Liquid Culture System for Mature Pollen in Hyuganatsu (Citrus tamurana hort. ex Tanaka)

飛香, 内田; 秋晴, 安部; 洋一郎, 星野; 久登, 國武

doi:10.2503/hrj.11.173

Cited by 6 publications

(2 citation statements)

References 19 publications

(1 reference statement)

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“…POLLEN CULTURE AND NANO-LC-MS ANALYSIS BY C-AND SI-LIKE TREATMENTS. Pollen grains of 'Banpeiyu' were cultured in the Citrus mature pollen culture system as described by Uchida et al (2012a), for 4-h initial cultivation in the dark at 25°C. Stylar crude protein extracts, derived from the styles of 'Hassaku' or 'Banpeiyu' were added into the medium to reach 50 mgÁmL -1 final concentration, as C-or SI-like treatment, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…In 'Hyuganatsu', protein expression in the pistils has been identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and has indicated that the pistils are converted from self-compatible to SI 3 to 5 d before anthesis (Uchida et al, 2012b). Moreover, for investigation of Citrus pollen tubes, we have established an in vitro pollen culture system (Uchida et al, 2012a) and an in vitro SI-like reaction system using the stylar crude protein extract treatment (Li et al, 2015). It has also been demonstrated that stylar crude protein extracts induced SI response in apple pollen tubes, as well as purified S-RNase protein in vitro (Meng et al, 2014).…”

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In Vitro Self-incompatible-like Response Applied for Protein Identification and Gene Expression Analysis in Citrus Cultivars, Banpeiyu and Hyuganatsu

Abe

Fuse

et al. 2015

J. Amer. Soc. Hort. Sci.

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Self-incompatibility (SI) is an important mechanism in higher plants that promotes outcrossing and prevents self-fertilization. ‘Banpeiyu’ (Citrus maxima) and ‘Hyuganatsu’ (Citrus tamurana), two of the Citrus cultivars distributed in Kyusyu, Japan, show gametophytic SI. In this study, we used the Citrus mature pollen culture system and stylar crude protein extracts to simulate compatible (C) and SI responses in ‘Banpeiyu’ pollen tubes. We analyzed the protein changes in pollen tubes with the C- and SI-like treatments by nano-liquid chromatography–mass spectrometry (nano-LC-MS); 14 and 27 proteins were identified in C- and SI-like treatments, respectively. We picked up some candidate genes that were particularly prevalent in SI-like treatment and analyzed their expression level changes during C- and SI-like treatments in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes. The expression levels of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese SOD (Mn SOD), catalase (CAT), and cysteine protease (CYP) increased after SI-like treatment. We used a fluorescent probe to visualize reactive oxygen species (ROS) level changes in ‘Banpeiyu’ and ‘Hyuganatsu’ pollen tubes after C- and SI-like treatments and found that 2-hour SI-like treatment induced ROS levels to increase in the pollen tubes of both cultivars. These results suggest that an ROS increase could be one of the key phenomena in the SI response of Citrus and that gene expression changes were responses to ROS generation.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

In Vitro Self-incompatible-like Response Applied for Protein Identification and Gene Expression Analysis in Citrus Cultivars, Banpeiyu and Hyuganatsu

Abe

Fuse

et al. 2015

J. Amer. Soc. Hort. Sci.

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Single-pollen genotyping to estimate mode of unreduced pollen formation in Citrus tamurana cv. Nishiuchi Konatsu

et al. 2016

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2n pollen formed by FDR in citrus. The Japanese local citrus cultivar, Nishiuchi Konatsu (Citrus tamurana hort. ex Tanaka; NK hereafter), has the ability to produce unreduced 2n pollen grains, allowing generation of polyploid progenies via sexual polyploidization. In this study, we developed a method of single-pollen genotyping for citrus and applied it to the analysis of transmission of heterozygosity in NK 2n pollen grains. Heterozygosity transmission was expressed as the percentage inheritance of a set of heterozygous alleles from the parent to the 2n gamete. The pathway of 2n pollen development was investigated by applying the observed heterozygosity transmission and genetic distance to two different map functions, for first division restitution (FDR) and second division restitution (SDR). The fit of the values observed for both functions was calculated, while virtually moving the centromere position. We screened for six heterozygous SSR (codominant microsatellite marker loci) in NK, all of which were expected to lie within the same linkage group. Pollen germination prior to DNA extraction was essential for this work, and 6-h incubation proved to be optimal for subsequent PCR amplification. Single-pollen genotyping unreduced NK 2n pollen grains revealed that heterozygosity transmission exceeded 50 % in all six alleles, and fitness tests indicated that the FDR map function better fitted the heterozygosity transmission observed rather than the SDR function. Our data thus strongly indicate that 2n pollen in NK is a result of first division restitution.

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Deep learning-based high-throughput detection of in vitro germination to assess pollen viability from microscopic images

Zhang

Zhao

Hoshino

2023

Journal of Experimental Botany

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In vitro pollen germination is considered the most efficient method to assess pollen viability. The pollen germination frequency and pollen tube length, which are key indicators of pollen viability, should be accurately measured during in vitro culture. In this study, a Mask R-CNN model trained using microscopic images of tree peony (Paeonia suffruticosa) pollen has been proposed to rapidly detect pollen germination rate and pollen tube length. To reduce the workload during image acquisition, images of synthesized crossed pollen tube were added to the training dataset, significantly improving the model accuracy in recognizing crossed pollen tubes. At an Intersection over Union threshold of 50%, a mean average precision of 0.949 was achieved. The performance of the model was verified using 120 testing images. The R 2 value of the linear regression model using detected pollen germination frequency against the ground truth was 0.909 and that using average pollen tube length was 0.958. Further, the model was successfully applied to two other plant species indicating a good generalizability and potential to be applied widely.

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Liquid Culture System for Mature Pollen in Hyuganatsu (Citrus tamurana hort. ex Tanaka)

Cited by 6 publications

References 19 publications

In Vitro Self-incompatible-like Response Applied for Protein Identification and Gene Expression Analysis in Citrus Cultivars, Banpeiyu and Hyuganatsu

In Vitro Self-incompatible-like Response Applied for Protein Identification and Gene Expression Analysis in Citrus Cultivars, Banpeiyu and Hyuganatsu

Single-pollen genotyping to estimate mode of unreduced pollen formation in Citrus tamurana cv. Nishiuchi Konatsu

Deep learning-based high-throughput detection of in vitro germination to assess pollen viability from microscopic images

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