Validation of nucellus-based direct somatic embryogenesis mutagenesis and phenotypic assessment of M1 mutants in kinnow mandarin (Citrus nobilis × Citrus deliciosa)

AMINA  SHUKOOR; OM PRAKASH  AWASTHI; R M  SHARMA; N V  SINGH; BHUPINDER  SINGH; THEIVANAI  M; JAGDISH  CHAWLA; ANAMIKA  RAI

doi:10.56093/ijas.v96i1.172242

Authors

AMINA SHUKOOR ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
OM PRAKASH AWASTHI ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
R M SHARMA ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
N V SINGH ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
BHUPINDER SINGH ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
THEIVANAI M ICAR-Central Coastal Agricultural Research Institute, Ela, Old Goa, Dist. North Goa, Goa, India
JAGDISH CHAWLA ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
ANAMIKA RAI ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India

https://doi.org/10.56093/ijas.v96i1.172242

Keywords:

Cluster analysis, Correlation, Direct somatic embryogenesis, EMS, Gamma, In ovulo nucellus, Regenerants

Abstract

In ovulo nucellus based direct somatic embryogenesis (DSE) is an efficient tool to assess mutagenic efficiency in perennial crops. A study was carried out in 2022–23 and 2023–24 at ICAR-Indian Agricultural Research Institute, New Delhi to compare the influence of 0.1% ethyl methane sulphonate (EMS) for 5 h and gamma irradiation (80 Gy) on embryogenesis, plantlet regeneration, and growth traits in kinnow mandarin (Citrus nobilis × Citrus deliciosa). Both treatments significantly reduced ovule survival compared with control (86.67%). The survival declined to 48.00% under EMS (0.1% for 5 h) and 35.78% following gamma irradiation (80 Gy for 10.9 min). The frequency of direct somatic embryogenesis declined to 49.07% under EMS and 43.85% under gamma irradiation. In contrast, post embryogenic parameters, including germination and bipolar conversion efficiency, were more severely affected, decreasing to 39.06–41.89% under EMS and 32.87–30.14%, under gamma treatment. Regenerants displayed short stature, reduced internodal length, and smaller leaves, though leaf number remained unaffected, suggesting that stress mainly altered expansion and elongation rather than leaf initiation. Correlation analysis indicated that biomass under EMS was closely linked to leaf area, while gamma treatment weakened relationships between height related traits and biomass. Cluster analysis revealed moderate variability under EMS but greater divergence among gamma irradiated populations. Overall, EMS generated point mutations with moderate phenotypic effects, whereas gamma rays exerted stronger disruptive impacts on regeneration and variability. These findings demonstrate that in ovulo nucellus-derived DSE is a robust system for evaluating mutagenic efficiency and optimizing treatments for generating targeted variability in perennial fruit crops.

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References

Agisimanto D, Noor N M, Ibrahim R and Mohamad A. 2016. Gamma irradiation effect on embryogenic callus growth of Citrus reticulata cv. Limau Madu. Sains Malaysiana 45(3): 329–37.

Apio H B, Elegba W, Nunekpeku W, Otu S A, Baguma J K, Alicai T, Danso K E, Bimpong I K and Ogwok E. 2024. Effect of gamma irradiation on proliferation and growth of friable embryogenic callus and in vitro nodal cuttings of Ugandan cassava genotypes. Frontiers in Plant Science 15: 1414128. DOI: https://doi.org/10.3389/fpls.2024.1414128

Bhojwani S S and Razdan M K. 1996. Plant Tissue Culture: Theory and Practice, Revised edition, Vol. 5. Elsevier, Amsterdam, The Netherlands. DOI: https://doi.org/10.1016/S0928-3420(96)80002-4

Bisht V, Rawat J M, Gaira K S, Purohit S, Anand J, Sinha S and Rawat B. 2024. Assessment of genetic homogeneity of in vitro propagated apple root stock MM 104 using ISSR and SCoT primers. BMC Plant Biology 24(1): 240. DOI: https://doi.org/10.1186/s12870-024-04939-3

Carra A, Wijerathna-Yapa A, Pathirana R and Carimi F. 2024. Development and applications of somatic embryogenesis in grapevine (Vitis spp.). Plants 13(22): 3131. DOI: https://doi.org/10.3390/plants13223131

Cimen B, Yesiloglu T, Incesu M and Yilmaz B. 2021. Studies on mutation breeding in citrus: Improving seedless types of ‘Kozan’ common orange by gamma irradiation. Scientia Horticulturae 278: 109857. DOI: https://doi.org/10.1016/j.scienta.2020.109857

Elnahal A S, El-Aref H M, Zayed B A, Hammad M A, Soliman A S and Abdelkhalik A F. 2025. Evaluation of EMS-induced variation in processed tomato: Morphological traits and yield associations. Plants 14: 2531.

Ferrari I F, Marques G A, Junior W L S, Biazotti B B, Pena Passos M, de Almeida J A S and Mayer J L S. 2021. Comparative ontogenesis of Coffea arabica L. somatic embryos reveals the efficiency of regeneration modulated by the explant source and the embryogenesis pathway. In Vitro Cellular and Developmental Biology-Plant 57(5): 796–810. DOI: https://doi.org/10.1007/s11627-021-10200-5

Gautam A S and Sood K C. 1992. Mutagenic effectiveness and efficiency of gamma rays, EMS, and synergistic effects in black gram (Vigna mungo). Cytologia 57: 85–89. DOI: https://doi.org/10.1508/cytologia.57.85

Ghasemi-Soloklui A A, Kordrostami M and Karimi R. 2023. Determination of optimum dose based of biological responses of lethal dose (LD25, 50, 75) and growth reduction (GR25, 50, 75) in ‘Yaghouti’grape due to gamma radiation. Scientific Reports 13(1): 2713. DOI: https://doi.org/10.1038/s41598-023-29896-z

Ghasemi-Soloklui AA, Kordrostami M and Jafari M. 2025. Deter-mination of optimal gamma radiation dose for mutation breeding in ’Sabz’ fig (Ficus carica L.) cuttings based on radiosensitivity and phenotypic changes. PLoS One 20(1): e0313017. DOI: https://doi.org/10.1371/journal.pone.0313017

Ikeuchi M, Favero D S, Sakamoto Y, Iwase A, Coleman D, Rymen B and Sugimoto K. 2019. Molecular mechanisms of plant

regeneration. Annual Review of Plant Biology 70: 377–406.

Konzak C F, Nilan R A, Wagner J and Foster R J. 1965. Efficient chemical mutagenesis. (In) The Use of Induced Mutations in Plant Breeding, pp. 49–70.

Pergamon Press/FAO/IAEA, Vienna. Kumar S, Awasthi O P, Sharma R M and Pradhan S. 2021. Physiological and biochemical responses of kinnow mandarin (Citrus nobilis × Citrus deliciosa) to EMS induced mutagenesis. The Indian Journal of Agricultural Sciences 91: 1015–19. DOI: https://doi.org/10.56093/ijas.v91i7.115116

Kumar A, Singh H, Pathania S, Bajaj K, Sangwan A and Sharda R. 2025. Effect of varying nitrogen and phosphorus rates under deficit irrigations on the performance of ‘kinnow’ mandarin (Citrus reticulata Blanco). Applied Fruit Science 67(4): 182. DOI: https://doi.org/10.1007/s10341-025-01411-w

Maluszynski M, Nichterlein K, Van Zanten L and Ahloowalia B S. 2000. Officially released mutant varieties–The FAO/IAEA Database. Mutation Breeding Review 12: 1–84.

Murugan T, Awasthi O P, Singh S K, Chawla G, Solanke A U, Kumar S and Jha G K. 2023. Molecular and histological validation of modified in ovulo nucellus culture based high-competency direct somatic embryogenesis and amplitude true-to-the-type plantlet recovery in kinnow mandarin. Frontiers in Plant Science 14: 1116151. DOI: https://doi.org/10.3389/fpls.2023.1116151

Murugan T, Awasthi O P, Chellapilla B and Shankar K. 2024. EMS induced in vitro mutagenesis for solid mutant induction and validation of mutants using morphological and molecular tools in kinnow mandarin (Citrus nobilis × Citrus deliciosa). The Indian Journal of Agricultural Sciences 94(6): 644–49. DOI: https://doi.org/10.56093/ijas.v94i6.145347

Neeraj B K, Singh M K, Singh R K, Singh A K and Pal A K. 2021. Mutagenic effectiveness and efficiency of gamma rays and EMS on chilli (Capsicum annum L.). International Journal of Chemical Studies 9(1): 2127–29. DOI: https://doi.org/10.22271/chemi.2021.v9.i1ad.11535

Nkurunziza R, Jankowicz-Cieslak J, Bocianowski J, Bhatnagar-Mathur P, Werbrouck S P and Ingelbrecht I L. 2025. Enhancing genetic diversity in Coffea arabica L. through induced mutagenesis. Current Plant Biology 43: 100514. DOI: https://doi.org/10.1016/j.cpb.2025.100514

Penna S, Vitthal S B and Yadav P V. 2012. In vitro mutagenesis and selection in plant tissue cultures and their prospects for crop improvement. Bioremediation Biodiversity Bioavailability 6: 6–14.

Piccioni E, Barcaccia G, Falcinelli M and Standardi A. 1997. Estimating alfalfa somaclonal variation in axillary branching propagation and indirect somatic embryogenesis by RAPD fingerprinting. International Journal of Plant Sciences 158(5): 556–62. DOI: https://doi.org/10.1086/297467

Riyadi I and Sumaryono. 2017. Effect of gamma irradiation on in vitro shoot regeneration and somaclonal variation in banana (Musa spp.). Indonesian Journal of Agricultural Science 18(2): 63–70.

Roose M L and Williams T E. 2007. Mutation Breeding. (In) Citrus Genetics, Breeding and Biotechnology, pp. 345–52. Khan I A (Ed). CABI International, Wallingford. DOI: https://doi.org/10.1079/9780851990194.0345

Singh S, Gupta M, Rattanpal H S, Singh G and Kaur N. 2023. Studies on fruit pedicel concerning fruitlet abscission in kinnow mandarin. Indian Journal of Horticulture 80(1): 80–85. DOI: https://doi.org/10.58993/ijh/2023.80.1.12

Theivanai M. 2023. ‘In vitro mutagenesis and validation of mutants using molecular markers in Kinnow mandarin’. PhD Thesis, ICAR-Indian Agricultural Research Institute, New Delhi.

Van Harten A M. 1998. Mutation Breeding: Theory and Practical Applications, pp. 353. Cambridge University Press, Cambridge. Witjaksono and Litz R E. 2004. Effect of gamma irradiation on embryogenic avocado cultures and somatic embryo develop-

ment. Plant Cell, Tissue and Organ Culture 77(2): 139–47.