Challenges of in vitro conservation of Сitrus germplasm resources

The main problems of establishment a slow growth in vitro collection of citrus and other tree crops cultivars are high degree of fungal contamination of bud explants and low growth potential of shoots. In this regard, the aim of current research is to assess the efficiency of decontamination procedure and the possibility of tissue culture initiation and slow growth conservation of valuable lemon cultivars. The best results of surface sterilization were obtained using immersion solutions of 0.3 % Veltolen – 25 minutes or 10 % Domestos – 25–30 minutes. In these treatments, 27.7–33.0 % of aseptic explants were obtained, respectively. However, after the third subculture, the yield of aseptic viable explants decreased till 10 % as a result of secondary contamination by endophytic fungi. The addition of biocide (“Gavrish”) in a nutrient medium at a concentration of 1 ml/l helped to increase the yield of aseptic viable explants till 50 %. However, after the third subculture the photosynthetic activity and the pigments content as well as growth rate decreased. Plants dropped yellowish leaves and eventually died. Thus, 37.35 % of plantlets survived after 8 months of conservation, and only 14.6 % survived after 10 months. Even after the third month of conservation significant decrease in the viability index and the coefficient of photosynthetic activity occurred in plants. Chlorophyll a in leaves decreased from 1.59 to 1.14 mg/g during 12 months in vitro conservation. The similar tendency observed on clorophyll b and carotenoids content. The experiments were carried out for 5 years using different lemon cultivars and other citrus varieties and cultivars. Thus, micropropagation and slow growth in vitro conservation of valuable lemon cultivars are still problematic and requires new technical solutions due to the low growth potential of plantlets raised from the mature buds that is consistent with the data of other researchers.

Citrus, nodal explant, contamination, micropropagation, slow growth conservation, culture media, trueto-type, endophytes

1. Belous O.G., Maliukova L.S., Kozlova N.V. Effect of mineral fertilizers on the pigment composition of leaves of tea plants in the conditions of subtropics of Russia. Subtropicheskoe i Dekorativnoe Sadovodstvo = Subtropical and Ornamental Horticulture. 2011;44:135-143. (in Russian)

2. Budagovskaya O.N., BudagovskyA.V., Budagovsky I.A., Solovyh N.V. A method for assessing the functional state of plants in vitro without braking sterility. Patent RU No. 0002604302 of 10.12.2016. (in Russian)

3. Çölgeçen H., Koca U., Toker G. Influence of different sterilization methods on callus initiation and production of pigmented callus in Arnebia densiflora Ledeb. Turk. J. Biol. 2009;35:513-520. DOI 10.3906/biy-0911-161.

4. Engelmann F. In vitro conservation methods. In: Callow J.A., FordLloyd B.V., Newbury H.J. (Eds.). Biotechnology and Plant Genetic Resources: Conservation and Use. CAB International, Wellingford, 1997;119-162.

5. Gogoi G., Borua P.K. Standardization parameters for critical problems encountered in plant in vitro culture technique. Int. J. Curr. Res. 2014;6(12):10964-10973.

6. Kloepper J.W., McInroy J.A., Hu C.-H. Association of plant damage with increased populations of deleterious endophytes following use of Benlate systemic fungicide. In: Schneider C., Leifert C., Feldmann F. (Eds.). Proc. 5th Int. Symp. “Endophytes for Plant Protection: the State of the Art” (Humboldt University, Berlin, 26–29 May, 2013). Berlin-Dahlem, 2013;56-69.

7. Kolomiets T.M., Malyarovskaya V.I., Gvasaliya M.V., Samarina L.S., Sokolov R.N. Micropropagation in vitro of subtropical, ornamental crops and endemics of the western Caucasus: original and optimized protocols. Selskokhozyastvennaya Biologia = Agricultural Biology. 2014;3:49-58. (in Russian)

8. Krishna H., Alizadeh M., Singh D., Singh U., Chauhan N., Eftekhari M., Sadh R.K. Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech. 2016;6(1):54. DOI 10.1007/s13205-016-0389-7.

9. Krueger R.R., Navarro L. Citrus Germplasm Resources. In: Khan I.A. (Ed.). Citrus: Genetics, Breeding and Biotechnology. CAB International, 2007;45-150.

10. Kulyan R.V. The genetic collection of citrus fruits is the basis for isolating the sources of economically valuable traits. Bulleten Michurinskogo Gosudarstvennogo Agrarnogo Universiteta = Bulletin of Michurin State Agrarian University. 2015;4:52-56. (in Russian)

11. Marin M.L., Duran-Vila N. Conservation of Citrus germplasm in vitro. Am. Soc. Hortic. Sci. 1991;116:740-746.

12. Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962;15:473-497.

13. Niedz R.P., Bausher M.G. Control of in vitro contamination of explants from greenhouse- and field-grown trees. In Vitro Cell. Dev. Biol. Plant. 2002;38:468-471.

14. Pence V.C. The possibilities and challenges of in vitro methods for plant conservation. Kew Bull. 2010;65:539-547.

15. Perez-Tornerо C.I., Tallon I., Porras I. An efficient protocol for micropropagation of lemon (Citrus limon) from mature nodal segments. Plant Cell Tiss. Organ Cult. 2009;100:263-271.

16. Rathore J.S., Rathore M.S., Singh M., Singh R.P., Shekhawat N.S. Micropropagation of mature tree of Citrus limon. Indian J. Biotechnol. 2007;6:239-244.

17. Ryndin A.V., Kulyan R.V. Collection of citrus cultures in humid subtropics of Russia. Sadovodstvo i Vinogradarstvo = Horticulture and Viticulture. 2016;5:24-30. (in Russian)

18. Samanhudi A.T.S., Muji R. In vitro axillary bud multiplication of Citrus nobilis Lour. in Indonesia. J. Life Sci. 2010;4(4):39-44.

19. Samarina L.S., Kolomiets Т.М., Gorshkov V.M. Evaluation of the regeneration capacity of Citrus explants in vitro. Sadovodstvo i Vinogradarstvo = Horticulture and Viticulture. 2012;6:27-30. (in Russian)

20. Shlyk A.A. Biosynthesis of chlorophyll and formation of photosynthetic systems. In: Theoretical Foundations of Photosynthetic Productivity. Moscow, 1971. (in Russian)