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Establishment of an efficient protocol for in vitro disinfection of seeds of seven Agave spp. species

By María Guadalupe Aguilar Rito, Amaury Martín Arzate Fernández*, Hilda Guadalupe García Núñez, Tomas Héctor Norman Mondragón

* Corresponding Author. Email: - / Institution: Universidad Autónoma del Estado de México

Accepted: 29/November/2023 – Published: 20/December/2023DOI: https://doi.org/10.18781/R.MEX.FIT.2310-1

Abstract Background. Disinfection of Agave seeds is a crucial step in in vitro culture to prevent contamination, which can be caused by microorganisms such as bacteria, fungi and viruses that can affect seedling growth and reduce seed germination rate. Therefore, proper seed disinfection is essential to ensure vigorous and healthy plant growth. Objective. Generate an efficient seed disinfection protocol in seven species of Agave; Agave marmorata, A. karwinskii, A. potatorum, A. angustifolia, A. cupreata, A. horrida and A. salmiana to reduce pollution levels.

Materials and Methods. A total of 12 disinfection treatments with disinfectants and different combinations were evaluated. The disinfectants used were; 3 % Hydrogen Peroxide for 24 h, Commercial Sodium Hypochlorite 5 % (v/v) for 5 min, Calcium Hypochlorite 8 % (w/v) for 15 min, Copper Sulfate 30 % (v/v) for 10 min, Mercury Chloride II 0.1 % (w/v) for 10 min. Before each treatment was tested, the seeds were pre-washed with liquid soap and subjected to the treatments, Subsequently, they were sown in DM medium and the percentage of germination and contamination for each treatment was evaluated weekly for a period of 30 days. Additionally, the contaminating microorganisms found were identified.

Results. The best treatment for seed disinfection was 30 % copper sulfate (v/v) for 10 min, 0.1 % mercuric chloride II for 10 min and 3 % hydrogen peroxide for 24 h, obtaining 100 % disinfection. Four genera of fungi were identified: Monilinia sp., Aspergillus sp., Penicillium sp., and Alternaria alternata, a bacterium; Bacillus sp., and a yeast, Schizosaccharomyces sp.

Keywords: Seeds, agave, disinfection methods, contamination, germination

Figure 1. A. salmiana seed germination. A) root emergence and elongation. (14 dap) B) Elongation of the plumule. (21 dap) C) Diferenciation of the root cotyledons and development. (30 dap) D) Full germination and development. (14-30 dap) E) Normal, completely developed A. salmiana seedlings. (50 dap); dap days after planting
Figure 1. A. salmiana seed germination. A) root emergence and elongation. (14 dap) B) Elongation of the plumule. (21 dap) C) Diferenciation of the root cotyledons and development. (30 dap) D) Full germination and development. (14-30 dap) E) Normal, completely developed A. salmiana seedlings. (50 dap); dap days after planting
Figure 2. Bacterial and fungal strains found in the seeds of Agave spp. A) <em>Schizosaccharomyces sp.</em> B) <em>Bacillus</em> sp. C) Penicilllium sp. D) <em>Alternaria alternata</em>. E) <em>Aspergillus sp.</em> F) <em>Monilinia sp.</em>
Figure 2. Bacterial and fungal strains found in the seeds of Agave spp. A) Schizosaccharomyces sp. B) Bacillus sp. C) Penicilllium sp. D) Alternaria alternata. E) Aspergillus sp. F) Monilinia sp.
Table 1. Agave species and seed collection data
Table 1. Agave species and seed collection data
Table 2. Disinfection treatments evaluated in seven Agave species
Table 2. Disinfection treatments evaluated in seven Agave species
Table 3. Analysis of variance for the variables contamination and germination, evaluating 12 disinfection treatments and seven agave species.
Table 3. Analysis of variance for the variables contamination and germination, evaluating 12 disinfection treatments and seven agave species.
Table 4. Analysis of variance for the variables contamination and germination, evaluatinMeans comparison for the variables contamination and germination in seven agave species, according to Tukey’s test (p<0.05).g 12 disinfection treatments and seven agave species.
Table 4. Analysis of variance for the variables contamination and germination, evaluatinMeans comparison for the variables contamination and germination in seven agave species, according to Tukey’s test (p<0.05).g 12 disinfection treatments and seven agave species.
Table 5. Contaminant microorganisms found in the Agave seeds after applying the treamtnets
Table 5. Contaminant microorganisms found in the Agave seeds after applying the treamtnets