A protocol for Chenopodium quinoa pollen germination

Abstract

Background Quinoa is an increasingly popular seed crop frequently studied for its tolerance to various abiotic stresses as well as its susceptibility to heat. Estimations of quinoa pollen viability through staining methods have resulted in conflicting results. A more effective alternative to stains is to estimate pollen viability through in vitro germination. Here we report a method for in vitro quinoa pollen germination that could be used to understand the impact of various stresses on quinoa fertility and therefore seed yield or to identify male-sterile lines for breeding. Results A semi-automated method to count germinating pollen was developed in PlantCV, which can be widely used by the community. Pollen collected on day 4 after first anthesis at zeitgeber time 5 was optimum for pollen germination with an average germination of 68% for accession QQ74 (PI 614886). The optimal length of pollen incubation was found to be 48 h, because it maximizes germination rates while minimizing contamination. The pollen germination medium’s pH, boric acid, and sucrose concentrations were optimized. The highest germination rates were obtained with 16% sucrose, 0.03% boric acid, 0.007% calcium nitrate, and pH 5.5. This medium was tested on quinoa accessions QQ74, and cherry vanilla with 68%, and 64% germination efficiencies, respectively. Conclusions We provide an in vitro pollen germination method for quinoa with average germination rates of 64 and 68% on the two accessions tested. This method is a valuable tool to estimate pollen viability in quinoa, and to test how stress affects quinoa fertility. We also developed an image analysis tool to semi-automate the process of counting germinating pollen. Quinoa produces many new flowers during most of its panicle development period, leading to significant variation in pollen maturity and viability between different flowers of the same panicle. Therefore, collecting pollen at 4 days after first anthesis is very important to collect more uniformly developed pollen and to obtain high germination rates.

Automated monitoring of pollen quality in Douglas fir using digital images: applications for handling and storage

“…Each type is differentiated by changes in size and circularity, allowing the software to easily distinguish and count between the elongated shapes of the viable pollen (type 1 and 2) from the rounded shapes of non-viable pollen (type 3 and 4) (Webber & Bonnet-Masimbert 1993). This represents an advantage over what is observed in most species, where pollen tube elongation implies a challenge for the creation of a reproducible macro, since under in vitro conditions individual tubes grow at different speeds and can take irregular shapes (Webber & Painter 1996); these irregularities may necessitate extensive manual corrections during analysis (Castillo et al 2022). This limitation may be the main reason for the lack of general macros to quantify germination in other species; however, the results presented here may be applicable to larch (Larix) pollen, which also follows a pre-germinative pattern similar to Pseudotsuga, as well as for other members of the genus (Ho & Rouse 1970, Takaso & Owens 1994, von Aderkas et al 2012.…”

“…This code was subsequently optimized to assess pollen viability in heat-stressed tomatoes (Ayenan et al 2020), and a significant correlation was found with results obtained from manual processing, indicating that the analysis can be adjusted to different species. However, image-based analysis for estimating germinability is mostly underwhelming; neither Fiji nor PlantCV, a semi-automated software designed for this purpose (Castillo et al 2022), allow full automation due to the additional complexity conferred by pollen tube growth. Notwithstanding, such software may be useful for automating evaluation in species where pollen morphology during germination follows a more uniform or predictable pattern.…”

Screening and optimisation of in vitro pollen germination medium for sweetpotato (Ipomoea batatas)

“…To screen a suitable initial medium formulation for in vitro sweetpotato pollen germination, extensive pre-experiments were conducted using many pollen germination media used in other plants. We screened 10 media used in different plants [ 40 – 49 ] (Table 1 , M1–M10) and simplified the composition of M1 (Table 1 , M11), which had the best germination efficiency in the pre-experiments. Pollen germination rates were compared between different media and pure water (Table 1 , CK), and the medium with the highest germination rate was selected for optimisation as the initial medium for in vitro sweetpotato pollen germination.…”

Genetic analysis of cassava brown streak disease root necrosis using image analysis and genome-wide association studies

“…PlantCV Version 2.1 is a Python-based package comprised of modular functions, which has remarkable flexibility, usability, and functionality in processing images from multiple platforms including red, green, and blue (RGB), Near-infrared (NIR), PSII fluorescence, thermal, and hyperspectral sensors. PlantCV has found application in the phenotypic characterization of a diversity of plant species including pennycress, Arabidopsis , wheat, teff, rice, common bean, quinoa, and more ( Castillo et al., 2022 ; Knapp et al., 2022 ; Griffiths et al., 2023 ; Pierz et al., 2023 ), underscoring its significance as a valuable tool for quantitative classification of plant phenotypes.…”