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Embling Production in Althaea officinalis L., Through Somatic Embryogenesis and Their Appraisal via Histological and Scanning Electron Microscopical Studies

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Abstract

In vitro propagation of a medicinally important plant, Althaea officinalis, has been achieved through somatic embryogenesis. Somatic embryos (globular to torpedo-shaped embryos) were induced on Murashige and Skoog’s (MS) medium augmented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D, 5.0, 10.0, 15.0, 20.0, and 25.0) alone or combined with N6-benzylaminopurine (BA, 0.1, 0.5, 1.0, 1.5, and 2.0 μM). These were directly formed from the cut ends and subsequently spread on the whole surface of internodal explants. For embryo maturation, torpedo embryos were transferred on a medium containing different levels of BA (0.1, 0.5, or 1.0 μM) and abscisic acid (ABA) (0.5, 1.0, or 1.5 μM) or α-naphthalene acetic acid (NAA) (0.1, 0.5 or 1.0 μM). Among the different concentrations tested, 0.5 μM BA along with 1.0 μM ABA was found most effective, on which a highest yield (58.0%) with an optimum number (35.0) of mature embryos (cotyledonary stage) was observed after 2 weeks of transfer. Germination of cotyledonary embryos into plantlets with 68% were observed on ½ MS medium. Histological and scanning electron microscopical (SEM) studies proved that the regenerated structures were somatic embryos and not shoot primordia. Plants grew vigorously when transferred to a greenhouse.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxy acetic acid

ABA:

Abscisic acid

BA:

6-Benzyladenine

MS:

Murashige and Skoog (1962)

NAA:

α-Naphthalene acetic acid

SE:

Somatic embryo

References

  1. Anonymous. (2003). The wealth of India. A dictionary of Indian raw materials and industrial products (Vol. 1, pp. 207–208). New Delhi: Council of Scientific and Industrial Research.

    Google Scholar 

  2. Sutovska, M., Nosalova, G., Sutovsky, J., Franova, S., Prisenznakova, L., & Capek, P. (2009). Possible mechanisms of dose-dependent cough suppressive effect of Althaea officinalis rhamnogalacturonan in guinea pigs test system. International Journal of Biological Macromolecules., 45, 27–32.

    Article  CAS  Google Scholar 

  3. Hage-Sleiman, R., Mroueh, M., & Daher, C. R. (2011). Pharmacological evaluation of aqueous extract of Althaea officinalis flower grown in Lebanon. Pharmaceutical Biology, 49, 327–333.

    Article  Google Scholar 

  4. Blumenthal, M., Goldberg, A., & Brinckmann, J. (2000). Herbal medicine: expanded commission E monographs (pp. 244–248). Austin: American Botanical Council.

    Google Scholar 

  5. Naz, R., & Anis, M. (2012). Acceleration of adventitious shoots by interaction between exogenous hormone and adenine sulphate in Althaea officinalis L. Applied Biochemistry and Biotechnology, 168, 1239–1255.

    Article  CAS  Google Scholar 

  6. Naz, R., Anis, M., & Aref, I. M. (2015). Management of cytokinin–auxin interactions for in vitro shoot proliferation of Althaea officinalis L.: a valuable medicinal plant. Rend Fis Acc Lincei, 26, 323–334.

    Article  Google Scholar 

  7. Morel, A., Trontin, J. F., Corbineau, F., Lomenech, A. M., Beaufour, M., Reymond, I., et al. (2014). Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses. Planta, 240, 1075–1095.

    Article  CAS  Google Scholar 

  8. Merkle, S. A., Bailey, R. L., Pauley, B. A., Neu, K. A., Kim, M. K., Rugu, C. L., & Montello, P. M. (1997). Somatic embryogenesis from tissues of mature sweetgum trees. Canadian Journal of Forest Research, 27, 959–964.

    Article  Google Scholar 

  9. Pedroso, C. M., & Pais, M. S. (1995). Factors controlling somatic embryogenesis. Plant Cell, Tissue and Organ Culture, 43, 147–154.

    Article  Google Scholar 

  10. Chen, J. T., & Chang, W. C. (2006). Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis ambilis. Biologia Plantarum, 50, 169–173.

    Article  Google Scholar 

  11. You, X. L., Han, J. Y., & Choi, Y. E. (2007). Plant regeneration via direct somatic embryogenesis in Panax japonicas. Plant Biotechnological Reports, 1, 5–9.

    Article  Google Scholar 

  12. Mahendran, G. V., & Bai, V. N. (2012). Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor Lindl. Scientia Horticulturae, 135, 40–44.

    Article  CAS  Google Scholar 

  13. Dhir, R., Shekhawat, G. S., & Alam, A. (2014). Improved protocol for somatic embryogenesis and calcium alginate encapsulation in Anethum graveolens L.: a medicinal herb. Applied Biochemistry and Biotechnology, 173, 2267–2278.

    Article  CAS  Google Scholar 

  14. Singh, R., Raj, M. K., & Kumari, N. (2015). Somatic embryogenesis and plant regeneration in Sapindus mukorossi Gaertn. from leaf-derived callus induced with 6-benzylaminopurine. Applied Biochemistry and Biotechnology, 177, 498–510.

    Article  CAS  Google Scholar 

  15. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays for tobacco tissue cultures. Physiologiae Plantarum, 15, 473–497.

    Article  CAS  Google Scholar 

  16. Johansen, D. A. (1940). Plant microtechnique. New York: Mc Graw-Hill Book Co. Inc..

    Google Scholar 

  17. Vasil, V., & Vasil, I. K. (1984). Preparation of cultured tissues for scanning electron microscopy. In I. K. Vasil (Ed.), Cell culture and somatic cell genetics of plants (Vol. I, pp. 738–743). Orlando: Academic Press.

    Google Scholar 

  18. Kanita, A., & Kothari, S. I. (2002). High efficiency adventitious shoot bud formation and plant regeneration from leaf explants of Dianthus chinensis L. Scientia Horticulturae, 96, 205–212.

    Article  Google Scholar 

  19. Nishiwaki, M., Fujino, K., Koda, Y., Masuda, K., & Kikuta, Y. (2000). Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture. Planta, 211, 756–759.

    Article  CAS  Google Scholar 

  20. Feher, A. (2005). Why somatic plant cells startto form embryos? In A. Mujib & J. Samaj (Eds.), Somatic embryogenesis (pp. 85–101). Berlin, Heidelberg: Springer.

    Google Scholar 

  21. Ivanova, A., Velcheva, M., Denchev, P., Atanassov, A., & Van Onckelen, H. (1994). Endogenous hormone levels during direct somatic embryogenesis in Medicago falcata. Physiologiae Plantarum, 92, 85–89.

    Article  CAS  Google Scholar 

  22. Guilfoyle, T. J., & Hagen, G. (2007). Auxin response factors. Current Opinion in Plant Biology, 10, 453–460.

    Article  CAS  Google Scholar 

  23. Tan, X., Calderon-Villalobos, L. I. A., Sharon, M., Zheng, C. X., Robinson, C. V., Estelle, M., & Zheng, N. (2007). Mechanism of perception by the TIR1 ubiquitin ligase. Nature, 446, 640–645.

    Article  CAS  Google Scholar 

  24. Prange, A. N. S., Serek, M., Bartsch, M., & Winkelmann, T. (2010). Efficient and stable regeneration from protoplasts of Cyclamen coum Miller via somatic embryogenesis. Plant Cell Tissue and Organ Culture, 101, 171–182.

    Article  Google Scholar 

  25. Chai, M., Jia, Y., Chen, S., Gao, Z., Wang, H., Liu, L., Wang, P., & Hou, D. (2011). Callus induction, plant regeneration, and long-term maintenance of embryogenic cultures in Zoysia matrella [L.] Merr. Plant Cell Tissue and Organ Culture, 104, 187–192.

    Article  CAS  Google Scholar 

  26. Azad, M. A. K., Yokota, S., Begum, F., & Yoshizawa, N. (2009). Plant regeneration through somatic embryogenesis of a medicinal plant, Phellodendron amurense Rupr. In Vitro Cellular Developmental Biology–Plant, 45, 441–449.

    Article  Google Scholar 

  27. Zimmerman, J. L. (1993). Somatic embryogenesis: a model for early development in higher plants. Plant Cell, 5, 1411–1423.

    Article  Google Scholar 

  28. Wakhlu, A. K., & Sharma, R. K. (1998). Somatic embryogenesis and plant regeneration in Heracleum candicans wall. Plant Cell Reports, 7, 866–869.

    Article  Google Scholar 

  29. Raemakers, C. J. J. M., Jacobsen, E., & Visser, R. G. F. (1995). Secondary somatic embryogenesis and application in plant breeding. Euphytica, 81, 93–107.

    Article  Google Scholar 

  30. Scholten, H. J., & Pierik, R. L. M. (1998). Agar as a gelling agent: chemical and physical analysis. Plant Cell Reports, 17, 230–235.

    Article  CAS  Google Scholar 

  31. Mujib, A., Ilah, A., Aslam, J., Fatima, S., Siddiqui, Z. H., & Maqsood, M. (2012). Catharanthus roseus alkaloids: application of biotechnology for improving yield. Plant Growth Reg., 68(2), 111–127.

    Article  CAS  Google Scholar 

  32. Elhag, H., El-Olemy, M. M., & Al-Said, M. S. (2004). Enhancement of somatic embryogenesis and production of developmentally arrested embryo in Nigella sativa L. Horticultural Science, 39, 321–323.

    CAS  Google Scholar 

  33. Nhut, D. T., Hanh, N. T. M., Tuan, P. Q., Nguyet, L. T. M., Tram, N. T. H., Chinh, N. C., Nguyen, N. H., & Vinh, N. D. (2006). Liquid culture as a positive condition to induce and enhance quality and quantity of somatic embryogenesis of Lilium longiflorum. Scientia Horticulturae, 110, 93–97.

    Article  Google Scholar 

  34. Ahmed, A. B. A., Rao, A. S., & Rao, M. V. (2009). Somatic embryogenesis and plant regeneration from cell suspension culture of Gymnema sylvestre (Retz) R. Br. Ex Roemer & Schultes. KMITL Science Technology Journal, 9, 18–26.

    Google Scholar 

  35. Davies, W. J., & Jones, H. G. (1991). Abscisic acid: physiology and biochemistry (pp. 39–52). Oxford: Bios.

    Google Scholar 

  36. Senger, S., Mokc, H. P., Conrad, U., & Manteuffel, R. (2001). Immunomodulation of ABA function affects early events in somatic embryo development. Plant Cell Reports, 20, 112–120.

    Article  CAS  Google Scholar 

  37. Ghanti, S. K., Sujata, K. G., Rao, M. S., & Kishor, P. V. K. (2010). Direct somatic embryogenesis and plant regeneration from immature explants of chickpea. Biologia Plantarum, 54, 121–125.

    Article  Google Scholar 

  38. Devi, C. B., & Narmathabai, V. (2011). Somatic embryogenesis in the medicinal legume Desmodium motorium (Houtt.) Merr. Plant Cell Tissue and Organ Culture, 106, 409–418.

    Article  Google Scholar 

  39. Sahrawat, A. K., & Chand, S. (2001). Continuous somatic embryogenesis and plant regeneration from hypocotyls segments of Psoralea corylifolia L., an endangered and medicinally important fabaceae plant. Current Science, 81, 1328–1331.

    CAS  Google Scholar 

  40. Agrawal, V., & Sardar, P. R. (2007). In vitro regeneration through somatic embryogenesis and organogenesis using cotyledons of Cassia angustifolia Vahl. In Vitro Cellular Developmental Biology–Plant, 43, 585–592.

    Article  CAS  Google Scholar 

  41. He, T., Yang, L., & Zhao, Z. (2011). Embryogenesis of Gentiana straminea and assessment of genetic stability of regenerated plants using inter simple sequence repeat (ISSR) marker. African Journal of Biotechnology, 10, 7604–7610.

    CAS  Google Scholar 

  42. Anbazhagan, V. R., & Ganapathi, A. (1999). Somatic embryogenesis in cell suspension cultures of pigeonpea (Cajanus cajan). Plant Cell Tissue and Organ Culture, 56, 179–184.

    Article  Google Scholar 

  43. Rout, G. R., & Nanda, R. M. (2005). Protocol of somatic embryogenesis in Acacia Arabica (Lamk) Willd. In S. Jain & P. Gupta (Eds.), Protocol for somatic embyogenesis in woody plants (pp. 401–412). Netherlands: Springer.

    Chapter  Google Scholar 

  44. Chalupa, V. (2005). Protocol of somatic embryogenesis: penunculate oak (Quercus robur L) and sessile oak (Quercus petraea/Matt./Liebl.). In P. Gupta & S. Jain (Eds.), Protocol for somatic embryogenesis in woody plants (pp. 369–378). Netherlands: Springer.

    Chapter  Google Scholar 

  45. Choi, Y. E., Kim, J. W., & Yoon, E. S. (1999). High frequency of plant production via somatic embryogenesis from callus or cell suspension cultures in Eleutherococcus senticosus. Annals of Botany, 83, 309–314.

    Article  CAS  Google Scholar 

  46. Samaj, J., Baluska, F., Bobak, M., & Volkmann, D. (1999). Extracellular matrix surface network of embryogenic units of friable maize callus contains arabinogalactan-proteins recognized by monoclonal antibody JIM4. Plant Cell Reports., 18, 369–374.

    Article  CAS  Google Scholar 

  47. Samaj, J., Bobak, M., Blehova, A., Kristin, J., & Auxtova Samajova, O. (1995). Developmental SEM observations on an extracellular matrix in embryogenic calli of Drosera rotundufolia and Zea mays. Protoplasma, 186, 45–49.

    Article  Google Scholar 

  48. Chapman, A., Blervacq, A. S., Tissier, J. P., Delbreil, B., Vasseur, J., & Hilbert, J. L. (2000a). Cell wall differentiation during early somatic embryogenesis in plants. I. Scanning and transmission electron microscopy study on embryos originating from direct, indirect, and adventitious pathways. Canadian Journal of Botany, 78, 816–823.

    Google Scholar 

  49. Paul, S., Dam, A., Bhattacharyya, A., & Bandyopadhyay, T. K. (2011). An efficient regeneration system via direct and indirect somatic embryogenesis for the medicinal tree Murraya koenigii. Plant Cell Tissue and Organ Culture, 105, 271–283.

    Article  Google Scholar 

  50. Bobak, M., Samaj, J., Pretova, A., Blehova, A., Hlinkova, E., Ovecka, M., Hlavacka, A., & Kutarnova, Z. (2004). The histological analysis of in direct somatic embryogenesis on Drosera spathulata Labill. Acta Physiologiae Plantarum, 26, 353–361.

    Article  Google Scholar 

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Acknowledgements

Research support from the Department of Science and Technology (DST) and the University Grants Commission (UGC), New Delhi, Government of India, under the DST-FIST-II (2012–2017) and UGC-DRS II (2016–2021) Programs, respectively, is greatly acknowledged. The authors extend their appreciation to the International Scientific Partnership Program (ISPP) at King Saud University for partial assistance. RN is grateful to the University Grants Commission, New Delhi, for the award of Dr. DS Kothari Postdoctoral Fellowship.

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Authors and Affiliations

  1. Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, 202 002, India

    Ruphi Naz & Mohammad Anis

  2. Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia

    Abdulrahman A. Alatar

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  1. Ruphi Naz
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  2. Mohammad Anis
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Correspondence to Mohammad Anis.

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Naz, R., Anis, M. & Alatar, A.A. Embling Production in Althaea officinalis L., Through Somatic Embryogenesis and Their Appraisal via Histological and Scanning Electron Microscopical Studies. Appl Biochem Biotechnol 182, 1182–1197 (2017). https://doi.org/10.1007/s12010-016-2391-2

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