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. 2022 May 19;11(10):1347.
doi: 10.3390/plants11101347.

Induction of Somatic Embryogenesis in Tamarillo (Solanum betaceum Cav.) Involves Increases in the Endogenous Auxin Indole-3-Acetic Acid

André Caeiro  1 , Sandra Caeiro  1 , Sandra Correia  1 , Jorge Canhoto  1
Affiliations

Induction of Somatic Embryogenesis in Tamarillo (Solanum betaceum Cav.) Involves Increases in the Endogenous Auxin Indole-3-Acetic Acid

André Caeiro et al. Plants (Basel). .

Abstract

Somatic embryogenesis (SE) is a complex biological process regulated by several factors, such as the action of plant growth regulators, namely auxins, of which the most physiologically relevant is indole-3-acetic acid (IAA). In tamarillo, an optimized system for induction of SE creates, after an induction process, embryogenic (EC) and non-embryogenic callus (NEC). In this work the endogenous levels of auxin along the induction phase and in the calli samples were investigated using chemical quantifications by colorimetric reactions and HPLC as well as immunohistochemistry approaches. Differential gene expression (IAA 11, IAA 14, IAA 17, TIR 1, and AFB3) analysis during the induction phase was also carried out. The results showed that the endogenous IAA content is considerably higher in embryogenic than in non-embryogenic calli, with a tendency to increase as the dedifferentiation of the original explant (leaf segments) evolves. Furthermore, the degradation rates of IAA seem to be related to these levels, as non-embryogenic tissue presents a higher degradation rate. The immunohistochemical results support the quantifications made, with higher observable labeling on embryogenic tissue that tends to increase along the induction phase. Differential gene expression also suggests a distinct molecular response between EC and NEC.

Keywords: HPLC; IAA; auxins; embryogenic calli; gene expression; immunohistochemistry.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Plant material. (A) Time course of leaf explant SE induction; leaves from in vitro established plantlets were used. EC 2 and NEC 2 are the embryogenic and non-embryogenic calli resulting from the induction process. (B) EC 2 and NEC 2 were previously established from zygotic embryos and used in the IAA quantifications. The bars in each figure represent 1 mm with exception of the leaves in which the length is 1 cm.
Figure 2
Figure 2
IAA assays in time courses from leaf segment induction. NEC and EC are non-embryogenic and embryogenic calli, respectively, similar to EC 1 and NEC 1 represented in Figure 1. (A) Immunohistochemistry observations. Fluorescence, transmission, and composed image of the different tissues, unlabeled tissue samples from EC were used as control (C—control; 8 w—8 weeks induction; 10 w—10 weeks induction; 12 w—12 weeks induction; NEC—non-embryogenic callus; EC—embryogenic callus). (B) Raw integrated intensity for each sample. (C) IAA quantification by HPLC. Results are presented as mean ± SD. Different letters are significantly different according to Tukey test (p < 0.05).
Figure 3
Figure 3
IAA quantification on induced embryogenic (EC) and non-embryogenic (NEC) calli, where EC 1 and NEC 1 are induced from zygotic embryos in 2,4-D supplemented medium and NEC 2, EC 2, and EC 3 are from leaf segments in a picloram-supplemented medium. (A) IAA quantification by Ehrlich reaction. (B) Statistical analysis of IAA in embryogenic calli assayed by Ehrlich reaction. (C) Statistical analysis of IAA in non-embryogenic calli assayed by Ehrlich reaction. (D) IAA degradation measured by discontinuous assay. (E) IAA quantification by HPLC. (F) Statistical analysis of IAA in embryogenic calli assayed by HPLC. (G) Statistical analysis of IAA in non-embryogenic calli assayed by HPLC. (H) Comparison of IAA measurement Ehrlich reaction and HPLC comparison between HPLC and Ehrlich reaction. Results of HPLC quantification (x axis) were plotted against the results of Ehrlich quantification (y axis) for each callus tissue tested. The linear fit equation is y = 4.818x − 0.3963 (R2 = 0.969). Results are presented as mean ± SD. Different letters are significantly different according to Tukey test or by the unpaired t-test in the case of non-embryogenic calli analysis (p < 0.05; n = 3).
Figure 4
Figure 4
HPLC quantification parameters. (A) Retention time of IPA (peak 1, Rt = 5.34 min) and IAA (peak 2, Rt = 6.00 min) in a sample. (B) IAA standard (Rt = 5.98 min). (C) UV-spectrum of IAA. (D) Calibration curve used in the quantification of IAA with the linear equation Area = 2.633 × 107[IAA] + 2.094 × 106 (R2 = 0.9984).
Figure 5
Figure 5
Relative gene expression. Relative expression was calculated in relation to reference genes previously validated for tamarillo. Results are presented as mean ± SD. Different letters are significantly different according to Tukey test (p < 0.05).
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