A rapid method for identifying ploidy level in Isatis indigotica Fortune and Isatis tinctoria Linnaeus

The genus Isatis is widely distributed throughout the world. In this work, thirty-two Isatis indigotica Fortune germplasms, collected from different regions and geographical locations in China, were analyzed the ploidy levels by ow cytometry. I. indigotica Fort. and Isatis tinctoria Linnaeus distinguished with root tip chromosome compression staining and cell ow cytometry. Microscopic observation showed that the chromosome numbers of I. indigotica Fort. and I. tinctoria L. were 2n = 14 and 2n = 28, respectively. In order to establish a ow cytometric nuclear experiment system suitable for I. indigotica Fort., the leaves of I. indigotica Fort. were used to prepare nuclear suspension with LB01, OTTO, Tris·MgCl 2 , patent of Luochang and Galbraith extraction buffer. It was found that the histogram generated by LB01, OTTO, Tris·MgCl 2 and Patent of Luochang dissociation solution has poor peak shapes and large CV values, and Galbraith dissociation solution extraction buffer was suitable for extracting nuclei from most germplasms. Flow cytometry proved to be a simple, rapid, and highly accurate method for identifying ploidy levels Isatis species.


Introduction
Isatis indigotica Fort., a biennial herbaceous plant of the family Cruciferae (Chen et al. 2014), it was used as traditional Chinese medicine with roots (Isatidis radix) and leaves (Isatidis folium) (Sun et al. 2021). As a medicinal plant for more than 2000 years from Shen Nong's Materia Medica, Isatidis radix has a widely useful activities including anti-virus, anti-bacterial, anti-endotoxic, anti-in ammatory, and immune regulatory effects (Ding and Zhu 2020;Kong et al. 2008). However, I.indigotica Fort. has been merged into Isatis tinctoria Linnaeus in Flora of China (2001) (Zhou et al. 2001). However, there is a transition between I. indigotica Fort. and I. tinctoria L., and the two have big differences in morphology. Due to the chaos of the original plants of I. indigotica Fort., the use of medicinal materials has also been restricted (Muzayyinah et al. 2016). This may lead to a series of inconsistent treatment effects and quality problems in the herbal medicine industry. Clarifying the germplasms source of medicinal materials is an important prerequisite to ensure that the medicinal materials are safe and effective (Xu et al. 2011). Accurating identi cation plays an important role in the rst key step for developing quality security assurance measures for Traditional Chinese Medicine (Chen et al. 2021). The development of accurate and reliable method for the identi cation I. indigotica Fort. appears to be extremely urgent.
The DNA encoding the genetic information of most organisms is located in the nucleus, and the detection technology of DNA content is one of the earliest developed, most widely used, and most basic ow cytometry (Bennett and Leitch 2005). Galbraith and the Hare developed a method for DNA content by ow cytometry plant cell nucleus, thus promoting botany unprecedented development (Galbraith et al. 2001;Hare and Johnston 2011). The principle of DNA content detection by ow cytometry is to use special uorescent dyes (propidium iodide, 4',6-diamidino-2-phenylindole, Acridine Orange, etc.) in series with the DNA inside the cell, enter the ow chamber of the ow cytometer, and emit after laser irradiation uorescence, to be collected through the respective lter sheets uorescence intensity per cell, and then the uorescence intensity of the stained cells to calculate the cellular DNA content (Ddg et al. 2021).
Flow cytometry provides powerful tools and detection methods for plant cell research (Olszewski et al. 2015). It can not only count plant cells, detect genome size, DNA content and ploidy level, but also sort cells in different periods and cycles, and construct chromosome DNA library. The use of ow cytometry to detect plant nuclear DNA content and ploidy is very important for the description of species characteristics, classi cation and genetic research. We often encounter a lot of interference in the detection of DNA fragments Isatis samples, mainly due to the plant cell structure (cell wall central vacuole) and component (secondary metabolites) particularity and complexity. At present, there are not many researches on the identi cation methods of I. indigotica Fort. (Kang et al. 2021). The chromosomes of I. indigotica Fort. are small and di cult to observe, which hinders ploidy analysis. Flow cytometry technology can solve this problem.

Materials And Methods
Plant material, culture medium and conditions The seeds consisted of 32 I. indigotica Fort. germplasms collected from different locations ( Table 1). The seeds were surface-sterilized with 70% ethanol for 1 min followed by 2.5% sodium hypochlorite for 8 min. The sterilized seeds were rinsed with double distilled water ve times and then dried by blotting on sterilized paper prior to testing.
Murashige and Skoog (MS) medium was used at full strength for callus induction and shoot regeneration and at half-strength for in vitro rooting (Duan et al. 2018). The culture medium contained 3% (w/v) sucrose and 0.7% agar or 0.3% phytagel. The pH of each medium was adjusted to 5.8 using potassium hydroxide (KOH) prior to autoclaving (103 kPa and 121°C for 20 min). Unless otherwise noted, cultures were maintained at 25±1°C under a 16 h/8 h light/dark photoperiod, provided by cool-white uorescent light with an irradiance of 35 µmol photons/(m 2 /s), and at 60% relative humidity. The sucrose used was the products of Solarbio (Beijing, China).
The seedling after seed germination become strong in 7 d. Planted in 30 cm pots in a greenhouse under natural light at 25℃ (16 h/8 h of light/dark).

Sample preparation
Take about 0.5 g of the young leaf sample, chop it with a sharp scalpel blade in a petri dish dripped with 1.5 mL extraction buffer (Table 2), then lter with a 500 mesh nylon mesh (Yan and Shao 2014). The puri ed nuclei were stained with 250 μL propidium iodide (PI; 50 μg/mL; Fluka, Buchs, Switzerland). RNase (50 μg/mL; Fluka) was then added to the nuclear suspension to prevent staining of double-stranded RNA. The samples were incubated on ice in the dark for 10 min before analysis by ow cytometry. All stages of extraction and staining were performed at 4℃. Young leaf sample from I. indigotica Fort. (Altay, Xinjiang, China) were used as a diploid reference. 10,000 nuclei were analyzed per sample.

Flow cytometric analysis
Ploidy identi cation: using CytoFLEX-ow cytometry sorter (Beckman Coulter, S.Kraemer Boulevard Brea, CA , USA), excitation light source is 15 argon ions, excitation wavelength is 488 nm, irradiated by the excitation light source can excite the uorescent dyed molecules to promote uorescence, and measure the uorescence intensity and signal intensity. It is directly proportional to the DNA content. At the same time, it is theoretically believed that as the number of chromosomes doubles, the DNA content of the cell nucleus must also double, so the DNA content can be used to estimate the ploidy of the cell chromosomes (Stanisic et al. 2015).
For each measurement, PI uorescence area signals (phycoerythrin area, PE-A) from 10,000 nuclei were collected using CytoFLEX. A gate was set using the PE-A and FSC parameters, allowing the uorescence measurements fromnuclei to generate a histogram of PE-A. Acquisition parameter settings (gain): FSC, 700; SSC, 250; FITC, 126; PE, 250; PB450, 101; KO525, 51. The mean values PE-A of the G1/G2 (nuclei) peak for the sample and theexternal standard were determined by analyzing the datausing CytExpert 2.3 software. To estimate ploidy level, the position of the G1 peak on the histogram obtained for each individual plant was compared with that of I. indigotica Fort. (Altay, Xinjiang, China).

Sample preparation
The young and fresh leaf samples can be uesd in Flow cytometer (De et al. 2010). After aseptic operation, the seeds are inoculated on MS medium and can be detected by Flow cytometry. Then the leaf bases of 7 d seedlings were used as initial samples (Fig. 1). At this time, the leaves were relatively fresh, chopped with a double blade, mixed with the dissociation solution, and then added PI staining, which will show a better experimental results.

Determination of blank control
In the MS medium, the seedlings of I. indigotica Fort. were well developed. The meristem of the root tip of the 7d seedling was used for chromosome counting (Liu and Zhang 2001). The chromosomes number of I. indigotica Fort. and I. tinctoria L. were determined to be 14, 28 (Fig. 2), indicating that I. indigotica Fort was diploid, and I. tinctoria L. was tetraploid. This was consistent with the description in the literature (Tuo et al. 2012).

Screening extraction buffer
Excellent preparation of nuclei is crucial for ow cytometry analysis (Rainer et al. 2014). However, obtaining nuclei from different plant species requires different extraction buffers suited to a particular plant tissue and metabolic components. We chose the ve extraction buffers including LB01 OTTO Tris·MgCl 2 Galbraith and Patent of Luo chang (Bhardwaj et al. 2014). Not enough cell nuclei can be detected in the sample, and no obvious DNA peaks can be generated in the histogram including LB01, OTTO, Tris·MgCl 2 and Patent of Luochang. We found that Galbraith extraction buffer was suitable for examining most germplasms (Galbraith 2004), resulting in a complete histogram of the peak shape (Fig. 3).

Data analysis
In the ow cytometer, the Mean PE-A of 32 germplasms of I. indigotica Fort. ranges from 927837~1026530, and the CV value ranges from 3.50%~ 4.74%, which is less than 5%, indicating that the data is true and reliable. The Mean PE-A±SD of I. tinctoria L. is (1863935±6150), and the CV±SD is (2.53±0.19)%, which is 1.5-2.0 times that of I. indigotica Fort. (Fig 4). The results showed that the 32 germplasms were I. indigotica Fort. instead of I. tinctoria L..

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The yield of cell nuclei in ow cytometry samples depends on the leaf age. The nucleus yield of old leaves is lower than that of young leaves. In addition, some plant leaves are prone to produce sticky substances when shredded, which adsorbs cell nuclei, resulting in a small number of cell nuclei (Pfosser et al. 2010). Therefore, the seedlings grown on the 7d after seed germination have young leaves and are applicable to this experiment.
Obtaining nuclei from different plant species requires different extraction buffers. Inappropriate dissociation uid will accelerate the rupture of the nucleus, resulting in excessive fragments of the nucleus suspension, poor quality, and failure to form peaks or poor peak shapes in the histogram. This can explain the fact that the samples prepared by Tris·MgCl 2 , LB01, OTTO and Luochang patent extraction buffer have too few nuclei and poor quality, resulting in poor histogram peak shapes (Galbraith 2004;Galbraith et al. 2001).
The CV value of the DNA peak is an important indicator to measure the reliability of the measurement result (Backovié et al. 1992). Generally, the recognized CV value of plants is about 3%~5% (Dolezel et al. 1998); and for the plants whose cytoplasm is rich in staining inhibitors, the recognized CV is not more than 7% (Ca L et al. 2005). Using the Galbraith extraction buffer, the measured CV value is 3-5%, indicating that the measured data is credible, and it also shows that the Galbraith extraction buffer is suitable for the analysis of these 32 germplasms of cell nuclei in I. indigotica Fort.
The experimental results show that the ploidy of I. indigotica Fort. was different from I. tinctoria L.. By Root tip chromosome compression staining and cell ow cytometry, distinguish I. indigotica Fortune and I. tinctoria Linnaeus. Root tip chromosome compression shows that the number of chromosomes in I. indigotica Fort. is 2n = 14, the number of chromosomes in I. tinctoria L. is 2n = 28. In ow cytometry 32 germplasms of I. indigotica Fort. all showed the same ploidy level. And different from I. tinctoria L. There were no signi cant differences in the different germplasms within the specie of I. indigotica Fort. collected from different locations with ploidy level, which indicates that ploidy level is not associated with geographic location.
In conclusion, we successfully used ow cytometry to determine ploidy levels in I. indigotica Fort. The high sensitivity and accuracy of ow cytometry enabled us to rapidly estimate the ploidy levels of Isatis species. This e cient technique helps to further elucidate the genetic diversity of I. indigotica Fort. and I. tinctoria L.