Optimizing hardening substrates and homozygosity testing for anther culture derived doubled haploid sweet pepper (Capsicum annuum var. grossum L.) lines

Authors

DOI:

https://doi.org/10.14719/pst.5391

Keywords:

doubled haploid (DH), hardening, substrate, survival frequency, vermicompost

Abstract

Sweet pepper (Capsicum annuum var. grossum L.), a valuable vegetable crop, faces significant challenges in large-scale doubled haploid (DH) production due to high mortality rates during the hardening phase. This study aimed to optimize substrate conditions for successful DH hardening and to validate genetic uniformity using molecular markers. The research focused on four genotypes of sweet pepper: Asha, Sympathy, Namelite, and Indra. Various substrate combinations were evaluated, including coco peat, perlite, vermiculite, yard manure (FYM), and vermicompost. The results show that substrates containing coco peat, perlite, vermiculite, and vermicompost consistently achieved high survival rates and superior growth parameters. Specifically, substrate T6 (coco peat + perlite + vermiculite + vermicompost, (1:1:1:1)) achieved the highest survival rate, with the Asha genotype reaching 97.64%. This substrate also promoted optimal plant height and leaf production, with Asha reaching 11.75 cm and producing 14.5 leaves on average. Homozygosity testing using SSR markers (CAM-8, CAM-19, CAM-54, CAM-79) confirmed that 447 (97.0%) of the 461 DH plants were accurate homozygous lines, with minimal diploid contamination across all genotypes. Among the genotypes, Indra exhibited the highest efficiency, with 98.4% of plants identified as proper DH lines. Chi-square analysis showed no significant deviation from expected Mendelian ratios, affirming genetic stability. These findings highlight the importance of substrate optimization in enhancing the acclimatization and survival of DH lines and demonstrate the effectiveness of SSR markers in ensuring genetic uniformity. This study provides valuable insights for improving DH production protocols in sweet pepper breeding programs.

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References

Pruthi JS. Capsicums or chillies. In: Pruthi JS, editor. Spices and condiments. New Delhi: National Book Trust; 1976. p. 40-49

Krishna De A, editor. Capsicum: The genus Capsicum. 1st ed. CRC Press; 2003 https://doi.org/10.1201/9780203381151

Bhalabhai JG, Rajhans S, Pandya H, Mankad A. A comprehensive review on Capsicum spp. Inter J Res Anal Rev. 2021;8(4):581-99.

Bharti H, Singh MC, Singh PK, Sahana KP, Mishra IM. Hi-Tech cultivation of Capsicum. Indian Horticulture. 2021;66(2). https://epubs.icar.org.in/index.php/IndHort/article/view/114654

Sood S, Kumar N. Heterosis for fruit yield and related horticultural traits in bell pepper. Inter J Veg Sci. 2010;16(4):361-73. https://doi.org/10.1080/19315260.2010.490576

Agarwal A, Gupta S, Ahmed Z. Influence of plant densities on productivity of bell pepper (Capsicum annuum L.) under greenhouse in high altitude cold desert of Ladakh. Acta Hortic. 2007;756:309-14. https://doi.org/10.17660/ActaHortic.2007.756.32

Chassy AW, Bui L, Renaud EN, Van-Horn M, Mitchell AE. Threeyear comparison of the content of antioxidant microconstituents and several quality characteristics in organic and conventionally managed tomatoes and bell peppers. J Agric Food Chem. 2006;54(21):8244-52. https://doi.org/10.1021/jf060950p

Seguí-Simarro JM. Androgenesis revisited. Botanical Review. 2010;76(3):377-404. https://doi.org/10.1007/s12229-010-9056-6

Comlekcioglu N, Ellialtioglu S. Review on the research carried out on in vitro androgenesis of peppers (Capsicum annuum L.). Res J Biotech. 2018;6:75-84.

Palacios MA, Seguí-Simarro JM. Anther culture in sweet pepper (Capsicum annuum L.). Methods Mol Biol. 2021;2288:279-91. https://doi.org/10.1007/978-1-0716-1335-1_17

Seguí-Simarro JM. Androgenesis in Solanaceae. In: Germana MA, Lambardi M, editors. In vitro embryogenesis in higher plants. New York: Humana Press; 2016. p. 209-44

Çiner DÖ, Tipirdamaz R. The effects of cold treatment and charcoal on the in vitro androgenesis of pepper (Capsicum annuum L.). Turk J Bot. 2001;26(3):131-39.

Nowaczyk P, Kisia?a A. Effect of selected factors on the effectiveness of Capsicum annuum L. anther culture. J Appl Genet. 2006;47(2):113-17.

Hazarika BN. Acclimatization of tissue-cultured plants. Curr Sci. 2003;85(12):1704-12. http://www.jstor.org/stable/24109975

Valadez-Bustos MG, Aguado-Santacruz GA, Carrillo-Castaneda G, Aguilar-Rincon VH, Espitia-Rangel E, Montes-Hernandez S, et al. In vitro propagation and agronomic performance of regenerated chili pepper (Capsicum spp.) plants from commercially important genotypes. In Vitro Cell Dev Biol Plant. 2009;45(5):650-58. https://doi.org/10.1007/s11627-009-9193-y

Kozai T. Micropropagation under photoautotrophic conditions. In: Debergh PC, Zimmerman RH, editors. Micropropagation technology and application. Dordrecht: Kluwer Academic Publishers; 1991. p. 447-69 https://doi.org/10.1007/978-94-009-2075-0_26

Buddendorf-Joosten JMC, Woltering EJ. Components of the gaseous environment and their effects on plant growth and development in vitro. Plant Growth Regul. 1994;15(1):1-16. https://doi.org/10.1007/BF00024671

Pospisilova J, Solarova J, Catsky J. Photosynthetic responses to stresses during in vitro cultivation. Photosynthetica. 1992;26(1):3-18.

Kozai T, Smith MAL. Environmental control in plant tissue culture - general introduction and overview. In: Aitken-Christie J, Kozai T, Smith ML, editors. Automation and environmental control in plant tissue culture. Dordrecht: Kluwer Academic Publishers; 1995. p. 301-18 https://doi.org/10.1007/978-94-015-8461-6_1

Desjardins Y. Photosynthesis in vitro - on the factors regulating CO2 assimilation in micropropagation systems. Acta Hortic. 1995;393:45-61. https://doi.org/10.17660/ActaHortic.1995.393.5

Pospíšilová J, Tichá I, Kadle?ek P, Haisel D, Plzáková Š. Acclimatization of micropropagated plants to ex vitro conditions. Biologia Plantarum. 1999;42(4):481-97.

Preece JE, Sutter EG. Acclimatization of micropropagated plants to the greenhouse and field. In: Debergh PC, Zimmerman RH, editors. Micropropagation technology and application. Dordrecht: Kluwer Academic Publishers; 1991. p. 71-93. https://doi.org/10.1007/978-94-009-2075-0_5

Kadlecek P. Effect of pretreatment by irradiance and exogenous saccharose under in vitro conditions on photosynthesis and growth of tobacco (Nicotiana tabacum L.) plants during acclimatization after transfer to soil [Diploma Thesis]. Charles University, Department of Plant Physiology; 1997

Bolar JP, Norelli JL, Aldwinckle HS. An efficient method for rooting and acclimation of micropropagated apple cultivars. Hort Science. 1998;33(7):1251-52. https://doi.org/10.21273/HORTSCI.33.7.1251

Fila G, Ghashghaie J, Hoarau J, Cornic G. Photosynthesis, leaf conductance and water relations of in vitro cultured grapevine rootstock in relation to acclimatization. Physiol Plant. 1998;102(3):411-18. https://doi.org/10.1034/j.1399-3054.1998.1020309.x

Diettrich B, Mertinat H, Luckner M. Reduction of water loss during ex vitro acclimatization of micropropagated Digitalis lanata clone plants. Biochem Physiol Pflanz. 1992;188:23-31. https://doi.org/10.1016/S0015-3796(11)80255-6

Marin JA, Gella R, Herrero M. Stomatal structure and functioning as a response to environmental changes in acclimatized micropropagated Prunus cerasus L. Ann Bot. 1988;62(6):663-70. http://www.jstor.org/stable/42765013

Pospisilova J, Synkova H, Haisel D, Catsky J, Wilhelmova N, Sramek F. Effect of elevated CO2 concentration on acclimation of tobacco plantlets to ex vitro conditions. J Exp Bot. 1999;50(330):119. https://doi.org/10.1093/jxb/50.330.119

Adi NKAP, Astarini IA, Astiti NPA. Acclimatization black orchid (Coelogyne pandurata lindl.) propagated in vitro on different media. Jurnal Simbiosis. 2014;2:203-14.

Andriani V, Pramushinta IAK. Pengaruh media tanam terhadap respon fisiologi aklimatisasi anggrek Cattleya. STIGMA: Jurnal Matematika dan Ilmu Pengetahuan Alam Unipa. 2017;10(1):17-19. https://doi.org/10.36456/stigma.vol10.no1.a1469

Kurniasih W, Nabiila A, Karimah SN, Fauzan MF, Riyanto A, Putra RR. Utilization of zeolite as a media of acclimatization to optimize growth of Phalaenopsis sp. hybrid. Bioma: Jurnal Ilmiah Biologi; 2017. https://doi.org/10.26877/bioma.v6i2.1713

Julhendri J, Gultom H, Fathurrahman F. Acclimatization of anthurium (Anthurium sp.) with various growth media and grow quick fertilizer. Dinamika Pertanian. 2013;28(2):103-12.

Rachmawati F, Pramanik D, Winarto B. Adventitious shoots derived from leaf explants in in vitro mass propagation of indonesian selected Anthurium clones. Int J Agric For Life Sci. 2020;4(2):48-55.

Malabadi RB, Kumar SV, Mulgund GS, Nataraja K. Induction of somatic embryogenesis in papaya (Carica papaya L.). Res Biotechnol. 2011;2(1):40-55.

Nasution LZ, Hasibuan M, Manurung ED. Adaptability of tissuecultured Dendrobium orchid plantlets on planting media and its position during acclimatization process. EandES. 2020;454(1):012166. https://doi.org/10.1088/1755-1315/454/1/012166

Barrales-Lopez A, Robledo-Paz A, Trejo C, Espitia-Rangel E, Rodriguez-De La O JL. Improved in vitro rooting and acclimatization of Capsicum chinense Jacq. plantlets. In Vitro Cell Dev Biol Plant. 2015;51(3):274-83. https://doi.org/10.1007/s11627-015-9671-3

Published

29-12-2024 — Updated on 01-01-2025

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1.
Kaushik J, Pradeep CK, Arun A. Optimizing hardening substrates and homozygosity testing for anther culture derived doubled haploid sweet pepper (Capsicum annuum var. grossum L.) lines . Plant Sci. Today [Internet]. 2025 Jan. 1 [cited 2025 Jan. 6];12(1). Available from: https://horizonepublishing.com/journals/index.php/PST/article/view/5391

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