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An in vitro pollen storage study was conducted using pollen from three seeded citrus plant genotypes, viz., ‘Mexican lime’, ‘W’. Murcott’ and ‘Mosambi’ and five seedlesscitrus genotypes, viz., ‘Lisbon lemon’, ‘Jaffa’, ‘Clementine’, ‘Hamlin’ and ‘Mukaku Kishu’. Pollen viability and germination percentage were evaluated at different storage temperature treatments, i.e., at room temperature (in anhydrous calcium chloride) (control), in a refrigerator at 4°C, in a freezer (-20°C), and in a freeze drier (-80°C). The viability of the pollen plants was tested with an acetocarmine stain (2%). Among all the tested sucrose concentrations (0, 5, 10, 15, 20, and 25%) for in vitro pollen germination, the 15% sucrose concentration had the highest effect on pollen germination. The results showed significant differences in pollen viability and germination under different storage temperature conditions. The pooled data revealed that, among the seeded genotypes, W. Murcotts showed the maximum mean viability and germination percentage (67.86% and 60.88%, respectively) after 48 weeks of storage at -80°C, and the minimum values were observed for Mexican lime (46.57% and 33.71%, respectively). However, in the low-seeded genotype, Mukaku Kishu had the maximum mean pollen viability and germination (71.52% and 64.07%, respectively) after 48 weeks of storage at -80°C, and the lowest values were observed in Jaffa (39.36% and 28.08%, respectively). The results indicate that the freeze drier storage temperature (-80°C) had the greatest effect on retaining pollen viability and germination in both the seeded and low-seeded genotypes. However, a progressive decrease in pollen viability and germination rate was observed with increasing duration at all storage temperatures, reaching a minimum at 48 weeks after storage. However, the reduction in pollen storage ability was greatest at room temperature and 40°C. Pollen grains stored at low temperatures (-80°C and -20°C) showed good viability and germination percentage compared with those stored at room temperature and 4°C.


Citrus Freeze drier temperature Genotypes Germination Low seeded Pollen viability Seeded

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How to Cite
Lamo, K., Sangwan , A. K., Singh, N., & Batth, M. K. (2024). Optimization of in vitro pollen storage conditions in seeded and low-seeded citrus genotypes. Environment Conservation Journal, 25(2), 467–482.


  1. Ahmed, S., Rattanpal, H. S., Ahmad, E. & Singh, G. (2017). Influence of storage duration and storage temperature on in vitro pollen germination of citrus species.International Journal of Current Microbiology and Applied Science.,6 (1),892-902. DOI:
  2. Alburquerque, N., Montiel, G. & Burgos, L. (2007). Short communication. Influence of storage temperature on the viability of sweet cherry pollen. Spanish Journal of Agricultural Research.,5 (1), 86-90. DOI:
  3. Aslantus, R. & Pirlak. (2002). Storage and germination of Strawberry pollen. IV International Symposium on Strawberry, pollen. (Eds.): Hietaranta, M.-M Linn., Palonen and Parikka, P. ActaHortcultureae, 2, 567. DOI:
  4. Bhat, Z. A., Dhillon, W. S., Shafi, R. H. S., Rather, J. A., Mir, A. H., Shafi, W., Rashid, R. Bhat, J. A., Rather, T. R. & Wani, T. A. (2012). Influence of Storage Temperature on Viability and In Vitro Germination Capacity of Pear (Pyrus spp.) J of Agri Sci., 4 (1). DOI:
  5. Chander, S., Rajasekharan, P. E. & Kurian, R. M. (2019). Pollen storage studies in sugar apple (Annona squamosa L) cv. Balanagar. Israel Journal of Plant Sciences,66 (7), 196-202. DOI:
  6. Chaudhury, R., Malik, S., K. & Rajan, S. (2010). An improved pollen collection and cryopreservation method for highly recalcitrant tropical fruit species of mango (Mangifera indica L.) and litchi (Litchi chinensis Sonn.). CryoLetters, 31 (5), 268–278.
  7. Dutta, S. K., Srivastav, M., Chaudhary, R., Lal, K., Patil, P., Sing, S. K. & Sing, A., K. (2013). Low temperature storage of mango (Mangifera indica L.) pollen Scientia Horticulturae,161 (3), 193-197. DOI:
  8. Gomes, P. R., Raseira, M. C. B. & Baudet, L. L. (2003). Onion (Allium cepa L.) Pollen Storage. Revista Brasileira de Sementes, 25 (2), 14-17. DOI:
  9. Khan, S., A. & Perveen, K. (2006). Germination capacity of stored pollen of Solanum melongena L. (Solanaceae) and their maintenance. Pakistan Journal of Botany,38 (6), 921-923.
  10. Kundu, M, Dubey, A., Srivastav, M., Malik, S. & Singh, B. (2014). Effect of gamma ray irradiation and cryopreservation on pollen stainability, in vitro germination, and fruit set in Citrus. Turkish Journal of Biology, 38 (4),1-9. DOI:
  11. Lora, J., Garcia-Lor, A. & Aleza, P. (2022). Pollen development and viability in diploid and doubled diploid citrus species. Frontiers in Plant Science,13 (1). DOI:
  12. Lora, J., Perez, D. E., Oteyza, M, A., Fuentetaja, P. & Hormaza, J. I. (2006). Low temperature storage and in vitro germination of cherimoya (Annona cherimola Mill.) pollen. Scientia Horticulturae,108 (4), 91-94. DOI:
  13. Martinez-Gomez, P., Gradziel, T. M., Ortega, E. & Dicenta, F. (2001). Short-term storage of almond pollen. HortScience, 35 (2), 1151-52. DOI:
  14. Martinez-Gomez, P., Gradziel, T. M., Ortega, E. & Dicenta, F. (2002). Low temperature storage of almond pollen. HortScience.,37 (1), 691-692. DOI:
  15. Mishra, R. & Shivanna, K (1982). Efficacy of organic solvents for storing pollen grains of some leguminous taxa. Euphytica, 31 (1),991-995. DOI:
  16. Rouiss, H., Bakry, H., Froelicher, Y., Navarro, L., Aleza, P. & Ollitrault, P. (2018). Origin of C. latifolia and C. aurantiifolia triploid limes: the preferential disomic inheritance of doubled-diploid ‘Mexican’ lime is consistent with an interploid hybridization hypothesis. Annals of Botany, 121 (3), 571–585. DOI:
  17. Salles, P., L., A., Darlan, R. J., Junqueira, P. M., Santos, K. P., & Morais, J. C. (2007). Viability of citrus pollen in different storage conditions. Ciencia e Agrotecnologia,31 (3), 147- 153. DOI:
  18. Sharafi, Y. & Bahmani, A. (2010). Study of pollen germination and tube growth in some Iranian Loquat cultivars and genotypes. 3th International Symposium on Loquat, 22-25 May. Antakya. Turkey.
  19. Thaipong, K., Dalaeman, S. & Sethpakdee, R. (2008). An appropriate temperature for grape pollen storage. Indian Journal of Agricultural Sciences,39 (1), 36-39.
  20. Towil, L. E. (2010). Long-term pollen storage. Plant Breeding Reviews 13(1), 179–
  21. Weatherhead, M., A., Grout, B., W., W. & Henshaw, G. G. (2006). Advantages of Storage of Potato Pollen in Liquid Nitrogen. Biomedical and Life Sciences, 21, 331-334. DOI:
  22. Yamamoto, M., Kubo, T. & Tominaga, S. (2006). Self- and cross-incompatibility of various citrus accessions. Journal of the Japanese Society for Horticultural Science,75 (5), 372–78. DOI:
  23. Zeng-Yu, W., Yaxin, G., Scott, M. & Spangenberg, G. (2004). Viability & longevity of pollen from transgenic and non transgenic tall fescue (Festuca arundinacea) (Poaceae) plants. American Journal of Botany,91 (4), 523-30. DOI:
  24. Zheng, R, H., Su, S. D., Xiao, H. & Tian, H. Q. (2019). Calcium: A Critical Factor in Pollen Germination and Tube Elongation. International Journal of Molecular Sciences,20(2),420. DOI: