Document Type : Research paper
Authors
Department of Horticulture, College of Aburaihan, University of Tehran, Pakdasht, Tehran, Iran
Abstract
Light spectrum is one of the environmental cues that influence plant growth and development. Light is a stimulating factor for induction of somatic embryos during tissue culture practices. To accelerate the direct embryogenesis, six different light spectra including: white (W), red (R), blue (B), green (G), red + blue (R+B) and red + far red (R+FR) together with dark condition (D), in combination with thidiazuron (TDZ) in four concentrations (0, 0.5, 1.5 and 3 mg L-1) were used. Inter-simple sequence repeat was used for identification and genetic stability analysis of somatic regenerated plantlets. Intact protocorm explants showed higher potential for direct somatic embryogenesis (DSE) than the other explants. The rate of DSE was highly dependent on the concentration of TDZ and its interaction with light spectra. R and R + FR spectra with 3 mg L-1 TDZ on intact protocorms and R+FR with 3 mg L-1 TDZ were efficient treatments to induce DSE without somaclonal variation. G light spectrum has also significant effects on DSE of protocorm explants. The amplified products showed 26 scorable bands and regenerates were completely identical to the mother plant. In conclusion, this protocol provides way to regenerate plants through embryogenesis, and is a reliable protocol to obtain proper development and genetic stable Phalaenopsis embryos.
Keywords
Aalifar M, Arab M, Aliniaeifard S, Dianati S, Zare Mehrjerdi M, Limpens E, Serek M. 2019. Embryogenesis efficiency and genetic stability of Dianthus caryophyllus embryos in response to different light spectra and plant growth regulators. Plant Cell Tissue and Organ Culture 139: 479- 492.
2. Aliniaeifard S, Seif M, Arab M, Zare Mehrjerdi M, Li T, Lastochkina O. 2018. Growth and photosynthetic performance of Calendula officinalis under monochromatic red light.
International Journal of Horticultural Science and Technology 5(1):123-32.
3. Al-Khayri J. 2010. Somatic embryogenesis of date palm (phoenix dactylifera) improved by coconut water. Biotechnology 9(4): 477-484.
4. Arditti J. 2009. Micropropagation of orchids. John Wiley and Sons.
5. Ascencio-Cabral A, Gutierrez-Pulido H, Rodriguez-Garay B, Gutierrez-Mora A. 2008. Plant regeneration of Carica papaya through somatic embryogenesis in response to light quality, gelling agent and phloridzin. Scientia Horticulturae 118: 155-160.
6. Bakhshaie M, Babalar M, Mirmasoumi M, Khalighi A. 2010. Somatic embryogenesis and plant regeneration of (Lilium Ledebourii) boiss an endangered species. Plant Cell Tissue and Organ Culture 102: 229-235.
7. Bairu M.W, Aremu A.O, Staden J. 2011. Somaclonal variation in plants causes and detection methods. Plant Growth Regulation 63: 147-173.
8. Banerjee R, Schleicher E, Meier S, Viana RM, Pokorny R, Ahmad M, Bittl R, Batschauer A. 2007. The signaling state of arabidopsis cryptochrome 2 contains flavin semiquinone. Journal of Biological Chemistry 282: 14916-14922.
9. Bayat L, Arab M, Aliniaeifard S, Seif M, Lastochkina O, Li T. 2018. Effects of growth under different light spectra on the subsequent high light tolerance in rose plants. AoB PLANTS 10, ply052.
10. Bhattachryya P, Kumaria S, Diengdoh R, Tandon P. 2014. Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile an endangered medicinal orchid. Meta gene 2: 489-504.
11. Bhatia P, Ashwath N, Midmore D.J. 2005. Effects of genotype, explant orientation, and wounding on shoot regeneration in tomato. In Vitro Cellular and Developmental Biology Plant 41: 457-464.
12. Burritt D.J, Leung D.W. 2003. Adventitious shoot regeneration from begonia × erythrophylla petiole sections is developmentally sensitive to light quality. Physiologia plantarum 118: 289-296.
13. Chan A. 2013. Light induction of somatic embryogenesis and dark germination of Arabidopsis thaliana, MSc Thesis, Monitoba University.
48 Int. J. Hort. Sci. Technol; Vol. 8, No. 1; March 2021
14. Chen J.T, Chang W.C. 2001. Effects of auxins and cytokinins on direct somatic embryogenesison leaf explants of oncidium 'gower ramsey’. Plant Growth Regulation 34: 229-232.
15. Chen J.T, Chang W.C. 2004. Tiba affects the induction of direct somatic embryogenesis from leaf explants of oncidium. Plant Cell Tissue and Organ Culture 79: 315-320.
16. Chen J, Chang W.C. 2006. Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biologia Plantarum 50: 169-173.
17. Chen J.R, Lian Wu, Bo-Wen Hu, Xing Y, Rong L, Zi-Niu D, Xing-Yao X. 2014. The influence of plant growth regulators and light quality on somatic embryogenesis in China rose (Rosa chinensis Jacq). Journal of Plant Growth Regulation 33: 295-304.
18. Chung J.P, Huang C.Y, Dai TE. 2010. Spectral effects on embryogenesis and plantlet growth of oncidium ‘gower ramsey’. Scientia Horticulturae 124: 511-516.
19. Chung H, Chen J, Chang W. 2007. Plant regeneration through direct somatic embryogenesis from leaf explants of dendrobium. Biologia Plantarum 51: 346-350.
20. Deo P.C, Tyagi A.P, Taylor M, Harding B. 2010. Factors affecting somatic embryogenesis and transformation in modern plant breeding. The South Pacific Journal of Natural and Applied Sciences 28: 27-40.
21. D'onofrio C, Morini S, Bellocchi G. 1998. Effect of light quality on somatic embryogenesis of quince leaves. Plant Cell Tissue and Organ Culture 53: 91-98.
22. Feher A, Pasternak T.P, Dudits D. 2003. Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue and Organ Culture 74: 201-228.
23. Feng J.H, Chen J.T. 2014. A novel in vitro protocol for inducing direct somatic embryogenesis in Phalaenopsis aphrodite without taking explants. The Scientific World Journal 131: 855-862.
24. Finer JJ. 1995. Direct somatic embryogenesis. Plant Cell Tissue and Organ Culture 72: 91-102.
25. Gow W.P, Chen J.T, Chang W.C. 2009. Effects of genotype, light regime, explant position and orientation on direct somatic embryogenesis from leaf explants of phalaenopsis orchids. Acta Physiologiae Plantarum 31: 330-363.
26. Gow W.P, Chen J.T, Chang W.C. 2010. Enhancement of direct somatic embryogenesis and plantlet growth from leaf explants of phalaenopsis by adjusting culture period and explant length. Acta Physiologiae Plantarum 32: 621-627.
27. Grzyb M, Kalandyk A, Mikuła A. 2018. Effect of tiba, fluridone and salicylic acid on somatic embryogenesis and endogenous hormone and sugar contents in the tree fern Cyathea Delgadii sternb. Acta Physiologiae Plantarum 40: 1-9.
28. Guo W.L, Chang Y.C.A, Kao C.Y. 2010. Protocorm-like bodies initiation from root tips of Cyrtopodium paranaense (orchidaceae). Hortscience 45: 1365-1368.
29. Harini I, Sita G.L. 1993. Direct somatic embryogenesis and plant regeneration from immature embryos of chilli (Capsicum annuum). Plant Science 89: 107-112.
30. Hartmann K.M. 1967. Phytochrome 730 (pfr), the effector of the “high energy photomorphogenic reaction” in the far-red region. Naturwissenschaften 54: 544-544.
31. Hasebe M, Iwatsuki K. 1990. Adiantum capillus-veneris chloroplast DNA clone bank As useful heterologous probes in the systematics of the leptosporangiate ferns. American Fern Journal 80: 20-25.
32. Hosseini A, Zare Mehrjerdi M, Aliniaeifard S, Seif M. 2019. Photosynthetic and growth responses of green and purple basil plants under different spectral compositions. Physiology and Molecular Biology of Plants 25, 741-752.
33. Huan L.V.T, Tanaka M. 2004. Callus induction from protocorm-like body segments and plant regeneration in Cymbidium (Orchidaceae). The Journal of Horticulture Science and Biotechnology 79: 406-410.
34. Huang Y, Tsai Y, Chen F. 2014. Characterization and expression analysis of somatic embryogenesis receptor-like kinase genes from Phalaenopsis. Genetics and Molecular Reserch 13: 10690-10703.
35. Husaini A.M, Abdin M. 2007. Interactive effect of light, temperature and TDZ on the regeneration potential of leaf discs of Fragaria x ananassa duch. In Vitro Cellular and Developmental Biology Plant 43: 576-584.
36. Khoddamzadeh A.A, Sinniah U, Kadir M, Kadzimin S, Mahmood M, Sreeramanan S. 2011. In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf
Efficient Method for Direct Embryogenesis in Phalaenopsis Orchid 49
segments of Phalaenopsis bellina Christenson. Plant Growth Regulation 65: 381-390.
37. Kraepiel Y, Miginiac E. 1997. Photomorphogenesis and phyfohormones. Plant Cell and Environment 20: 807-812.
38. Kuo H.L, Chen J.T, Chang W.C. 2005. Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis ‘little steve’. In Vitro Cellular and Developmental Biology Plant 41: 453-460.
39. Li H, Tang C, Xu Z. 2013. The effects of different light qualities on rapeseed (Brassica Napus l.) plantlet growth and morphogenesis in vitro. Scientia Horticulturae 150: 117-124.
40. Ludevid M.D, Ruiz-Avila L, Valles M.P, Stiefel V, Torrent M, Torne JM, Puigdomenech P. 1990. Expression of genes for cell-wall proteins in dividing and wounded tissues of (Zea mays L). Planta 180: 524-530.
41. Mahendran G, Bai V.N. 2012. Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor. Scientia Horticulturae 135: 40-44.
42. Mayer J.L, Stancato G.C, Appezzato B. 2010. Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of oncidium flexuosum sims (orchidaceae). Plant Cell Tissue and Organ Culture 103: 411-416.
43. Meilasari D, Iriawati I. 2016. Regeneration of Plantlets Through PLB Formation in Phalaenopsis ‘Join Angle X Sogo Musadian’. Journal of Mathematical and Fundamental Sciences 48: 204 212.
44. Mengxi L, Zhigang X, Yang Y, Yijie F. 2011. Effects of different spectral lights on oncidium PLB induction, proliferation, and plant regeneration. Plant Cell Tissue and Organ
Culture 106: 1-10.
45. Michler C.H, Lineberger R.D. 1987. Effects of light on somatic embryo development and abscisic levels in carrot suspension cultures. Plant Cell Tissue and Organ Culture 11: 189-207.
46. Moradi S, Dianati Daylami S, Arab M, Vahdati K. 2017. Direct somatic embryogenesis in Epipactis veratrifolia a temperate terrestrial orchid. The Journal of Horticulture Science and Biotechnology 92: 88-97. 47. Moghare Abed S, Naderi D. 2020. Stimuli Effects of Different LEDs on Some Morphological and Biochemical Traits of Two Varieties of Calendula officinalis. International Journal of Horticultural Science and Technology 7(2):139-151.
48. Morel GM. 1960. Producing virus-free cymbidium. American Orchid Society Bulletin 98: 202-2011.
49. Mose W, Indrianto A, Purwantoro A, Semiarti E. 2017. The influence of thidiazuron on direct somatic embryo formation from various types of explant in Phalaenopsis amabilis blume orchid. Hayati Journal of Biosciences 24: 201-205.
50. Mulgund G.S, Nataraja K, Malabadi R.B, Kumar S.V. 2011. TDZ induced in vitro propagation of an epiphytic orchid xenikophyton smeeanum. Reserch in Plant Biology 68: 312-320.
51. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.
52. Naing A.H, Kim C.K, Yun B.J, Jin J.Y, Lim K.B. 2013. Primary and secondary somatic embryogenesis in chrysanthemum. Plant Cell Tissue and Organ Culture 112: 361-368.
53. Ng C.Y, Saleh N.M. 2011. In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies. Plant Cell Tissue and Organ Culture 105: 193-202.
54. Nazari F, Khosh-Khui M, Azadi P. 2016. A simple and efficient direct shoot organogenesis method using leafy petiole explants in Gerbera jamesonii ‘Royal Soft Pink’. International Journal of Horticultural Science and Technology 3(1): 51-58.
55. Pant B, Thapa D. 2012. In vitro mass propagation of an epiphytic orchid, Dendrobium primulinum lindl through shoot tip culture. African Journal of Biotechnology 11: 9970-9974.
56. Park S.Y, Murthy H.N, Paek K.Y. 2002. Rapid propagation of Phalaenopsis from floral stalk- derived leaves. In Vitro Cellular and Developmental Biology Plant 38: 168-172.
57. Park S.Y, Yeung E.C, Paek K.Y. 2010. Endoreduplication in Phalaenopsis is affected by light quality from light-emitting diodes during somatic embryogenesis. Plant Biotechnology Reports 4: 303-309.
58. Raji R, Lotfi M, Tohidfar M, Zahedi B, Carra A, Abbate L, Carimi F. 2018. Somatic embryogenesis of muskmelon (Cucumis melo) and genetic stability assessment of regenerants using flow cytometry and ISSR markers. Protoplasma 255: 873-883.
59. Rodríguez-Sahagún A, Acevedo-Hernández G, Rodríguez-Domínguez JM, Rodríguez-Garay B,
50 Int. J. Hort. Sci. Technol; Vol. 8, No. 1; March 2021
Cervantes-Martínez J, Castellanos-Hernández O.A. 2011. Effect of light quality and culture medium on somatic embryogenesis of Agave tequilana. Plant Cell Tissue and Organ Culture 104: 271-275.
60. Rojas-Herrera R, Loyola-Vargas V. 2002. Induction of a class acidic chitinase in foliar explants of (Coffea arabica L) during somatic embryogenesis and wounding. Plant Science 163: 705-711.
61. Samarfard S, Kadir MA, Kadzimin SB, Ravanfar S. 2013. Genetic stability of in vitro multiplied Phalaenopsis gigantea protocorm-like bodies as affected by chitosan. Notulae Botanicae Horti Agrobotanici Cluj Napoca 41: 177-183.
62. Santarem E.R, Pelissier B, Finer J.J. 1997. Effect of explant orientation, Ph, solidifying agent and wounding on initiation of soybean somatic embryos. In Vitro Cellular and Developmental Biology Plant 33: 13-19.
63. Shinomura T, Nagatani A, Hanzawa H, Kubota M, Watanabe M, Furuya M. 1996. Action for spectra phytochrome and specific photoinduction of seed germination in Arabidopsis thaliana. Proceedings of the National Academy of Sciences 93: 8129-8133.
64. Siragusa M, Carra A, Salvia L, Puglia AM, De-Pasquale F, Carimi F. 2007. Genetic instability in calamondin (Citrus madurensis lour) plants derived from somatic embryogenesis induced by
diphenylurea derivatives. Plant Cell Reports 26: 1289-1296.
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2. Aliniaeifard S, Seif M, Arab M, Zare Mehrjerdi M, Li T, Lastochkina O. 2018. Growth and photosynthetic performance of Calendula officinalis under monochromatic red light.
International Journal of Horticultural Science and Technology 5(1):123-32.
3. Al-Khayri J. 2010. Somatic embryogenesis of date palm (phoenix dactylifera) improved by coconut water. Biotechnology 9(4): 477-484.
4. Arditti J. 2009. Micropropagation of orchids. John Wiley and Sons.
5. Ascencio-Cabral A, Gutierrez-Pulido H, Rodriguez-Garay B, Gutierrez-Mora A. 2008. Plant regeneration of Carica papaya through somatic embryogenesis in response to light quality, gelling agent and phloridzin. Scientia Horticulturae 118: 155-160.
6. Bakhshaie M, Babalar M, Mirmasoumi M, Khalighi A. 2010. Somatic embryogenesis and plant regeneration of (Lilium Ledebourii) boiss an endangered species. Plant Cell Tissue and Organ Culture 102: 229-235.
7. Bairu M.W, Aremu A.O, Staden J. 2011. Somaclonal variation in plants causes and detection methods. Plant Growth Regulation 63: 147-173.
8. Banerjee R, Schleicher E, Meier S, Viana RM, Pokorny R, Ahmad M, Bittl R, Batschauer A. 2007. The signaling state of arabidopsis cryptochrome 2 contains flavin semiquinone. Journal of Biological Chemistry 282: 14916-14922.
9. Bayat L, Arab M, Aliniaeifard S, Seif M, Lastochkina O, Li T. 2018. Effects of growth under different light spectra on the subsequent high light tolerance in rose plants. AoB PLANTS 10, ply052.
10. Bhattachryya P, Kumaria S, Diengdoh R, Tandon P. 2014. Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile an endangered medicinal orchid. Meta gene 2: 489-504.
11. Bhatia P, Ashwath N, Midmore D.J. 2005. Effects of genotype, explant orientation, and wounding on shoot regeneration in tomato. In Vitro Cellular and Developmental Biology Plant 41: 457-464.
12. Burritt D.J, Leung D.W. 2003. Adventitious shoot regeneration from begonia × erythrophylla petiole sections is developmentally sensitive to light quality. Physiologia plantarum 118: 289-296.
13. Chan A. 2013. Light induction of somatic embryogenesis and dark germination of Arabidopsis thaliana, MSc Thesis, Monitoba University.
48 Int. J. Hort. Sci. Technol; Vol. 8, No. 1; March 2021
14. Chen J.T, Chang W.C. 2001. Effects of auxins and cytokinins on direct somatic embryogenesison leaf explants of oncidium 'gower ramsey’. Plant Growth Regulation 34: 229-232.
15. Chen J.T, Chang W.C. 2004. Tiba affects the induction of direct somatic embryogenesis from leaf explants of oncidium. Plant Cell Tissue and Organ Culture 79: 315-320.
16. Chen J, Chang W.C. 2006. Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biologia Plantarum 50: 169-173.
17. Chen J.R, Lian Wu, Bo-Wen Hu, Xing Y, Rong L, Zi-Niu D, Xing-Yao X. 2014. The influence of plant growth regulators and light quality on somatic embryogenesis in China rose (Rosa chinensis Jacq). Journal of Plant Growth Regulation 33: 295-304.
18. Chung J.P, Huang C.Y, Dai TE. 2010. Spectral effects on embryogenesis and plantlet growth of oncidium ‘gower ramsey’. Scientia Horticulturae 124: 511-516.
19. Chung H, Chen J, Chang W. 2007. Plant regeneration through direct somatic embryogenesis from leaf explants of dendrobium. Biologia Plantarum 51: 346-350.
20. Deo P.C, Tyagi A.P, Taylor M, Harding B. 2010. Factors affecting somatic embryogenesis and transformation in modern plant breeding. The South Pacific Journal of Natural and Applied Sciences 28: 27-40.
21. D'onofrio C, Morini S, Bellocchi G. 1998. Effect of light quality on somatic embryogenesis of quince leaves. Plant Cell Tissue and Organ Culture 53: 91-98.
22. Feher A, Pasternak T.P, Dudits D. 2003. Transition of somatic plant cells to an embryogenic state. Plant Cell Tissue and Organ Culture 74: 201-228.
23. Feng J.H, Chen J.T. 2014. A novel in vitro protocol for inducing direct somatic embryogenesis in Phalaenopsis aphrodite without taking explants. The Scientific World Journal 131: 855-862.
24. Finer JJ. 1995. Direct somatic embryogenesis. Plant Cell Tissue and Organ Culture 72: 91-102.
25. Gow W.P, Chen J.T, Chang W.C. 2009. Effects of genotype, light regime, explant position and orientation on direct somatic embryogenesis from leaf explants of phalaenopsis orchids. Acta Physiologiae Plantarum 31: 330-363.
26. Gow W.P, Chen J.T, Chang W.C. 2010. Enhancement of direct somatic embryogenesis and plantlet growth from leaf explants of phalaenopsis by adjusting culture period and explant length. Acta Physiologiae Plantarum 32: 621-627.
27. Grzyb M, Kalandyk A, Mikuła A. 2018. Effect of tiba, fluridone and salicylic acid on somatic embryogenesis and endogenous hormone and sugar contents in the tree fern Cyathea Delgadii sternb. Acta Physiologiae Plantarum 40: 1-9.
28. Guo W.L, Chang Y.C.A, Kao C.Y. 2010. Protocorm-like bodies initiation from root tips of Cyrtopodium paranaense (orchidaceae). Hortscience 45: 1365-1368.
29. Harini I, Sita G.L. 1993. Direct somatic embryogenesis and plant regeneration from immature embryos of chilli (Capsicum annuum). Plant Science 89: 107-112.
30. Hartmann K.M. 1967. Phytochrome 730 (pfr), the effector of the “high energy photomorphogenic reaction” in the far-red region. Naturwissenschaften 54: 544-544.
31. Hasebe M, Iwatsuki K. 1990. Adiantum capillus-veneris chloroplast DNA clone bank As useful heterologous probes in the systematics of the leptosporangiate ferns. American Fern Journal 80: 20-25.
32. Hosseini A, Zare Mehrjerdi M, Aliniaeifard S, Seif M. 2019. Photosynthetic and growth responses of green and purple basil plants under different spectral compositions. Physiology and Molecular Biology of Plants 25, 741-752.
33. Huan L.V.T, Tanaka M. 2004. Callus induction from protocorm-like body segments and plant regeneration in Cymbidium (Orchidaceae). The Journal of Horticulture Science and Biotechnology 79: 406-410.
34. Huang Y, Tsai Y, Chen F. 2014. Characterization and expression analysis of somatic embryogenesis receptor-like kinase genes from Phalaenopsis. Genetics and Molecular Reserch 13: 10690-10703.
35. Husaini A.M, Abdin M. 2007. Interactive effect of light, temperature and TDZ on the regeneration potential of leaf discs of Fragaria x ananassa duch. In Vitro Cellular and Developmental Biology Plant 43: 576-584.
36. Khoddamzadeh A.A, Sinniah U, Kadir M, Kadzimin S, Mahmood M, Sreeramanan S. 2011. In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf
Efficient Method for Direct Embryogenesis in Phalaenopsis Orchid 49
segments of Phalaenopsis bellina Christenson. Plant Growth Regulation 65: 381-390.
37. Kraepiel Y, Miginiac E. 1997. Photomorphogenesis and phyfohormones. Plant Cell and Environment 20: 807-812.
38. Kuo H.L, Chen J.T, Chang W.C. 2005. Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis ‘little steve’. In Vitro Cellular and Developmental Biology Plant 41: 453-460.
39. Li H, Tang C, Xu Z. 2013. The effects of different light qualities on rapeseed (Brassica Napus l.) plantlet growth and morphogenesis in vitro. Scientia Horticulturae 150: 117-124.
40. Ludevid M.D, Ruiz-Avila L, Valles M.P, Stiefel V, Torrent M, Torne JM, Puigdomenech P. 1990. Expression of genes for cell-wall proteins in dividing and wounded tissues of (Zea mays L). Planta 180: 524-530.
41. Mahendran G, Bai V.N. 2012. Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor. Scientia Horticulturae 135: 40-44.
42. Mayer J.L, Stancato G.C, Appezzato B. 2010. Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of oncidium flexuosum sims (orchidaceae). Plant Cell Tissue and Organ Culture 103: 411-416.
43. Meilasari D, Iriawati I. 2016. Regeneration of Plantlets Through PLB Formation in Phalaenopsis ‘Join Angle X Sogo Musadian’. Journal of Mathematical and Fundamental Sciences 48: 204 212.
44. Mengxi L, Zhigang X, Yang Y, Yijie F. 2011. Effects of different spectral lights on oncidium PLB induction, proliferation, and plant regeneration. Plant Cell Tissue and Organ
Culture 106: 1-10.
45. Michler C.H, Lineberger R.D. 1987. Effects of light on somatic embryo development and abscisic levels in carrot suspension cultures. Plant Cell Tissue and Organ Culture 11: 189-207.
46. Moradi S, Dianati Daylami S, Arab M, Vahdati K. 2017. Direct somatic embryogenesis in Epipactis veratrifolia a temperate terrestrial orchid. The Journal of Horticulture Science and Biotechnology 92: 88-97. 47. Moghare Abed S, Naderi D. 2020. Stimuli Effects of Different LEDs on Some Morphological and Biochemical Traits of Two Varieties of Calendula officinalis. International Journal of Horticultural Science and Technology 7(2):139-151.
48. Morel GM. 1960. Producing virus-free cymbidium. American Orchid Society Bulletin 98: 202-2011.
49. Mose W, Indrianto A, Purwantoro A, Semiarti E. 2017. The influence of thidiazuron on direct somatic embryo formation from various types of explant in Phalaenopsis amabilis blume orchid. Hayati Journal of Biosciences 24: 201-205.
50. Mulgund G.S, Nataraja K, Malabadi R.B, Kumar S.V. 2011. TDZ induced in vitro propagation of an epiphytic orchid xenikophyton smeeanum. Reserch in Plant Biology 68: 312-320.
51. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.
52. Naing A.H, Kim C.K, Yun B.J, Jin J.Y, Lim K.B. 2013. Primary and secondary somatic embryogenesis in chrysanthemum. Plant Cell Tissue and Organ Culture 112: 361-368.
53. Ng C.Y, Saleh N.M. 2011. In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies. Plant Cell Tissue and Organ Culture 105: 193-202.
54. Nazari F, Khosh-Khui M, Azadi P. 2016. A simple and efficient direct shoot organogenesis method using leafy petiole explants in Gerbera jamesonii ‘Royal Soft Pink’. International Journal of Horticultural Science and Technology 3(1): 51-58.
55. Pant B, Thapa D. 2012. In vitro mass propagation of an epiphytic orchid, Dendrobium primulinum lindl through shoot tip culture. African Journal of Biotechnology 11: 9970-9974.
56. Park S.Y, Murthy H.N, Paek K.Y. 2002. Rapid propagation of Phalaenopsis from floral stalk- derived leaves. In Vitro Cellular and Developmental Biology Plant 38: 168-172.
57. Park S.Y, Yeung E.C, Paek K.Y. 2010. Endoreduplication in Phalaenopsis is affected by light quality from light-emitting diodes during somatic embryogenesis. Plant Biotechnology Reports 4: 303-309.
58. Raji R, Lotfi M, Tohidfar M, Zahedi B, Carra A, Abbate L, Carimi F. 2018. Somatic embryogenesis of muskmelon (Cucumis melo) and genetic stability assessment of regenerants using flow cytometry and ISSR markers. Protoplasma 255: 873-883.
59. Rodríguez-Sahagún A, Acevedo-Hernández G, Rodríguez-Domínguez JM, Rodríguez-Garay B,
50 Int. J. Hort. Sci. Technol; Vol. 8, No. 1; March 2021
Cervantes-Martínez J, Castellanos-Hernández O.A. 2011. Effect of light quality and culture medium on somatic embryogenesis of Agave tequilana. Plant Cell Tissue and Organ Culture 104: 271-275.
60. Rojas-Herrera R, Loyola-Vargas V. 2002. Induction of a class acidic chitinase in foliar explants of (Coffea arabica L) during somatic embryogenesis and wounding. Plant Science 163: 705-711.
61. Samarfard S, Kadir MA, Kadzimin SB, Ravanfar S. 2013. Genetic stability of in vitro multiplied Phalaenopsis gigantea protocorm-like bodies as affected by chitosan. Notulae Botanicae Horti Agrobotanici Cluj Napoca 41: 177-183.
62. Santarem E.R, Pelissier B, Finer J.J. 1997. Effect of explant orientation, Ph, solidifying agent and wounding on initiation of soybean somatic embryos. In Vitro Cellular and Developmental Biology Plant 33: 13-19.
63. Shinomura T, Nagatani A, Hanzawa H, Kubota M, Watanabe M, Furuya M. 1996. Action for spectra phytochrome and specific photoinduction of seed germination in Arabidopsis thaliana. Proceedings of the National Academy of Sciences 93: 8129-8133.
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