. 1, 2012, pp. 127-136.
 Y. Lee, E. C. Yeung, N. Lee and M. C. Chung, “Embryo Development in the Lady’s Slipper Orchid, Paphiopedilum delenatii, with Emphasis on the Ultrastructure of the Suspensor,” Annals of Botany, Vol. 98, No. 6, 2006, pp. 1311-1319. doi:10.1093/aob/mcl222
 M. A. P. C. Costa, F. A. A. Mourao Filho and B. M. J. Mendes, “Isolamento e Eficiência de Plaqueamento de Protoplastos de Citros,” Revista Brasileira de Fruticultura, Vol. 24, No. 2, 2002, pp. 472-476.
 K. Kanwar, A. Bhardwaj and R. Deepika, “Efficient Regeneration of Plantlets from Callus and Mesophyll Derived Protoplasts of Robinia pseudoacacia L.,” Plant Cell, Tissue and Organ Culture, Vol. 96, No. 1, 2009, pp. 95-103.
 K. Kanchanapooma, S. Jantarob and D. Rakchadb, “Isolation and Fusion of Protoplasts from Mesophyll Cells of Dendrobium pompadour,” Science Asia, Vol. 27, No. 1, 2001, pp. 29-34.
 J. M. Da Silva Júnior, P. Renato, R. Paiva, A. C. A. L. Campos, M. Rodrigues, M. A de F. Carvalho and W. C. Otoni, “Protoplast Production and Isolation from Etlingera elatior,” Science Asia, Vol. 34, No. 1, 2012.
 M. Monteiro, B. Appezzato-Da-Glória, M. J. Valarini, C. A. Oliveira and M. L. C. Vieira, “Plant Regeneration from Protoplasts of Alfalfa (Medicago sativa) via Somatic Embryogenesis,” Scientia Agricola, Vol. 60, No. 4, 2003, pp. 683-689.
 C. Kativat, O. Poolsawat and P. Tantasawat, “Optimization of Factors for Efficient Isolation of Protoplasts in Sunflower (Helianthus annuus L.),” Australian Journal of Crop Science, Vol. 6, No. 6, 2012, pp. 1004-1010.
 G. Kaur, S.K. Banga, K. P. S. Gogna, S. Joshi and S. S. Banga, “Moricandia Arvensis Cytoplasm-Based System of Cytoplasmic Male Sterility in Brassica juncea: Reappraisal of Fertility Restoration and Agronomic Potential,” Euphytica, Vol. 138, No. 2, 2004, pp. 271-276.
 D. Li and M. Z. Bao, “Plant Regeneration from Protoplasts Isolated from Embryogenic Suspension Cultured Cells of Cinnamomum camphora L.,” Plant Cell Reports, Vol. 24, No. 8, 2005, pp. 462-467.
 M. Elhiti and C. Stasolla, “The Use of Zygotic Embryos as Explants for In Vitro Propagation: An Overview,” Methods in Molecular Biology, Vol. 710, No. 4, 2011, pp. 229-255.