Fenotipagem de alta performance, indução de haploides, germinação in vitro e microenxertia em Opuntia spp.

Abstract

Opuntia spp. has significant economic relevance in semi-arid regions but faces challenges in genetic characterization, efficient propagation, and compatibility between micrografts. Thus, this study aimed to perform high-performance phenotyping for genetic divergence analysis, correlate sepal length with microspore development, induce embryogenesis through androgenesis and gynogenesis, evaluate the effect of gibberellic acid (GA₃) on in vitro germination, conduct micropropagation, and finally, test the compatibility of micrografts with different ploidy levels using antioxidants to reduce oxidation in the micrograft region of Opuntia spp. For high-performance phenotyping, seeds from 13 accessions were evaluated for color and eight quantitative characteristics using ImageJ® software. Tocher clustering was based on Mahalanobis distance, while the graphical dispersion of accessions was analyzed through canonical variables. The relative importance of the variables was determined using Singh’s method and canonical variables. In the experiment correlating sepal length with microspore development, floral buds were classified into four stages (5.0–5.9 mm, 6.0–6.9 mm, 7.0–7.9 mm, 8.0–8.9 mm). Pearson’s correlation and a completely randomized design were used. For embryogenesis induction, 25 anthers and 25 ovules were inoculated in Murashige & Skoog (1962) medium with four sucrose concentrations (0, 30, 60, and 90 g.L⁻¹) and fixed doses of benzylaminopurine (BAP) and indoleacetic acid (IAA) (2 mg.L⁻¹). In the evaluation of GA₃ effects on germination, seeds were disinfected and inoculated in MS medium with different GA₃ concentrations (0, 15, 25, and 30 mg.L⁻¹). The germination percentage, germination speed index (GSI), seedling length, stem diameter, and root length were analyzed. For micropropagation, the number of shoots per accession was assessed. The experiments followed a completely randomized design in a 13 × 4 factorial scheme, with 52 treatments and four replications. In micrografting, three Opuntia spp. accessions with different ploidy levels were used: Opuntia ficus indica (2n = 8x = 88), Opuntia undulata (2n = 4x = 44), and Opuntia cochenillifera (2n = 2x = 22). Homo- and heterograft combinations were evaluated. To reduce oxidation, three treatments were applied: T1 – autoclaved distilled water (control), T2 – 5.68 µM ascorbic acid, and T3 – 5.68 µM cysteine. Compatibility was assessed after 45 days. The results of all experiments were subjected to variance analysis, and means were compared using Tukey’s test (5% and 1% probability) and Scott Knott (5% and 1% probability) in GENES software. The results showed that Opuntia cochenillifera accessions had the highest values for the evaluated characteristics. Tocher clustering formed four groups, with the variables area, perimeter, roundness, solidity, and seed fresh mass contributing to accession differentiation. Phenotyping proved to be an efficient tool for genetic characterization. Significant inverse correlations were observed between sepal length and microspore stage. Lengths between 5.0 and 6.9 mm were ideal for androgenesis induction. The MS + 2.0 mg.L⁻¹ BAP + 2.0 mg.L⁻¹ IAA + 60 g.L⁻¹ sucrose treatment was optimal for embryo induction. GA₃ concentrations of 15 and 25 mg.L⁻¹ promoted higher GSI, while 30 mg.L⁻¹ reduced germination. Accessions 5, 45, 57, and 81 showed high germination rates without GA₃. Accessions 5, 57, and 81 demonstrated potential for micropropagation. The use of ascorbic acid favored micrograft establishment by reducing oxidation. After 45 days, compatible grafts exhibited callus formation and vascular connection. Under drought conditions, Opuntia cochenillifera is recommended as a scion and Opuntia ficus-indica as a rootstock. This study contributes to the propagation and selection of Opuntia spp.