Effect of photoperiod on somatic embryogenesis in different organs of chickpea (Cicer arietinum L.)

پژوهش‌های حبوبات ایران

Article 2, Volume 7, Issue 1, June 2016, Pages 25-36   

Document Type: Original Articles

DOI: 10.22067/ijpr.v7i1.34385

Authors

ALI AKBAR MOZAFARI^{* 1}; Kazhal Kamangar²

¹University of Kurdistan

²Islamic Azad University of Saveh, Saveh

Abstract

Introduction
Somatic embryogenesis is an efficient platform for the generation of transgenic plants and synthetic seeds (Kiran et al., 2005). Somatic embryo’s growth and development were influenced by different factors, including photoperiod, genotype as well as acidity, plant growth regulators (PGRs) and nutrient content of tissue culture media (May & Trigiano, 1991). Darkness is one of the important and vital affecting variables on somatic embryogenesis (Angoshtari et al., 2009). In some studies, positive effects of darkness on some plant species have been reported. This study was carried out to investigate the potential of somatic embryogenesis from leaf, hypocotyl and epicotyl explants of two chickpea cultivars (Piruz and Kaka) on basal Murashige and Skoog (MS) medium in darkness and light conditions.

Materials and Methods
In this study, hypocotyl, epicotyl and young leaf segments of two chickpea cultivars “Piruz” and “Kaka” were used as explant. Surface sterilization was performed by soaking chickpea seeds in 96% ethanol solution for 60 Seconds and immediate immersion in 2% sodium hypochlorite solution for 15 minutes. Following rinsing three times in sterile double-distilled water, the seeds were aseptically cultured on free-PGRs ½ MS medium (Murashige & Skoog, 1962). After 3-4 days, explants were excised from germinated seedlings and implanted on MS medium supplemented with different concentration of 2,4-D and NAA (2, 3, 4 and 5 mg/l) as well as different levels of TDZ and Picloram (1, 1.5, 2 and 2.5 mg/l) to the initiation of embryogenic callus. To produce globular embryos, 16 hormonal treatments were then incubated at 25±1 ºC under continuous darkness and photoperiod (16-h light and 8-h darkness) at a light intensity of 36 µmol m–2 s–1. In order to develop the embryogenesis, another three treatments were used under similar condition. For embryonic callus induction the following characteristics were studied: embryogenesis frequency, the frequency of globular, heart shape and cotyledonary embryos. Regenerated plantlets from mature embryos were washed thoroughly with sterile water and then transplanted to plug tray and fertilized with Hogland solution two times per week. Plants were transferred to the pot and kept under greenhouse condition. Plants were finally transferred to open-field condition. This study was conducted as the factorial design based on completely a randomized design with five replicate (jar) and four shoots per each jar. The collected data were analyzed by SAS ver. 9.1 software. Mean comparisons were carried out by Duncan test at the 1% level of significancy.

Results and Discussion
The results showed that auxins were more effective than cytokinins in terms of callus induction. The highest frequency of embryogenesis was achieved with hypocotyl explants in 2 mg/l 2,4-D in Kaka cultivar under constant darkness. For the development of embryos, callus with globular embryo were transferred to MS medium supplemented with different PGRs. The frequency of embryogenesis was higher in dark condition than that of in the light condition. During callogenesis, plant species require different physical (light and temperature) conditions e.g. some plant produces more callus in darkness while in the other species, callus induction and proliferation took place in a normal photoperiod (Suhasini et al., 1994). It probably depends on the genetic structure of the plant. The highest percentage of globular embryo development of the heart shape embryo and then to cotyledonary embryo was obtained from Kaka leaf explants growing on medium supplemented with 0.5 mg/l BA + 0.5 mg/l of 2,4-D. 2 mg/l of 2,4-D in both dark and light conditions was induced the highest rate of embryogenesis. For the initiation of embryogenesis, 2, 4-D play an undeniable role because of this synthetic auxin can lead to overexpression of different genes during stress as well as genes involved in initiation of somatic embryogenesis (Kitamiya et al., 2000; Quiroz-Figueroa, 2006). 2,4-D by its strong auxin activity, can influence metabolism of other phytohormones which in turn affect somatic embryogenesis directly or indirectly (Quiroz-Figueroa, 2006). A prerequisite to induce somatic embryo is a reorganization of cell physiology, metabolism and gene expression (Fehret et al., 2002). In this study, with increasing concentrations of the 2,4-D, embryogenesis was reduced. Additionally increasing of 2,4-D concentration could reduce frequency of embryogenesis.

Conclusion
Somatic embryo induction depends on various factors including cultivar, type, concentration and combination of PGRs as well as explant type. By comparison to the light condition, darkness is in favor of somatic embryo biogenesis. Between two cultivars, Kaka showed better ability in formation of somatic embryos. Response of a plant tissue or specific organ in somatic embryogenesis process is the outcome of endogenous hormones and exogenous growth regulators, and this response can be physiologically varied depending on the type of explant.

Keywords

Cultivar; Darkness; Growth regulators; In vitro

References

1. Ali, A., Naz, S., and Iqbal, J. 2007. Effect of different explants and media compositions for efficient somatic embryogenesis in sugarcane. Pakistan Botanical Society 39(6): 1961-1977.

2. Angoshtari, R., Tavakkol, R., Afshari, K.S., and Omidi, M. 2009. Effects of abscisic acid on somatic embryogenesis and induction of desiccation tolerance in Brassica napus. Asian Journal of Plant Sciences 8: 276-284.

3. Barna, K.S., and Wakhlu, A.K., 1993. Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.). Plant Cell Report 12: 521-524.

4. Durzan, D.J. 2012. Interpolated apomictic somatic embryogenesis, and sporogenesis, asexual heterospory, mito sporogenesis and genomic silencing in a gymnosperm artificial sporangium. Integrating vegetative propagation, biotechnologies and genetic improvement for tree production and sustainable forest management. June 25-28, 2012. Brno, Czech Republic. pp. 3-36.

5. Feher, A., Pasternak, T., Otvos, K., Miskolczi, P., and Dudits, D. 2002. Induction of embryogenic competence in somatic plant cells: a review. Biologia 57: 5-12.

6. Fernandez, H., Perez, C., and Sanchez-Tam, R. 2000. Modulation of the morphogenic potential of the embryonic axis of Juglans regia L. by cultural conditions. Plant Growth Regulators 30: 125-131.

7. Gaj, M.D. 2004. Factors influencing somatic embryogenesis induction and plant regeneration with particular reference to Arabidopsis thaliana L. Plant Growth Regulators 43: 27-47.

8. Gerdakaneh, M., Mozafari, A., Khalighi, A., and Sioseh-mardah, A. 2009. The effects of carbohydrate source and concentration on somatic embryogenesis of strawberry (Fragaria × ananassa Duch.). American-Eurasian Journal of Agricultural & Environmental Sciences 6(1): 76-80.

9. Iantcheva, A., Vllahova, M., Bakalova, E., Kondorosi, E., Elliott, M.C., and Atanassov, A. 1999. Regeneration of diploid annual medics via direct somatic embryogenesis promoted by thidiazuron and benzylaminopurine. Plant Cell Reports 18: 904-910.

10. Jimenez, V.M., and Thomas, C. 2005. Participation of Plant Hormones in Determination and Progression of Somatic Embryogenesis. In: A. Mujid and J. Samaj (Eds.). Somatic Embryogenesis Plant Cell Monographs. DOI 10.1007/7089_034/ Introduction to the Electronic Age. E-Published online. pp 103-118.availble at Web site molbiol.ru/forums/uploads/.../ Somatic_Embryogenesis.txt

11. ‎Karami, O., Deljou, A., Esna-Ashari, M., and Ostad-Ahmadi, P. 2006. Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.). Scientia Horticulturae 110: 340-344.

12. Karimi, K.G., and Karami, O. 2008. Picloram-induced somatic embryogenesis in leaves of strawberry (Fragaria ananassa L.). Acta Biologica Cracoviensia Series Botanic 50(1): 69-72.

13. Karkonen, A. 2001. Plant tissue cultures as models for tree physiology: Somatic embryogenesis of Tilia cordata and Lignin biosynthesis in Picea abies suspension cultures as case studies. Department of Biosciences Division of Plant Physiology University of Helsinki. 89 pp.

14. Kiran, G., Sujata, M., Srinathrao, P.B., and Kishor, K. 2010. Direct somatic embryogenesis and plant regeneration from immature explants of chickpea. Biological Plantarum 54(1): 121-125.

15. Kiran, C.P., Kaviraj, G., Jogeswar, P.B., Kavi, K., and Srinath, R. 2005. Direct and high frequency somatic embryogenesis and plant regeneration from hypocotyls of chickpea (Cicer arietinum L.) a grain legume. Current Science 89: 1012-1018.

16. Kitamiya, E., Suzuki, S., Sano, T., and Nagata, T. 2000. Isolation of two genes that were induced upon the initiation of somatic embryogenesis on carrot hypocotyls by high concentrations of 2,4-D. Plant Cell Reports 19: 551-557.

17. Khosravi, S., Vatanpour Azghandi, A., Hadad, R., and Mojtahedi, N. 2007. In vitro propagation of a commercial cultivar of Lilium (Lilium longiflorum var: Ceb-Dazzel) throught direct somatic embryogenesis. Seed and Plant Improvement Journal 23(3): 159-168.

18. Mashayekhi, K. 2000. The protein synthesis spectrum during the induction phase of somatic embryogenesis in carrot (Daucus carota L.) culture and the role of nitrogen forms for embryo development. Institute of Plant Nutrition Department of Tissue Culture Justus Liebig University, Giessen, Germany.199 pp.

19. May, R.A., and Trigiano, R.N. 1991. Somatic embryogenesis and plant regeneration from leaves of Dendranthema grandiflora. Journal of the American Society for Horticultural Science 116(2): 366-371.

20. Mozsar, J., and Viczian, O. 1996. Genotype effect on somatic embryogenesis and plant regeneration of Vitis spp. Vitis 35(4): 155-157.

21. Pintos, B., Martin, J.P., Centeno, M.L., Villalobos, N., Guerra, H., and Martin, L. 2002. Endogenous cytokinin levels in embryogenic and non-embryogenic calli of Medicago arborea L. Plant Science 163: 955-960.

22. Quiroz-igueroa, F.R., Rojas, R., Herrera, R.M., and Galaz-Avalos,V.M. 2006. Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell, Tissue and Organ Culture 86: 285-301.

23. Saelim, L., Phansiri, S., Supatcharee, N., Malinee, U., and Jarunya, N. 2006. Optimization of in vitro cyclic somatic emhryogenesis and regeneration of the Asian cultivars of Cassava (Manihot esculenta Crantz) for genetic manipulation system global. Journal of Biotechnology & Biochemistry 1(1): 07-15.

24. Shagufta, Naz, A.A., Fayyaz, A.S., and Javed, I. 2008. Somatic embryogenesis from immature cotyledons and leaf calli of chickpea (Cicer arietinum L.). Pakistan Journal of Botany 40(2): 523-531.

25. Suhasini, K., Sagare, A.P., and Krishnamurthy, K.V. 1994. Direct somatic embryogenes is from mature embryo axes in chickpea (Cicer arietinum L.). Plant Science 102: 189-194.

26. Thomas, C., Meyer, D., Himber, C., and Steinmetz, A. 2004. Spatial expression of a sunflower SERK gene during induction of somatic embryogenesis and shoot organogenesis. Plant Physiology & Biochemistry 42: 35-42.

27. Tokuji, Y., and Kuriyama, K. 2003. Involvement of gibberellins and cytokinin in the formation of embryogenic cell clumps in carrot (Daucus carota). Journal of Plant Physiology 160: 133-141.

28. Vengadesan, G., Paula, Æ., and Pijut, M. 2009. Somatic embryogenesis and plant regeneration of northern red oak (Quercus rubra L.). Plant Cell Tissue and Organ Culture 97: 141-149.

29. Victor, J.M.R., Murch, S.J., Krishna, Raj, S., and Saxena, P.K. 1999. Somatic embryogenesis and organogenesis in peanut: The role of thidiazuron and N6-benzylaminopurine in the induction of plant morphogenesis. Plant Growth Regulation 28: 9-15.

30. Von, Arnold, S., Sabala, I., Bozhkov, P., Dyachok, J., and Filonova, L. 2002. Developmental pathways of somatic embryogenesis. Plant Cell, Tissue and Organ Culture 69: 233-249.

31. Zare, A.R., Solouki, M., Omidi, M., and Irvani, N. 2010. Callus induction and plant regeneration in Ferula Assa Foetida L. Trakia Journal of Sciences 8(1): 11-18.