Polyploidization of Plumbago auriculata Lam. in vitro and its characterization including cold tolerance
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Plumbago auriculata Lam. (Plumbaginaceae) is an ornamental/medicinal flowering shrub. However, it could be stop growing under 5 °C resulting in its poor germplasm resources. Increasing the tolerance of polyploids to environmental stress will enrich its germplasm resources. In this study, we thus employed the stem segments and buds exposed to pendimethalin and trifluralin in vitro to conduct an effective polyploid protocol, in which buds were cultured for 7 days in MS medium with 800 μM. In this study, we have developed a novel polyploid identification method based on accuracy and efficiency. Early screening was performed based on the morphological characteristics of this plants, and then polyploidy was determined by flow cytometry. The number of chromosomes was determined by the chromosomal accounting. At the anatomical level, in comparison with diploids, the root parenchyma, stem collenchyma and tetraploid spongy tissues were thickened, the guard cell size and vascular bundles number were increased, and the stomata were widened; but the stomatal density was decreased. After 24 h of cold stress, the maximal quantum yield of photosystem II (Fv/Fm) and maximum primary yield of photochemistry of PSII (Fv/Fo) of diploids decreased significantly. Conversely, the minimal value of chlorophyll a fluorescence (Fo), relative electrical conductivity and malondialdehyde increased markedly. Accordingly, the cold tolerance of the tetraploids presented better than these of the diploids. Taken together, our study has first developed an effective protocol to obtain and identify the tetraploid of P. auriculate plants. Furthermore, it was proved that this polyploid has presented improving cold tolerance and enriching phenotypic properties. These findings could be useful for improving cold-tolerance breeding and enriching genetic diversity of P. auriculate plants.
We used stem segments and buds exposed to pendimethalin and trifluralin in vitro to conduct polyploids of Plumbago auriculata and compared characterizations including cold tolerance between diploids and polyploids.
KeywordsCold stress Chlorophyll fluorescence Pendimethalin Plumbago auriculata Polyploidization Trifluralin
Minimal value of chlorophyll a fluorescence
Variable fluorescence of photosystem II
The maximal quantum yield of photosystem II
Maximum primary yield of photochemistry of PSII
Relative electrical conductivity
This work was partial supported by the Breeding Project of the Sichuan Province 13th Five-Year Plan to Tackle Key Problems, China (No. 2016NYZ0038).
YJ wrote the manuscript. YJ, GH and WL performed the experiments. SG and TL assisted with the experimental design. SL, SG, JH, and YL helped to improve the manuscript. XC, QL, LY, DH and JL assisted with analyzing the experimental results. SL, YJ and SG revised the original manuscript based on the comments of reviewers and editors.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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