Chromosome doubling of Sedum alfredii Hance: A novel approach for improving phytoremediation efficiency
Graphical abstract
Introduction
Heavy metals including cadmium (Cd), lead (Pb), arsenic (As), chromium (Cr) and mercury (Hg), are major environmental pollutants and have caused serious metal contamination issues, especially in soils. Accumulation of heavy metals in soils and agricultural production are of great interest because of their side effects of food safety. Cd, as a non-essential element to plants (Shahid et al., 2016), tends to accumulate in plant tissues and is easily taken up by plant, finally posing health risks to humans and animals by entering into the food chain (Liu et al., 2011).
Sedum alfredii Hance, as a Cd/zinc (Zn) hyperaccumulator firstly found from an old Pb/Zn mining area in China, possesses an ability of accumulating up to 9000 μg/g Cd and 29,000 μg/g Zn in shoots without any toxic symptoms at hydroponic condition (Yang et al., 2004, Yang et al., 2006a). Besides, S. alfredii can reproduce for next generation within a short period of time due to its asexual propagation. However, the large-scale application of S. alfredii into phytoremediation was severely restricted because of its low biomass and limited remediation efficiency.
Genetic engineering provides a new idea for improving the phytoremediation efficiency. Transgenic technology mainly increases the phytoextraction efficiency of heavy metals by transferring some genes related to heavy metal absorption and accumulation. Our previous studies have shown that transferring transporting genes isolated from S. alfredii such as SaNRAMP3 and SaZIP4 to other plants can promote the absorption, transport and accumulation of heavy metals (Feng et al., 2018, Yang et al., 2018). However, due to its safety and related legal provisions, genetic engineering has been largely limited in practical applications. Unlike genetic engineering, chromosomal engineering technology can induce the increase of chromosome number of plants and resulted in a duplication of the plant genome (Corbeillie et al., 2018). Accompanied with the plant genome duplication in the nucleus were the increases in the gene dosage, which brought about significant changes on the morphology and physiology including thicker leaves, heavier stem and gaining plants with larger biomass (Kong et al., 2017). Therefore, chromosomal engineering technology was a promising method for improving remediation efficiency. Chromosomal engineering technology has been widely used in many different species since last century. Aleza et al. (2009) obtained stable tetraploid citrus for triploid seedless mandarins by grafting method combined with colchicine and oryzalin treatment. Colchicine-treated petioles led to an increase in both size and shape of Actinidia chinensis (Wu et al., 2012). Concentration of 0.2% colchicine was used for tissue culture and successfully produced autotetraploid plants of Sophora flavescens Aiton (Wei et al., 2010). Up to date, the prime objective that generating chromosome set doubling plants was increasing plants yields as well as producing new materials for plant breeding and no study has focused on phytoremediation with chromosome set doubling method. Thus, chromosomal engineering of S. alfredii is of great value which may result in the potential of much more biomass and larger amount of heavy metals, and leading to increased phytoremediation efficiency.
Therefore, the objectives of this research were: (1) obtaining the chromosome set doubling plants (CSD) of S. alfredii through colchicine treatment; (2) comparing the morphological and physiological characteristics of CSD with the wild type plants (WT); (3) studying the effects of chromosomal engineering on cadmium accumulation.
Section snippets
Plant material and in vitro multiplication
Sedum alfredii plants were obtained from the experimental field without Cd contamination in Zhejiang University. For in vitro culture, shoots were surface sterilized with 70% ethanol for 30 sec, then in a 0.1% solution of mercuric chloride for 10 min, followed by three rinses with sterile water. Then the shoots were cut into stems with axils and placed on a differential medium, which was consisted of MS culture medium (Murashige and Skoog, 1962) with an addition of 3.0 mg/L 6-benzyladenine
Effect of colchicine on S. alfredii chromosome set doubling
During the time when the shoot segments were growing into intact plants, survival number and mixoploid number were recorded in all treatments. The results revealed that the colchicine concentration and processing time affected survival rates and mixoploid induction rates (Table 2). Mixoploid plantlets were obtained by putting the explants into 0.1%–0.2% colchicine for 24–72 hr. When the explants were treated with the same concentration of colchicine, the survival rates of explants were declined
Discussion
Production of polyploid forms has been widely used and developed among various varieties such as Rosa (Kermani et al., 2003), mulberry (Chakraborti et al., 1998), maize (Barnabás et al., 1999, Battistelli et al., 2013), tomatillo (Robledo-Torres et al., 2011), melon (Lotfi et al., 2003), Brassica napus (Möllers et al., 1994, Chen et al., 1994), wheat (Hansen and Andersen, 1998, Chauhan and Khurana, 2011), Spanish onion (Fayos et al., 2015), Sophora flavescens Aiton (Wei et al., 2010) and so on.
Conclusions
In this study, the flow cytometry was used to determine the DNA ploidy and the results suggested that S. alfredii is a mixoploid. With colchicine treatment, we gained DNA ploidy obviously changed plants named as chromosome set doubling plants (CSD). Comparing with the wild type plants (WT) of S. alfredii, CSD exhibited typical “gigas” characteristics in both morphology and Cd accumulation. They have thicker and bigger leaf, darker leaf color, larger stomata, stronger stems, denser root hair and
Disclosure
The authors have no conflicts of interest to declare.
Acknowledgments
This work was supported by the National Key Research and Development Project of China (No. 2016YFD0800801), the National Natural Science Foundation of China (No. 41771345) and the Fundamental Research Funds for the Central Universities (2019FZJD007). The authors sincerely appreciate Dr. Xianyin Zhang from College of Agriculture & Biotechnology of Zhejiang University for his technical support of flow cytometry analysis.
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