Abstract
Doubled haploidy serves as an effective tool for enhancing genetic gain in plant breeding programs. Androgenesis, an extensively-employed form of doubled haploidy, involves totipotent induction within the immature microgametophyte followed by agamous embryogenesis and chromosome doubling. Protocol development for in vitro androgenesis is reliant upon the application of species- (and oftentimes genotype-) specific induction stimuli as well as phenotypic markers that reflect cellular reprogramming. In cell cultures, however, the latter is impeded readily by pseudo-embryogenic structures of somatic origin. In this study, gynoecium-derived trichomes of soybean were intentionally incorporated into isolated microspore cultures. Trichome morphology was then compared to early-dividing zygotic and apozygotic plant embryos previously reported, as were nuclei orientation and cross wall composition. Lastly, autofluorescence intensity was measured for trichomes and various microspore phenotypes, the results of which implied that autofluorescence post-light exposure may be a useful parameter for culture purification and/or quality control. The findings herein demonstrate that trichomes serve a pseudo-embryogenic role during in vitro androgenesis in soybean and necessitate extra caution when identifying sporophytic tissues in species with floral pubescence.
Key message
This manuscript draws morphological parallels between eudicot embryos and gynoecial trichomes, and may be used as a reference/cautionary guide for the identification of pseudo-embryos in vitro.
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs11240-021-02071-w/MediaObjects/11240_2021_2071_Fig1_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs11240-021-02071-w/MediaObjects/11240_2021_2071_Fig2_HTML.png)
Abbreviations
- CW:
-
Calcofluor white M2R
- DAPI:
-
4′6-Diamidino-2-phenylindole
- MCT:
-
Multicellular secretory Trichome
- IMC:
-
Isolated microspore culture
- PBS:
-
Phosphate-buffered saline
References
Arteaga N, Savic M, Méndez-Vigo B, Fuster-Pons A, Torres-Pérez R, Oliveros JC, Picó FX, Alonso-Blanco C (2021) MYB transcription factors drive evolutionary innovations in Arabidopsis fruit Trichome patterning. Plant Cell. https://doi.org/10.1093/plcell/koaa041
Bal U, Shariatpanahi ME, Castro AJ, Emery D, Clement C, Dehestani-Ardakani M, Touraev A (2012) Pseudo-embryogenic structures in anther and isolated microspore cultures in vitro: a cautionary guide. Czech J Gen Plant Breed 48:51–60
Beilstein MA, Al-Shehbaz IA, Kellogg EA (2006) Brassicaceae phylogeny and trichome evolution. Am J Bot 93:607–619
Cardoso MB, Bodanese-Zanettini MH, Mundstock EC, Kaltchuk-Santos E (2007) Evaluation of gelling agents on anther culture: response of two soybean cultivars. Braz Arch Biol Technol 50:933–939
Coumans M, Zhong D (1995) Doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 2. In vitro isolated microspore culture. Plant Cell Tiss Org Cult 41:203–209
Deslauriers C, Powell AD, Fuchs K, Pauls KP (1991) Flow cytometric characterization and sorting of cultured Brassica napus microspores. Biochim Biophys Acta (BBA) Mol Cell Res 1091:165–172
Fahn A (2000) Structure and function of secretory cells. Adv Bot Res 31:37–75
Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tiss Org Cult 104:301–309
Garda M, Hale B, Rao N, Lowe M, Bright M, Goodling S, Phillips GC (2020) Soybean androgenesis I: identification of pyramidal stressors in anther cultures that sustain cell divisions and embryo formation from isolated microspore cultures. In Vitro Cell Dev Biol Plant 56:415–429
Hale B, Phipps C, Rao N, Wijeratne A, Phillips GC (2020) Differential expression profiling reveals stress-induced cell fate divergence in soybean microspores. Plants 9:1510
Hale B, Phipps C, Rao N, Kelley C, Phillips GC (2021) Soybean androgenesis II: non-gametophytic morphologies in isolated microspore culture. In Vitro Cell Dev Biol Plant. https://doi.org/10.1007/s11627-020-10144-2
Hauser MT (2014) Molecular basis of natural variation and environmental control of trichome patterning. Front Plant Sci 5:320
Healy RA, Horner HT, Bailey TB, Palmer RG (2005) A microscopic study of trichomes on gynoecia of normal and tetraploid Clark cultivars of Glycine max and seven near-isogenic lines. Int J Plant Sci 166:415–425
Healy RA, Palmer RG, Horner HT (2009) Multicellular secretory trichome development on soybean and related Glycine gynoecia. Int J Plant Sci 170:444–456
Horner HT, Healy RA, Cervantes-Martinez T, Palmer RG (2003) Floral nectary fine structure and development in Glycine max L. (Fabaceae). Int J Plant Sci 164:675–690
Joosen R, Cordewener J, Supena EDJ, Vorst O, Lammers M, Maliepaard C, Boutilier K (2007) Combined transcriptome and proteome analysis identifies pathways and markers associated with the establishment of rapeseed microspore-derived embryo development. Plant Physiol 144:155–172
Pechan PM, Keller WA, Mandy F, Bergeron M (1988) Selection of Brassica napus L. embryogenic microspores by flow sorting. Plant Cell Rep 7:396–398
R Core Team (2017) R: a language and environment for statistical computing. R Found. Stat, Comp
Schulze D, Pauls KP (1998) Flow cytometric characterization of embryogenic and gametophytic development in Brassica napus microspore cultures. Plant Cell Physiol 39:226–234
Seguí-Simarro JM, Nuez F (2007) Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers. J Exp Bot 58:1119–1132
Sharma S, Sangwan NS, Sangwan RS (2003) Developmental process of essential oil glandular trichome collapsing in menthol mint. Curr Sci 84:544–550
Soriano M, Li H, Boutilier K (2013) Microspore embryogenesis: establishment of embryo identity and pattern in culture. Plant Reprod 26:181–196
Tang X, Liu Y, He Y, Ma L, Sun MX (2013) Exine dehiscing induces rape microspore polarity, which results in different daughter cell fate and fixes the apical-basal axis of the embryo. J Exp Bot 64:215–228
Werker E (2000) Trichome diversity and development. Adv Bot Res 31:1–35
Yu M, Zhao J (2012) The cytological changes of tobacco zygote and proembryo cells induced by beta-glucosyl Yariv reagent suggest the involvement of arabinogalactan proteins in cell division and cell plate formation. BMC Plant Biol 12:126
Acknowledgements
This study was funded through support from USDA-NIFA Non-Land Grant Colleges of Agriculture Capacity Building award number 2018-70001-28762, Arkansas Soybean Promotion Board, University of Arkansas System Division of Agriculture, Corteva Agriscience Open Innovation, and Corteva Agriscience Internship Program.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by Sergio J. Ochatt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Hale, B., Lor, P., Chellamma, S. et al. Gynoecium pubescence in soybean: a prevalent false-positive during in vitro androgenesis. Plant Cell Tiss Organ Cult 146, 417–421 (2021). https://doi.org/10.1007/s11240-021-02071-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-021-02071-w