Efficient Protocol for Protoplast Isolation and Plant Regeneration of Fritillaria imperialis L.

Authors
E. Chamani
S. K. Tahami
Department of Horticultural Science, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil, Islamic Republic of Iran.
Abstract
The present study reports an efficient protocol for isolation and regeneration of protoplasts from callus of Fritillaria imperialis L. There is no published method recommended for protoplast isolation and regeneration from Fritillaria imperialis L. A range of factors, which influence the success of isolation and regeneration of F. imperialis protoplasts, were investigated. From the results obtained, callus Fresh Weight (FW) of 0.4 g produced the highest number of viable protoplasts, which was 1.12×105 protoplasts g-1 FW. The highest amount of viable protoplasts (1.01×105 protoplasts g-1 FW) was obtained when the mannitol concentration was maintained at 9% (w/v). The best treatment for isolation of F. imperialis protoplast (1.37×105 protoplasts g-1 FW) was treatment with 2% cellulase and 0.1% pectinase with 9% mannitol for 8 h. For enhancement of the protoplasts division and the percentage of colony formation, different concentrations from Casein Hydrolysate (CH), 2,4-Dichlorophenoxyacetic acid (2,4-D) and Benzyl-Adenine (BA) were used. The results revealed that cell wall and colony formation were better in liquid medium than those on semi-solid medium. The highest plating efficiency (1.26×106 per g FW) and highest callus formation were obtained using the medium containing 0.5 mg L–1 2,4-D, 1 mg L–1 BA, and 200 mg L–1 CH. Micro-calli were formed after one month of culture. Many plantlets were formed on the calli after transfer of the proliferated calli to regeneration medium. The highest plantlet regeneration (100%) was obtained using the medium containing 0.5 mg L–1 (NaphthaleneAcetic Acid) NAA and 1.5 mg L–1 BA.

Keywords

1. Arnalte, E., Perez, P., Segura, J. and Cornejo, M.1991. Protoplast Isolation from Digitalis obscura Microspores. Physiologia Plant, 1: 182:1.
2. Assani, A., Haicour, R., Wenzel, G., Cote, F., Bakry, F., Foroughi-Wehr, B., Ducreux, G., Aguillar, M. E. and Grapin, A.2001. Plant Regeneration from Protoplasts of Dessert Banana cv. Grande Naine (Musa spp., Cavendish Sub-group AAA) via Somatic Embryogenesis. Plant Cell Rep., 20: 482-488.
3. Assani, A., Haïcour, R., Wenzel, G., Foroughi-Wehr, B., Bakry, F. and Côte, F. X. 2002. Influence of Donor Material and Genotype on Protoplast Regeneration in Banana and Plantain Cultivars (Musa spp.). Plant Sci., 162: 355–362.
4. Azad, M., Yokota, S., Ishiguri, F. and Yoshizawa, N. 2006. Plant Regeneration from Mesophyll Protoplasts of a Medicinal Plant, Phellodendron amurense Rupr. In Vitro Cellular Dev. Biol. Plant., 42(6): 502-507.
5. Bodansky, O. 1954. Relationship of Enzyme Concentration to Substrate Change Derived from Time-Course of Reaction. J. Biol. Chem., 209(1): 281-284.
6. Chabane, D., Assani, A., Bouguedoura, N., Haïcour, R. and Ducreux, G. 2007. Induction of Callus Formation from Difficile Date Palm Protoplasts by Means of Nurse Culture. C. R. Biologie., 330: 392–401.
7. Chamani, E., Tahami, S. K., Zare, N., Asghari-Zakaria, R., Mohebodini, M. and Joyce, D. 2012. Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss. Not. Bot. Horti. Agrobo., 40(2): 123-128.
8. Chang, C., Tsai, Y. and Wei-Chin, C.1999. A Tissue Culture Protocol for Propagation of a Rare Plant, Lilium speciosum Thunb. var. Glorisoides Baker. Bot. Bull. Acad. Sinica, 41(2): 139-142.
9. Chen, L. J., Zhu, X. Y., Gu, L. and Wu, J. 2005. Efficient Callus Induction and Plant Regeneration from Anther of Chinese Narcissus (Narcissus tazetta L. var. Chinensis Roem). Plant Cell Rep., 24: 401-407.
10. Darvishi, E., Zarghami, R., Mishani, C. A., Omidi, M. and Sarkhosh, A. 2006. Investigation of the Best Time of Enzyme Treatment in Order to Isolate the Protoplast from Embryogenic Callus to Saffron (Crocus sativus L.) Biotechnol., 5(3): 284-286.
11. Davey, M. R., Anthony, P., Power, B. and Lowe, K. C. 2005. Plant protoplasts: Status and Biotechnological Perspectives, Biotechnol. Adv., 23: 131–171.
12. De Hertogh, A. and Le Nard, M. 1993. The Physiology of Flower Bulbs. Elsevier, Amsterdam.
13. Du, L. and Bao, M. Z. 2005. Plant Regeneration from Protoplasts Isolated from Embryogenic Suspension Cultured Cells of Cinnamomum camphora L. Plant Cell Rep., 24: 462-467.
14. Duquenne, B., Eeckhaut, T., Werbrouck, S. and Huylenbroeck, J. V. 2007. Effect of Enzyme Concentrations on Protoplast Isolation and Protoplast Culture of Spathiphyllum and Anthurium. Plant Cell Tiss. Org. Cult., 91(2): 165-173.
15. Dulieu, H. L., Bruneau, R. and Pelletier, A. 1983. Heritable Differences in In-vitro Regenerability in Petunia at the Protoplast and at the Seedling Stage. In: “Protoplasts1983", (Eds.): PotryLus, C. T., Harms, A., Hutter, R., King, P. J. and Shillito, R. D.. Birhauser, Basel, PP. 236-237.
16. Ebrahimie, E., Mohammadi-Dehcheshmeh, M. and Sardari, M. 2006a. Fritillaria Species Are at the Risk of Extinction in Iran: Study on Effective Factors and Necessity of International Attention. Hort. Sci., 41: 1002.
17. Fray, D. D and Wang, H. J. 2006. Adaptive One-factor-at-a-time Experimentation and Expected Value of Improvement. Technometrics, 48 (3): 418-431.
18. Hahne, B., Lorz, H. and Hahne, G. 1990. Oat Mesophyll Protoplasts: Their Response to Various Feeder Cultures. Plant Cell Rep., 8: 590–593.
19. Horita, M., Morohashi, H. and Komai, F. 2002. Regeneration from Flowering Plants from Difficile Lily Protoplasts by Means of a Nurse Culture. Planta, 215: 880–884.
20. Izhar, S. and Power, B. J. 1977. Genetical Studies with Petunia Leaf Protoplasts.1. Genetic Variation to Specific Growth Hormones and Possible Genetic Control on Stages of Protoplast Development in Culture. Plant Sci. Lett., 8: 375-383.
21. Jullien, F., Diemer, F., Colson, M. and Faure, O. 1998. An Optimizing Protocol Regeneration of Three Peppermint Cultivars (Mentha x piperita). Plant Cell Tiss. Org. Cult., 54(3): 153-159.
22. Karim, M. A. and Adachi, T. 1997. Cell Suspension, Isolation and Culture of Protoplasts of Allium cepa. Plant Cell Tissue Organo Culture, 51: 43-47.
23. Kanwar, K., Bhardwaj, A. and Deepika, R. 2009. Efficient Regeneration of Plantlets from Callus and Mesophyll Derived Protoplasts of Robinia pseudoacacia L. Plant Cell Tissue Tiss. Org. Cult., 96(1): 95-103.
24. Kashyap, H. V. 2001. Advanced Topic in Zoology. Orient Blackswam, United States of America.
25. Karp, G. 2005. Cell and Molecular Biology. John Wiley and Sons, United States of America.
26. Kisaka, H., Kisaka, M., Kanno, A. and Kameya, T. 1998. Intergeneric Somatic Hybridization of Rice (Oryza sativa L.) and Barley (Hordeum vulgare L.) by Protoplast Fusion. Plant Cell Rep., 17: 362-367.
27. Kremer, S. M. and Wood, P. M. 1992. Continuous Monitoring of Cellulose Action on Microcrystalline Cellulose. Appl. Microbiol. Biotechnol., 37(6): 750-755.
28. Lenting, H. B. M. and Warmoeskerken, M. M. C. G. 2001. Mechanism and Interaction between Cellulose Action and Applied Shear Force: A hypothesis. J. Biotechnol., 89(2): 217-226.
29. Linnaeus, C. 1753. Species Plantarum, Stockholm. pp. 303-304.
30. Marchant, R., Davey, M. R. and Power, J. B. 1997. Isolation and Culture of Mesophyll Protoplasts from Rosa hybrida. Plant Cell Tiss. Org. Cult., 50: 131–134.
31. Murashige, T. and Skoog, F. 1962. A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures. Plant Physiol., 15: 473-479.
32. Naik, P. K. and Nayak, S. 2005. Different Modes of Plant Regeneration and Factors Affecting In vitro Bulblet Production in Ornithogalum virens. Sci. Asia, 31: 409-414.
33. Nagata, T. and Takede, H. 1984. Isolation and Culture of Protoplast Tobacco, In: “Cell Culture and Somatic Cell Genetic of Plants”, (Ed.): Vasil, L.. Academic Press, New York, London, PP. 328-337.
34. Nelsestuen, G. L. and Martinez, M. B. 1999. Steady State Enzyme Velocities that Are Independent of Enzyme Concentration: An Important Behavior in Many Membrane and Particle-bound States. Biochem., 36(30): 9081-9086.
35. Ochatt, S. T. and Power, B. J. 1988a. Plant Regeneration from Mesophyl Protoplasts of Wiliams Bon Chretien (Syn. Bartlett Pear, Pyrus communis L.). Plant Cell Rep., 7: 587-589.
36. Ochatt, S. T. and Power, B. J. 1988b. Rhizogenesis in Callus from Conference Pear (Pyrus communis L.). Protoplasts. Plant Cell Tiss. Org. Cult., 13: 159-164.
37. Oh, M. H. and Kim, S. G. 1994. Plant Regeneration from Petal Protoplast Culture of Petunia hybrida. Plant Cell Tiss. Org. Cult., 36: 275-283.
38. Pierik, R. L. M. 1998. In vitro Culture of Higher Plants. Ferdowsi University Press, Mashhad, 406 PP.
39. Pongchawee, K., Na-nakorn, U., Lamseejan, S., Poompuang, S. and Phansiri, S. 2006. Factors Affecting the Protoplast Isolation and Culture of Anabias nana Engler. T. O. Bot., 2: 193-200.
40. Raikar, S. V., Braun, R. H., Bryant, C., Conner, A. J. and Christey, M. C. 2008. Efficient Isolation, Culture and Regeneration of Lotus corniculatus Protoplasts. Plant Biotechnol. Rep., 2(3): 171-177.
41. Rastogi, S. C. 2003. Cell and Molecular Biology. New Age International, United States of America.
42. Rao, K. S. and Prakash, A. H. 1995. A Simple Method for the Isolation of Plant Protoplasts. J. Biosci., 20(5): 645-655.
43. Rix, E. M. 2001. Fritillaria: A Revised Classification. The Fritillaria Group of the Alpine Garden Society, Edinburgh, United Kingdom.
44. Royal Society of Chemistry. 2005. Enzymes. Retrieved 16 August, 2009, from http://www.rsc.org/education/teachers/learnnet/cfb/enzymes.htm.
45. Saker, S. S., Neuman, K. H., Badawy, E. M., EL-Bahr, M. K. and Taha, H. S. 1999. Isolation and Culturing of Protoplasts from Hypericum perforatum L. Arab J. Biotech., 2: 227-234.
46. Saxena, P. K. and Gill, R. 1986. Removal of Browning and Growth Enhancement by Polyvinylpolypyrrolidone in Protoplast Cultures of Cyamopsis tetragonoloba L. Biol. Plant, 28: 313–315.
47. Sinha, A., Wetten, A. C. and Caligarim, P. D. S. 2003. Effect of Biotic Factors on the Isolation of Lupinus albus Protoplasts. Australian J. Bot., 51(1): 103- 109.
48. Tamura, M., Tao, R. and Akira, S. 1993. Improved Protoplast Culture and Plant Regeneration of Japanese Persimmon (Diospyrous Kaki L.). J. Breed., 43: 239-245.
49. Te-Chato, S. 1997. Isolation and Culture of Protoplast of Somkhag (Garcinia atroviridis Griff.) to Microcolony. Songklanakarin J. Sci. Technol., 19: 255-262.
50. Tian, D. and Rose, R. J. 1999. Asymmetric Somatic Hybridisation between the Annual Legumes Medicago truncatula and Medicago scutellata. Plant Cell Rep., 18: 989–96.
51. Umate, P., Rao, K. V., Kiranmayee, K., Sree, T. J. and Sadanandam, A. 2005. Plant Regeneration of Mulberry (Morus indica) from Mesophyll-derived Protoplasts. Plant Cell Tiss. Org. Cult., 82(3): 289-293.
Journal of Agricultural Science and Technology
Volume 18, Issue 2
March and April 2016
Pages 467-482