Callogenesis and regeneration of common bean (Phaseolus vulgaris L.) by using thin cell layer explants

Document Type : مقالات پژوهشی

Authors

• Atefeh Bakhshian
• Khadijeh Bagheri

Department of Plant Production and Genetics, Faculty of Agriculture, University of Zanjan, Zanjan, Iran

10.22067/ijpr.v10i2.62582

Abstract

Introduction
Common bean (Phaseolus vulgaris L.) is the most important species of the legume. This plant is a very important source of vegetable protein, especially in those regions of the world in which animal proteins are scarce. However, breeding can not add certain genes that do not exist naturally in the P. vulgaris gene pool. Due to this limitation of plant breeding, new trait improvement approaches such as interspecific horizontal gene transfer via genetic engineering need to be utilized in order to complement the limitations encountered by conventional breeding of this crop. Still, there is not an optimized protocol for beans regeneration and this is a big challenge for bean breeding through genetic engineering
 
Material & Methods
In this study, two experiments were conducted to evaluate callus induction and regeneration in bean. Seeds of P. vulgaris were washed thoroughly with distilled water and tween-20 (10%). Subsequently they were surface sterilized with 70% ethanol for 2 min followed by sodium hypochloride (3%) for 15 min. After five rinses with sterile distilled water, they were allowed to germinate aseptically on half strength Murashige and Skoog (MS) medium. In the first experiment, induction of callus and regeneration were investigated in six common bean cultivars with embryonic axis explants under the influence of TDZ, BAP and IAA plant growth regulators. The sterile seeds were soaked for 20 hours, embryogenic axes were isolated from seeds and cultured on MS medium with different growth regulators including BAP (11µM) and IAA (0/57µM), BAP (44/4µM) and TDZ (2/27µM), BAP (44/4µM). In the second experiment, sterile seeds germinated under the pretreatments medium (free TDZ, 10µM TDZ) and then, after 14 days, Transverse thin cell layer explants (with 0.3-0.5 mm thick) were excised from epicotyls. For induction of regeneration, explants were transferred to solid MS medium supplemented with 20 g/lit sucrose, B5 vitamins and 10µM TDZ  or 10µM BAP. After 14 days, TDZ concentration reduced to 1μM in the TDZ (10µM) treatment and BAP (10µM) treatment remained intact. After 28 days, all samples were transferred to medium MSB5+ BAP (10µM) + AgNO₃ (10 µM). After 48 days, all samples were transferred to MSB5+ BAP (1µM) + GA₃ (3 µM) + AgNO₃ (10 µM) and after 62 days,  all samples were transferred to MSB5+ NAA (1µM) + AgNO₃ (10 µM). During this period the callogenesis and regeneration rate were recorded.
 
Results & Discussion
The results of first experiment showed that the highest callus production (100%) related to BAP (44/4µM) + TDZ (2/27µM) and BAP (44/4µM) treatments in all cultivars except Line 4. Based on the results, callus induction in all varieties (except Line 4) were same in various growth regulator treatments. In embryogenic axes explant, the effect of BAP and TDZ on callus induction was much better than IAA. Second  experiment data showed that most callus induction in the pretreatment of the seedlings by TDZ (10µM), treatment by TDZ (10µM) and BAP (10µM) in Line 4 cultivar and Aligudarz was in next rank. Most regeneration achieved in the pretreatment TDZ (10µM) and treatment by TDZ (10µM) in Azna, Naz, Line 8 and  Aligudarz cultivars. The regeneration of Line 4 was weak despite the good callogenesis. From the 6 cultivars, Aligudarz showed better results in callogenesis and regeneration. It can be concluded that TDZ is better than BAP for regeneration in tTCLS explants of bean.  In both experiments, the positive effect of silver nitrate on the reduction of phenolic compounds were observed. Based on previous studies, the combination of BAP and AgNO3 at the same concentrations(10µM) significantly raised the number of developed shoots, but unfortunately in our study the same result was not observed and the effect of AgNO₃ on shoot development was weak.
 
Conclusion
Unlike previous studies, cytokinin used in this study, did not lead to direct regeneration of bean. The results showed that high concentrations of the cytokinin is lead to callus induction in tested varieties. It was observed that a prolonged exposure to high TDZ concentration had an inhibitory effect on further development of shoots and by reducing the concentrations of TDZ, regeneration will occur. The results also showed that transverse thin cell layers explants from epicotyl are able to regenerate under the influence of TDZ and BAP better than embryonic axis explants. Regeneration severely is affected by genotype, maybe that's why still, there is not an optimized protocol for bean regeneration.

Keywords

• Callogenesis
• Embryonic axis
• Phaseolus vulgaris
• Regeneration
• Thin Cell Laye

---

References

1. Angenon, G., and Thu, T. 2011. Genetic Transformation. In: A. Pratap and J. Kumar (Eds). Biology and Breeding of Food Legumes. CABI., UK, p. 178-192.

2. Capelle, S.C., Mok, D.W., Kirchner, S.C., and Mok, M.C. 1983. Effects of thidiazuron on cytokinin autonomy and the metabolism of N6-(Δ2-Isopentenyl)[8-14C] adenosine in callus tissues of Phaseolus lunatus L. Plant Physiology 73(3): 796-802.

3. Castillo, B.M., Rodríguez de la O, J.L., Gallardo, J.O.M and Iturriaga, G. 2015. In vitro plants of common bean (Phaseolus vulgaris L.) obtained by direct organogenesis. Journal of Agricultural Science 7(11): 169-179.

4. 4. Collado, R., Veitia, N., Bermudez-Caraballoso, I., Garcia, L., Torres, D., Romero, C., Lorenzo, J.R., and Angenon, G. 2013. Efficient in vitro plant regeneration via indirect organogenesis for different common bean cultivars. Scientia Horticulturae 153: 109-116.

5. 5. De Carvalho, M.H.C., Van Le, B., Zuily-Fodil, Y., Thi, A.T.P., and Van, K.T.T. 2000. Efficient whole plant regeneration of common bean (Phaseolus vulgaris) using thin-cell-layer culture and silver nitrate. Plant Science 159(2): 223-232.

6. 6. De Clercq, J., Zambre, M., Van Montagu, M., Dillen, W., and Angenon, G. 2002. An optimized Agrobacterium-mediated transformation procedure for Phaseolus acutifolius Gray. Plant Cell Reports 21(4): 333-340.

7. 7. Dillen, W., De Clercq, J., Goossens, A., Van Montagu, M., and Angenon, G. 1997. Agrobacterium-mediated transformation of Phaseolus acutifolius Gray. Theoretical and Applied Genetics 94(2): 151-158.

8. Estrada-Navarrete, G., Alvarado-Affantranger, X., Olivares, J., Guille´n, G., Dı´az-Camino, C., Campos, F., Quinto, C., Gresshoff, P.M., and Sanchez, F. 2007. Fast, efficient and reproducible genetictransformation of Phaseolus spp. by agrobacterium rhizogenes. Nature Protocols 2(7): 1819-1824.

9. 9. Gamborg, O.L.C., Miller, R.A., and Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50(1): 151-158.

10. 1 Lakshmanan, P., Loh, C.S., and Goh, C.J. 1995. An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture. Plant Cell Reports 14(8): 510-514.

11. 11. Malik, K.A., and Saxena, P.K. 1992. Regeneration in Phaseolus vulgaris: High-frequency induction of direct shoot formation in intact seedlings by N6-benzylaminopurine and thidiazuron. Planta 186(3): 384-389.

12. Ministry of Agriculture Jahad. 2016. Amarnameh Keshavarzi. Available at web site http://amar.maj.ir/Portal/Home/Default.aspx?CategoryID=117564e0-507c-4565-9659fbabfb4acb9b. (In Persian).

13. 13. Mukeshimana, G., Ma, Y., Walworth, A.E., Song, G., and Kelly, J.D. 2013. Factors influencing regeneration and Agrobacterium tumefaciens-mediated transformation of common bean (Phaseolus vulgaris L.). Plant Biotechnology Reports 7(1): 59-70.

14. 14. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497.

15. 15. Nhut, D.T., Van Le, B., Tanaka, M., and Van, K.T.T. 2001. Shoot induction and plant regeneration from receptacle tissues of Lilium longiflorum. Scientia Horticulturae 87(1): 131-138.

16. 16. Sabzikar, R., Sticklen, M.B., and Kelly, J.D. 2010. In vitro regeneration and morphogenesis studies in common bean. Plant Cell, Tissue and Organ Culture 100(1): 97-105.

17. 17. Thomas, J.C., and Katterman, F.R. 1986. Cytokinin activity induced by thidiazuron. Plant Physiology 81(2): 681-683.

18. 18. Van K, T.T. 1980. Control of morphogenesis by inherent and exogenously applied factors in thin cell layers [Plants, de novo buds, roots]. International Review of Cytology 32: 291-311.

19.Van, K.T.T., Lie-Schricke, H., Marcotte, J., and Trinh, T. 1986. Winged bean [Psophocarpus tetragonolobus (L.) DC.]. In: Y.P.S. Bajaj (Ed). Biotechnology in Agriculture and Forestry Vol. 2. Crop I. Springer-Verlag, Berlin, p. 556-567.

20.Van Le, B., Jeanneau, M., Sadik, S., Tu, S., Vidal, J., and Van, K.T.T. 1998. Rapid plant regeneration on a C4 dicot species: Amaranthus edulis. Plant Science 132(1): 145-154.

21.Veltcheva, M., Svetleva, D., Petkova, S., and Perl, A. 2005. In vitro regeneration and genetic transformation of common bean (Phaseolus vulgaris L.) problems and progress. Scientia Horticulturae 107(1): 2-10.

22. Zambre, M., Goossens, A., Cardona, C., Van Montagu, M., Terryn, N., and Angenon, G. 2005. A reproducible genetic transformation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the mexican bean weevil. Theoretical and Applied Genetics 110(5): 914-924.

22.