Karyotype analysis of obtained tetraploid in medicinal plant ( Platycodon grandiflorus )

Platycodon grandiflorus is an important medicinal plant in China. Induction of polyploidy was successfully attempted in this medicinal plant using 0.05% colchicine semi-solid under 72 h by apical shoot tip treatment of young seedlings, and then 50% mutant plants were obtained. It was testified by the chromosome number in mitosis and its karyotype analysis for diploid control and obtained mutants. The results showed that the chromosome number of the diploid plant was 2n=2x=18, whereas the chromosome number of the obtained mutants was 2n=4x=36. The karyotype formula of mutant plant and diploid control were K=2n=4X=36=24m+12sm (4SAT) and K=2n=2X=18=12m+6sm (2SAT), respectively by karyotype analysis and they belonged to 2B and 2A karyotype. It was confirmed that the obtained mutant plants were tetraploid.


INTRODUCTION
Platycodon grandiflorus, a species of perennial flowering plant of the family Campanulaceae, is native to East Asia (such as China, Korea, Japan, and East Siberia) and has been used as an important medicine or food for centuries in China.In Europe, it is cultivated mainly as an ornamental plant (Lee et al., 2002).However, its potential health care uses have become a hot research topic in Asia.As far as in 1970s, the root of this species (radix) was extensively used as an anti-inflammatory in the treatment of coughs and colds (Lee, 1973); a finding of Jang et al. (2013) suggested this effect in lipopolysaccharide stimulated BV2 microglial cells.In addition, according to some recent reports, saponins from the roots of P. grandiflorus (CKS), as one of the most essential functional components in radix, have been shown to exhibit many other pharmacological activities, including anti-cancer (Park et al., 2005;Lee et al., 2004Lee et al., , 2013;;Jeong et al., 2010;Chun et al., 2013), antioxidant (Jeong et al., 2010;Ryu et al., 2012), the inhibition of pancreatic lipase (Zhao and Kim, 2004), anti-allergic (Han et al., 2009), anti-hyperglycemiccholesterol (Ahn et al., 2012;Jang et al., 2010), anti-skin photoaging (Hwang et al., 2011), stimulate osteoblast differentiation (Jeong et al., 2010), lowering Cholesterol (Zhao, 2006), and antiobesity effects (Zhao et al., 2006;Hwang et al., 2013).
Moreover, another component of root, named homogalacturonan, was found having a property of antiangiogenesis activity (Xu et al., 2011), suggesting its potential to be developed as an anti-angiogenesis drug.Chen et al. (2013) also demonstrated the intervention of effective parts extracted from P. grandiflorus on microangiopathy of diabetic rats.Hence, P. grandiflorus has gradually developed into an effective remedy used in many kinds of medicines as a treatment for some intractable diseases, such as cancer, glycuresis, obesity, airway diseases (Choi et al., 2011), which is no longer restricted to traditional ills like cough.Due to its increased demand for this medicinal material, it is necessary to focus on its breeding program.So far, most researchers are much interested in collecting wild germplasm P. grandiflorus, resulting in the lack of natural resources.In order to keep sustainable state, polyploid breeding make it possible to increase new germplasm in this medicinal plant.In our former study, induction of polyploidy was attempted using 1% agar with 0.05% colchicine semi-solid under 72 h by apical shoot tip treatment of young seedlings.A single drop (2-4 μl) of the warm (~50°C) semi-solid was painted between cotyledons of each seedling to cover the apical bud.That was one application and three applications were conducted in every 24 h interval time for totally three times.Finally, 50% mutant plants were successfully obtained in P. grandiflorus by preliminary morphology identification (Wu et al., 2011).In this study, the chromosome number was counted in mitosis and comparative karyotype analysis was measured by the induced mutants and diploid control.The purpose was to further check its ploidy level for induced mutants and to determine its karyotype characteristics as compared with its diploid control.

Plant
P. grandiflorus came from Natural Medicinal Plant Garden in Shanxi province.Field experiments were finished in this garden and inside experiments were carried out in Herbal Breeding Laboratory of Shanxi Agricultural University.

Mitotic karyotype analysis
For Karyology, seeds of the obtained mutants and diploid controls were collected and germinated on moist filter paper lining petri dish at approximately 27°C.0.5 to 1 cm long freshly grown root tips were cut off at 8 to 9 am to obtain mitotic metaphases and determine karyotype characteristics, including chromosome numbers, chromosome length and total length of all chromosomes.Firstly, these root tips for pretreatment were placed into a saturated solution of p-Dichlorobenzene and aqueous α-bromonaphthalene for 2.5 h at 20°C, and then fixed in 3:1 v/v absolute ethanol:glacial acetic acid for a minimum of 24 h at 4°C.The fixed tips were then washed thoroughly in distilled water and meristems were hydrolysed in 1 M hydrogen chloride (HCl) for about 2 min at room temperature.1 to 2 mm length from the tips were cut and placed on clear glass slides with Carbol fuchsin solution as the staining.Squash technique was made for cytological studies.These scattered cells were were photographed through a 10×40 microscope with an OLYMPUS BX51 digital camera.10 best metaphases were selected for further measures by Photoshop 8.0 software to study the karyotype.The method for karyotypic analysis followed with Li and Chen (1985) and the karyotype classification was based on Stebbins (1971).

Chromosome numbers analysis
Both the numbers and the characters of chromosomes in mitosis are the most persuasive checking standards for the ploidy level.In this research, karyotypic characters, mitotic metaphase chromosomes of diploid control and the mutants are as shown in Tables 1 and 2 and Figures 1 to 4, respectively.Analysis of somatic metaphases showed that the chromosome numbers of mutants were 2n=4x=36, while the chromosome numbers of diploid control were 2n=2x=18.It is the direct evidence for this polyploid induction in P. grandiflorus.

Karyotype analysis of diploid control
Statistical observations were made for more than fifty root tip cells, all of which were at mitotic metaphase and could be counted for the chromosome number.The metaphase chromosomes of the diploid P. grandiflorus are as shown in Figure 1.The relative length of chromosomes in the diploid control was from 4.18 to 7.67.Arm ratios ranged from 1.08 to 2.00 and the average arm ratio was 1.481.The karyotypic formula was 2n=2x=18=12m+6sm (2SAT), consisting of 12 metacentric chromosomes and 3 pairs of submetacentric chromosomes (Table 1), and a satellite on the short arm of group VI chromosomes was observed.The karyotype was type 2A (Figure 2).

Karyotype analysis of obtained mutants
More than fifty root tip cells were statistically observed, all of these cells were at mitotic metaphase and could be counted the chromosome numbers for obtained mutants.The metaphase chromosomes of the mutants P. grandiflorus, which are as shown in Figure 3, were arranged into 9 groups of four chromosomes each.The chromosomes in each group had a similar morphology.Relative lengths ranged from 2.02 to 4.05, and arm ratios ranged from 1.18 to 2.45.The average arm ratio was 1.622 (Table 2).Four satellites were seen on the short arms of the eleventh and twelfth chromosomes (Figure 4).The karyotype was type 2B (Table 2) and the karyotypic formula was 2n=4x=36=24m+12sm (4SAT).

DISCUSSION
Our study has illuminated the karyotype characters of the medicinal plant P. grandiflorus through comparative karyotype analysis of the diploids and induced mutants, while demonstrating the previous study (Wu et al., 2011;Zhao et al., 2004) in which the somatic chromosome number of diploid P. grandiflorus was noted as 2n=2x=18 and the chromosome number of obtained tetraploids was 2n=4x=36, both of which with basic chromosome number x=9 by chromosome counting in root-tip cells.
According to Stebbins (1971), karyotype evolution is generally from symmetry to asymmetry in higher plants  generally from symmetry to asymmetry in higher plants during the biological evolution process.Karyotypes are classified into 12 types (1A-4A, 1B-4B, 1C-4C) based on analysis of plant materials.This theory suggests that primitive plants have symmetric karyotypes whereas asymmetric karyotypes occur in more evolutionary advanced taxa.In this study, the karyotype of the tetraploid showed that the ratio of the longest chromosome to the shortest chromosome was 2.00 and the percentage of chromosome with arm ratio exceeding 2 was 22%.It belonged to the karyotype of 2B, which was consistent with the result by Wang et al. (2006b).
While the diploid control belonged to 2A in this study and followed with the result (Wang et al., 2006a).This indicated that the intrachromosomal symmetry in the diploid was higher than in the tetraploids.In addition, Wang et al.

Chromosome number
Relative length (%) (2007) found a haploid karyotype of P. grandiflorus belonging to the type 1A, exhibited higher symmetric and primitive.This suggested that the tetraploids were advanced than its other types with low ploidy level.This phenomenon showed that a plant with the higher ploidy level may be advanced than those with lower ploidy levels.From this study we also confirmed that the tetraploid genome got rearranged and its fragments may be
In the present study, octoploid (8x), mixoploid (2x + 4x), except diploid and tetraploid in some shoot-tip meristem cells were found.This has been caused by abnormal mitosis or meiosis induced by doubling chromosome number, as was expected, it led to some abnormal phenomenon during the meiosis of its PMCs, such as quadrivalent, laggard chromosomes and irregular chromosome bridges (Wu et al., 2012).High-level polyploids can be stunted or infertile, possibly resulting from the extreme genetic redundancy and somatic instability that leads to chimera tissue (Ockendon, 2008).In this study, the occurrence of mixoploidy was compared between the second generations M 2 and M 1 , and found that the proportion of chimera was lower in the second generation M 2 with about 3% than M 1 in P. grandiflorus.In short, the results of the present work have improved the understanding of the organization, structure, and evolution of the genome of P. grandiflorus.Different types of polyploidy levels, especially autotetraploidy of this medicinal plant will provide abundant germplasm resources for treatments of diseases.Polyploid plant may have much more functional components in its vegetative organs and its function will be much improved.There will be continuation in the report of changes of the components of polyploidy plant and its new functions in later study.

Figure 1 .
Figure 1.The morphology of somatic chromosomes of diploid P. grandiflorus.

Figure 3 .
Figure 3.The morphology of somatic chromosomes of tetraploid P. grandiflorus.

Table 1 .
The coefficient of chromosomes of diploid P. grandiflorus.

Table 2 .
The coefficient of chromosomes of obtained tetraploid P. grandiflorus.