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A high-throughput flow cytometry system for early screening of in vitro made polyploids in Dendrobium hybrids

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Abstract

Orchids of the genus Dendrobium hold a high economical value in the international markets both as pot plants or cut flowers, and for the production of some metabolites with antioxidant and anti-tumoral activities. Manipulating ploidy levels of Dendrobium species is one of the possible methods to develop new varieties with increased ornamental value and a higher production of secondary metabolites. In this work, we present a new and fast flow cytometry approach to obtain and select Dendrobium phalaenopsis × Dendrobium loddigesii polyploids, through an early in vitro screening on protocorm like bodies (PLBs) after antimitotic treatment. Our approach allows the identification of the best time of treatment on control PLBs and the assessment of best conditions for polyploid recovery just one month after treatments, by using Cycle Value. We were able to discard about the two-third of the unchanged material by drastically reducing the explants to work with and the corresponding costs. Different conditions, regarding concentrations and exposition time, were tested using colchicine or amiprophos-methyl (APM). A high polyploids recovery, up to 80%, were obtained with both antimitotic agents, and those materials were further characterized by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), to identify independent polyploids explants with increased levels in high-value molecules as shihunidine and hircinol, together with stochastic events and genotype-specific metabolite fluctuations.

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Abbreviations

APM:

Amiprophos-methyl

COL:

Colchicine

FCM:

Flow cytometry

HESI:

Heated electrospray ionization

LC-HRMS:

Liquid chromatography-high resolution mass spectrometry

MS:

Murashige and Skoog medium

NDM:

New Dogashima medium

PDP:

Partially duplicated polyploids

PLBs:

Protocorm like bodies

PPE:

Progressively partially endoreduplication

TT:

Total tetraploid

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Acknowledgements

This work was supported by NOVAORCHID Project D. M. 11074/7643/09, funded by Italian Ministry of Agriculture, Food and Forestry Policies. The Authors thank Dimitri Paskulov and Benedetto Aracri (Floramiata SpA, Siena, Italy) for providing seeds of the Dendrobium hybrid. Authors are also very grateful to Raffaela Tavazza (ENEA Casaccia Research Center, Biotechnology and Agroindustry Division, Rome, Italy) for the reading of the manuscript and her valuable suggestions.

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  1. Green Biotechnology Laboratory, Biotechnology and Agroindustry Division, Casaccia Research Center, ENEA - Italian National Agency for New Technologies, Energy and Sustainable Economic Development, via Anguillarese 301, Rome, 00123, Italy

    V. Grosso, A. Farina, D. Giorgi, L. Nardi, G. Diretto & S. Lucretti

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  1. V. Grosso
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  2. A. Farina
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  3. D. Giorgi
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Contributions

VG, LN, SL: Conceived and designed the experiments. VG, AF, DG, LN, GD, SL: Performed the experiment. VG, LN, GD: Analysed the data. VG, DG, GD, SL: Wrote the paper.

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Correspondence to V. Grosso.

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Communicated by Danny Geelen.

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11240_2017_1310_MOESM1_ESM.xlsx

Supplemental Table 1. LC-HRMS of secondary metabolites in the control and in TT/PDP polyploids of Dendrobium PLBs. Data are expressed as fold on the internal standard level (formononetin). For details, see Materials and Methods (XLSX 44 KB)

11240_2017_1310_MOESM2_ESM.xlsx

Supplemental Table 2. LC-HRMS of secondary metabolites in the control and in TT/PDP polyploids of Dendrobium PLBs. Data are expressed as fold on the levels of the corresponding metabolites in control. Red and green boxed indicate, respectively, statistically significant over- and down-accumulated metabolites in a TT/PDP polyploid over the control. For details, see Materials and Methods (XLSX 46 KB)

11240_2017_1310_MOESM3_ESM.xlsx

Supplemental Table 3. Metabolite-metabolite correlation matrix in the control and in TT/PDP polyploids of Dendrobium PLBs. Each square represents the Pearson correlation coefficient between the metabolite heading the column with the metabolite heading the row. Hues of red and green indicate, respectively, the strength of the positive and negative correlation. For details, see Materials and Methods (XLSX 53 KB)

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Grosso, V., Farina, A., Giorgi, D. et al. A high-throughput flow cytometry system for early screening of in vitro made polyploids in Dendrobium hybrids. Plant Cell Tiss Organ Cult 132, 57–70 (2018). https://doi.org/10.1007/s11240-017-1310-8

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