Skip to main content
  • Research article
  • Open access
  • Published:

Induction of tetraploids in Paper Mulberry (Broussonetia papyrifera (L.) L’Hér. ex Vent.) by colchicine



Broussonetia papyrifera (L.) L’Hér. ex Vent. has the characteristics of strong stress resistance, high crude protein content, and pruning tolerance. It is an ecological, economic, and medicinal plant. Polyploid plants usually perform better than their corresponding diploid plants in terms of nutrients, active substances, and stress resistance.


In this study, the leaves, calli, and seeds of diploid B. papyrifera were used for tetraploid induction by colchicine. The induction effect of colchicine on B. papyrifera was summarized through the early morphology, chromosome count and flow cytometry. It was concluded that the best induction effect (18.6%) was obtained when the leaves of B. papyrifera were treated in liquid MS (Murashige and Skoog) medium containing 450 mg·L-1 colchicine for 3 d. The comparative analysis of the growth characteristics of diploid and tetraploid B. papyrifera showed that tetraploid B. papyrifera has larger ground diameter, larger stomata, thicker palisade tissue and thicker sponge tissue than diploid B. papyrifera. In addition, the measurement of photosynthetic features also showed that tetraploids had higher chlorophyll content and higher photosynthetic rates.


This study showed that tetraploid B. papyrifera could be obtained by treating leaves, callus and seeds with liquid and solid colchicine, but the induction efficiency was different. Moreover, there were differences in stomata, leaf cell structure and photosynthetic features between tetraploid B. papyrifera and its corresponding diploid. The induced tetraploid B. papyrifera can provide a technical basis and breeding material for the creation of B. papyrifera germplasm resources in the future.


Polyploidy refers to having more than two sets of chromosomes in each cell, and most polyploid chromosomes are even [1]. Polyploidy plays a key role in promoting phenotypic diversity and evolution [2, 3]. Fawcett et al. [4] found that many plant lineages had independent genome-wide replication events in the Cretaceous-Tertiary (KT) [5]. Nonetheless, previous estimates on the frequency of polyploid species formation have shown that the formation and establishment of new polyploid species in nature are rare [6]. The genetic diversity of polyploids increased relative to their diploid ancestors. In addition, this genetic diversity may show novelty at the biochemical, physiological, morphological and ecological levels, so polyploids are better than diploid parents at least in the short term [7]. The increase of polyploid genetic variation may lead to increased tolerance to a wider range of ecological and environmental conditions. For example, the total seed yield of tetraploid Themeda triandra Forsk is more than four times higher than that of diploids under drought and high-temperature stress [8]. Nowadays, global climate anomalies are frequent, and it is also a good strategy to improve the adaptability of plants by inducing polyploidy [9, 10]. Moreover, compared with diploid plants, polyploid plants have larger vegetative storage organs [11], higher contents of active substances [12, 13] and stronger disease [14]. For example, Xi et al. [15] found that polyploid Populus tomentosa grew rapidly in North China, and its 8-year volume was 2–3 times that of diploid control. Hu et al. [16] found that triploid and tetraploid carambola produced thicker and larger leaves, larger pollen grains and flowers, and larger fruits than diploid carambola; by comparing the differences between diploid and polyploid Rhododendron, Mo et al. [17] concluded that the leaves of polyploid Rhododendron were larger and thicker; Hias et al. [18] based on visual symptom assessment and real-time PCR to quantify the DNA of apple scab in apple leaves, and observed that tetraploid apples with a single gene resistant genotype had increased resistance compared to their diploids.

Ploidy breeding is the breeding process of obtaining offspring with changed chromosome multiples through artificial mutation technology. It has been widely used in breeding new varieties to improve their value [19]. For example, it can improve the content of active ingredients in some medicinal plants [20, 21] and the fruit quality of some fruit crops [22]. Polyploidy can be induced by sexual hybridization or somatic chromosome doubling. Sexual hybridization is based on the principle that male or female parents can produce unreduced gametes (2n). The main obstacle to the application of sexual polyploidy is the low frequency of unreduced gametes [23]. In contrast, somatic chromosome doubling is more widely used in plant polyploid breeding, which can be induced by extremely high or low-temperature upheaval, ionizing radiation, chemical reagents and so on. Among them, colchicine induction polyploidy is one of the common induction methods. Colchicine is a tubulin inhibitor and microtubule interfering agent. The principle of colchicine-induced polyploidy is to combine with heterodimers in mitotic cells, hinder the formation of spindle filaments, destroy spindle function, and inhibit the movement of chromosomes to the two poles of cells to form chromosome-doubled cells [24, 25]. Many plants have successfully induced polyploidy with colchicine. For example, with colchicine concentration of 150 mg·L−1 for 7 d, induced Zingiber Officinale Roscoe cv. ‘Fengtou’ ginger with chromosome doubling rate reached 18% [26]; 0.1%(w/v) colchicine for 48 h effectively induced polyploidy in Hyoscyamus reticulatus L. [27]; the seeds of Acacia mearnsii were treated with 0.01% colchicine for 6 h and successfully induced tetraploid [28].

B. papyrifera is a deciduous tree belonging to the mulberry family, which is widely distributed in Asia and the Pacific [29]. Its phloem fiber is long and has been a good material for papermaking since ancient times [30]. B. papyrifera contains flavonoids, terpenoids, alkaloids and other bioactive substances, which have antioxidant, antibacterial, anti-inflammatory and other effects [31,32,33,34]. In recent years, more and more scholars have found that B. papyrifera is a woody plant with high protein, high fat and low crude fiber, which has the potential to become an excellent feed raw material. For example, the production performance and meat quality of Hu sheep were improved after feeding with the feed added with B. papyrifera fermentation [35]. Adding B. papyrifera silage can enhance the immune and antioxidant functions of Holstein cows [36]. B. papyrifera grows rapidly and has strong stress resistance [37] and may adapt to various adverse conditions of heavy metal-polluted soil and karst soils [38]. Therefore, it is a good tree species for ecological restoration and the greening of urban settings [39].

In this study, the leaves, callus and seeds of diploid B. papyrifera were used as explant materials to induce polyploidy under different concentrations of colchicine and treatment durations, and the artificially induced tetraploid B. papyrifera was obtained for the first time. Meanwhile, by comparing the morphological characteristics and physiology of diploid and tetraploid B. papyrifera, we can more comprehensively understand the differences between B. papyrifera of different ploidies. The purpose of this study was to obtain polyploid B. papyrifera to improve its biomass, and content of protein and improve its feeding value and ecological restoration ability.

Materials and methods

Plant material and growth conditions

The diploid seeds of B. papyrifera were from South China Agricultural University (23° N, 113° E). Mature and healthy B. papyrifera seeds were selected, eroded in concentrated sulfuric acid for 9 min to soften the seed coat, then treated with 70% ethanol for 50 s and 2% sodium hypochlorite for 15 min, and rinsed with sterile water for 3–4 times. After that, put the seeds into a sterile seed culture medium and cultivated into plantlets in a tissue culture room. The leaf material comes from the leaves of sterile B. papyrifera seedlings, and the callus is induced from the leaves of seedlings in the MS (Murashige and Skoog) + 2.0 mg·L−1 6BA (6-benzyladenine) + 0.05 mg·L−1 IBA (indole-3-butyric acid).

The solid colchicine medium was colchicine + MS + 2.0 mg·L−1 6BA + 0.05 mg·L−1 IBA + 3% (w/v) sucrose + 0.6% (w/v) agar for adventitious shoot induction, and colchicine + 1/2 MS + 3% (w/v) sucrose + 0.4% (w/v) agar for seed culture. A liquid medium was used to remove agar based on a solid medium. All adventitious shoots induced root formation in rooting medium 1/2 MS (Half-strength Murashige and Skoog) + 0.05 mg·L−1 NAA (α-naphthalene acetic acid) + 3% (w/v) sucrose + 0.6% (w/v) agar. The pH of all media was adjusted to 5.8 – 6.2 with HCl or NaOH solution. Colchicine was purchased from Sigma-Aldrich (St. Louis, MO, USA), the plant growth regulators were purchased from Phyto Technology Laboratories (Lenexa, Kansas, USA), and agar, sucrose, concentrated sulfuric acid and hydrochloric acid were obtained from the Guangzhou reagent factory (Guangzhou, Guangdong). The materials were cultured in the tissue culture room with a 26 ± 2℃, 1500 lx light intensity and 12 h light/dark photoperiod.

Polyploidy induction and identification

Induction treatment of solid colchicine medium

Under aseptic conditions, the leaves of sterile B. papyrifera plantlets close to the stem tip were cut into 1.0 × 1.0 cm2 pieces, and the calli were cut into 1 × 1 × 1 cm3 pieces. The leaf pieces were precultured on MS medium for 3 d and then transferred to adventitious shoot induction medium supplemented with colchicine (0, 250, 350, 450, 550 mg·L−1) for 1, 2, 3, and 4 w in the dark. The calli were cultured in the dark for 1, 2, 3, and 4 w in the adventitious shoot induction medium supplemented with 0, 150, 250, 350 and 450 mg·L−1 colchicine. After colchicine treatment, the explants were transferred to an adventitious shoot induction medium without colchicine for normal culture. After 45 d, the number of induction adventitious shoot was counted and then the adventitious shoots were cut off and transferred to the rooting medium. When the plantlet height reached approximately 10 cm, its ploidy was identified, and the optimum time of tetraploid induction was recorded.

Induction treatment of liquid colchicine medium

The cut leaves were placed into the liquid medium for adventitious shoot induction with colchicine concentrations of 0, 250, 350, 450 and 550 mg·L−1, while the calli were induced in liquid medium with 0, 150, 250, 350 and 450 mg·L−1 colchicine. Treatments were bred for 1, 2, 3 and 4 d with continuous shaking at 80 rpm in the dark and then washed with sterile water. Subsequently, the treated explants were transferred to a solid adventitious shoot induction medium without colchicine. The number of induction adventitious shoot was counted after 45d. When the adventitious shoot grew to approximately 2 cm, they were transferred to the rooting medium.

Soak the seeds with colchicine

Put the sterilized seeds into the seed culture medium, when the seeds grew 2 mm radicle, they have soaked in colchicine solutions with concentrations of 0, 100, 200, 300 and 400 mg·L−1 for 1, 2 and 4 d. Thereafter, the seeds were washed with sterile water 3–4 times and then inoculated into the seed culture medium.

Chromosome counting

According to the method of Chen et al. [40], the chromosome number of the root tip was counted, and the chromosome number of tetraploid plants and control diploid plants was determined. At about 10:00 am, the root tips were cut from the regenerated plantlets and collected for chromosome counting. The samples were pretreated with ice water (4 °C) for 10 h and then fixed in fresh Carnoy’s solution (glacial acetic acid: 95% ethanol, 1:3) for 24 h. After the fixed root tips were rinsed with water for 15 min, the root tips were treated with 1 N HCl at a constant temperature of 60 °C for 8 min and then rinsed with water for 20 min. Root tip meristems about 1 mm long were stained with Modified Carbol-Fuchsin Solution (Solarbio, Beijing, China) for 20 min and then squashed on a microscope slide, and the chromosome number was observed with a microscope (DS-Ri2, Nikon, Tokyo, Japan).

Flow cytometry analysis

The DNA content of leaf cells in colchicine-treated and control diploid B. papyrifera (the control diploid B. papyrifera was used as the internal standard.) was measured by flow cytometry (Sysmex CyFlow® Ploidy Analyzer, Görlitz, Germany). The kit was a CyStain® UV Precise P (JIYUAN BIO-TECH, China). Fresh tender leaves were harvested, cut into 0.5 × 0.5 cm2, and vertically chopped with a blade in 400 μL extraction buffer for 30–60 s. After filtration through a 30 μm filter membrane, the samples were stained with 1600 μL 4',6-diamidino-2-phenylindole and dihydrochloride (DAPI) for 2 min and then tested on the machine.

Comparison of different ploidies of B. papyrifera

Morphological analysis

The regenerated plantlets of diploid and tetraploid B. papyrifera were transplanted into the substrate (perlite: peat soil = 1:1) and grown in the greenhouse (the temperature was controlled at 26 ± 2℃, and the light source was natural light) for two months. The plant height, ground diameter, leaf length, leaf width, petiole length and internode distance of tetraploid and diploid plantlets were measured.

Observation of stomatal and leaf cell structure

Tweezers were used to tear out the transparent epidermis of B. papyrifera leaves of different ploidy levels with the same plantlet stage and quickly placed into the water droplets on the prepared glass slide to make a temporary water sealing sheet. After production, 10 visual fields were observed and photographed under a microscope (DS-Ri2, Nikon, Tokyo, Japan), and the length of the stomata was measured.

The production of paraffin sections of leaves involves cutting the leaves into small pieces, fixing them with FAA (Formaldehyde Alcohol Acetic Acid) Fixative immediately for 24 h, embedding the materials with paraffin and slicing them. The sections were observed with a microscope (DS-Ri2, Nikon, Tokyo, Japan), and the thicknesses of the upper epidermis, lower epidermis, palisade tissue and sponge tissue were measured and photographed.

Photosynthetic features

From 9:30 am to 12:30 am on a sunny day, the second leaf of diploid and tetraploid plantlets was selected from top to bottom to measure the data of photosynthetic features. The chlorophyll content was measured by a portable chlorophyll content meter (SPAD 502 Plus, Osaka, Japan), and the net photosynthetic rate (Pn, μmol·m−2·s−1), stomatal conductance (Gs, mol·H2Om−2·s−1), intercellular CO2 concentration (Ci, μl·L−1) and transpiration rate of leaves (Tr, mmol·m−2·s−1) were measured by Portable photosynthetic apparatus LI-6400XT (Li-Cor BioScience, Lincoln, NE, USA), and the light intensity was set to 1000 μmol·s−1.

Statistical analyses

Each colchicine induction treatment was repeated 3 times, and each repetition contained 15 experimental materials. The number of explants inducing buds was counted and the induction rate of the adventitious shoot was calculated. All the adventitious roots were analyzed by flow cytometry after chromosome observation and counting. Then according to the results, the chimera induction rate and tetraploid induction rate were counted. Data were analyzed by using SPSS version 25.0 (SPSS, Inc., Chicago, IL, USA) and Excel (Microsoft Corp., Redmond, WA, USA).

$$\text{Adventitious shoot induction rate}\ (\%)=\frac{\text{Number of budding explants}}{\text{Number of explants}}\times100$$
$$\text{Seed survival rate}\ (\%)=\frac{\text{Number of germinated seeds}}{\text{Number of seeds}}\times100$$
$$\text{Tetraploid induction rate}\ (\%)=\frac{\text{Number of tetraploid plantlets}}{\text{Number of adventitious shoots}}\times100$$
$$\text{Mixed ploidy induction rate}\ (\%)=\frac{\text{Number of chimeric plantlets}}{\text{Number of adventitious shoots}}\times100$$
$$\text{CTR}\ (\text{cell tension ratio})\ (\%)=\frac{\text{Thickness of palisade tissue}}{\text{Thickness of leaf}}\times100$$


Tetraploid induction and verification

Induction efficiency of solid colchicine medium

Table 1 and Table S1 showed that there were differences in adventitious shoot induction of B. papyrifera leaves under the conditions of different colchicine concentrations and treatment times. When the concentration of colchicine was constant, the induction rate of adventitious shoot decreased gradually with increasing treatment time. When the concentration of colchicine was 350 mg·L−1 and the treatment time was 3 w, the induction rate of tetraploids was the highest, which was 13.6%. Table 2 (Table S2) indicates that although the adventitious shoot induction rate of the callus was high under the treatment of a solid colchicine medium, tetraploids were not induced.

Table 1 Effect of solid colchicine medium on leaf induced polyploidy
Table 2 Effect of solid colchicine medium on callus induced polyploidy

Induction efficiency of liquid colchicine medium

Browning occurred in some materials treated with liquid colchicine during regeneration, and the induction rate of the adventitious shoot was low, but the ploidy induction rate was high. The results (Table 3, Table S3) indicated that when the leaves were treated with 450 mg·L−1 liquid colchicine for 3 d, the tetraploid induction rate was the largest, which was 18.7%. Although the adventitious shoot induction rate was higher when the colchicine concentration was 250 mg·L−1 and the treatment time was less than 3 d, tetraploidy was not induced. It can be seen from Table 4 (Table S4) that the induction rate of tetraploids obtained by treating the B. papyrifera callus with 350 mg·L−1 liquid colchicine for 2 d was the highest at 10.0%.

Table 3 Effect of liquid colchicine medium on leaf induced polyploidy
Table 4 Effect of liquid colchicine medium on callus induced polyploidy

Soaking the seeds with colchicine

Although the survival rate of seeds treated with 100 mg·L−1 and 200 mg·L−1 liquid colchicine was relatively high, the chimerism induction rate and tetraploid induction rate were very low. The tetraploid induction rate was the highest (11.7%) in the 4 d and 400 mg·L−1 treatments, although in this treatment, the survival rate decreased by 22.2%, and the radicle was expanded, the root hair was thick, and the growth was slow (Table 5, Table S5).

Table 5 Polyploidy induced by soaking seeds in liquid colchicine

Ploidy identification

Chromosome counting is an intuitive method to identify ploidy, which can directly observe the number of plant chromosomes. The results showed that the chromosome number of root tip cells of diploid control plants was 2n = 2x = 26 (Fig. 1a), while that of induced tetraploid plants was 2n = 4x = 52 (Fig. 1b).

Fig. 1
figure 1

Chromosome count of diploid and tetraploid. a diploid; b tetraploid

The detection results of the relative content of leaf nuclei of diploid, mixed ploid and tetraploid plants by flow cytometry showed that the main peak of diploid appeared at about 13,000 (Fig. 2a), the mixed ploids had two peaks, 13,000 and 26,000 respectively (Fig. 2b). And the main peak of tetraploid appeared at about 26,000 (Fig. 2c). The results of tetraploid induction showed that the tetraploid trees were obtained.

Fig. 2
figure 2

Flow cytometry results of diploid, mixed ploid and tetraploid. a diploid; b mixed ploid; c tetraploid, the diploid is the reference standard

Comparison of diploid and tetraploid characteristics


It can be seen from Table 6 that there are phenotypic differences between tetraploid B. papyrifera and diploid. The stem of tetraploid B. papyrifera is stronger than that of diploid, the length and width of leaves are increased and the node spacing and plant height are shorter (Fig. 3).

Table 6 Morphology of B. papyrifera with different ploidy
Fig. 3
figure 3

Different ploidy plantlets. a diploid, b tetraploid

Stomatal and leaf cell structure

The stomata of B. papyrifera at different ploidy levels are shown in Fig. 4. According to Table 7, the differences in stomatal length and density between diploid and tetraploid were observed under an optical microscope. The length of stomata in tetraploid was significantly longer than that in diploid, and the density was significantly lower. The observation of leaf anatomical structure showed that the thickness of palisade tissue, the thickness of spongy tissue and the CTR of tetraploid were significantly greater than those of diploid, while the thickness of the upper and lower epidermis of the leaf was lower (Fig. 4).

Fig. 4
figure 4

Observation on stomata and leaf cell structure of different ploidy. a, c diploid; b, d tetraploid. UP: Upper epidermis thickness; LO: Lower epidermis thickness; SPT: Sponge tissue thickness; PTT: Palisade tissue thickness

Table 7 Difference analysis of stomata and leaf tissue structure of different ploidy

Photosynthetic features

The observations (Table 8) showed that there were significant differences in the photosynthetic features of B. papyrifera with different ploidies. The chlorophyll content (SPAD value) and leaf nitrogen content of tetraploids were higher than diploids. The net photosynthetic rate (P < 0.001), stomatal conductance (P < 0.001), intercellular CO2 concentration (P < 0.001) and transpiration rate (P < 0.001) of tetraploid were significantly higher than those of diploid, which were 9.61 μmol·m−2·s−1, 0.13 mol·H2Om−2·s−1, 287.87 μl·L−1, 2.78 mmol·m−2·s−1 respectively.

Table 8 Photosynthetic features of different ploidy


In this paper, the tetraploid of B. papyrifera was successfully induced from leaves, callus and seeds treated with colchicine for the first time, the ploidy was identified by chromosome counting and flow cytometry, and it was found that the induction rate of leaves treated with liquid colchicine was better. It may be that in liquid colchicine, the leaves of B. papyrifera are more fully exposed to colchicine, which increases the probability of cell mutation. Previously, polyploid induction of mulberry (trees of the same family as B. papyrifera) was reported. Wang et al. [41] treated mulberry regenerated shoots with colchicine by a dip (0.0%, 0.1%, 0.15% and 0.2%) and drip (0.0%, 0.15%, 0.2% and 0.25%), and successfully induced mulberry polyploids.

Moreover, the results showed that the tetraploid induction rate of B. papyrifera leaves was higher than that of callus and seeds. This may be the callus and seeds are too sensitive to colchicine, high concentrations of colchicine easily lead to callus and seed inactivation. Relatively, the tolerance of leaves is higher. Of course, different plants have different types of explants. For example, different explants of Vitis x Muscadinia hybrids were treated with colchicine, and the highest tetraploid induction rate was obtained when the shoot tips were treated with 625 μM liquid colchicine medium for 72 h [42]. Although colchicine can effectively induce polyploidy, it is toxic to plants [43]. The results of this study showed that the regeneration ability of the material decreased significantly with the increase of colchicine concentration and treatment time. Meanwhile, the growth of B. papyrifera explants treated with colchicine was relatively slow, which may be due to the physiological interference of colchicine, resulting in a decrease in cell division rate [44]. This is consistent with the research results of polyploid induction of Gerbera (Gerbera jamesonii Bolus cv. Sciella) [45], yacon (Smallanthus sonchifolius) [46] and Patchouli (Pogostemon cablin Benth.) [47]. Colchicine can obtain polyploid by inhibiting the formation of microtubules, but this can also lead to the disintegration of the cytoskeleton in plant cells, resulting in cell death [48]. This paper also found that in addition to tetraploid, colchicine induction also induced chimeras. Because the cell division period of the plant material meristem is not synchronous, the effect of colchicine on the cells of the apical meristem is not consistent, so mixed ploidy plants with diploid cells and tetraploid cells will be formed [49]. Although mixed ploidy may be better than diploid in some characteristics, the ploidy level is unstable, which is not conducive to production and application [50].

The morphological indexes of ground diameter, leaf length, leaf width and petiole length of tetraploid B. papyrifera were larger than those of diploid B. papyrifera. Studies have shown that after chromosome doubling, gene expression will change due to dose and interaction effects, resulting in morphological and physiological changes. Due to the increase in chromosomes and DNA, cells in polyploid plants enlarge [51]. Therefore, compared with the morphology of diploid plants, polyploid plants have larger vegetative organs, which can be used to screen and distinguish plants with different diploid levels. Compared with diploid plants, many polyploid plants have larger organs, such as larger pollen grains of tetraploid basil [52] and larger leaf mass of tetraploid European pear [53]. However, we also found that the internode length of tetraploid B.papyrifera was shorter than that of diploid B.papyrifera, and the plant height was shorter. Some scholars have pointed out that this may be because polyploid plants need to consume more materials to grow and maintain DNA, resulting in a decline in some indicators [54]. Our results confirmed that the stomata of polyploid plants were significantly greater than diploid plants, while the stomatal density was the opposite. This is consistent with the research results of Brachiaria ruziziensis [55], Passiflora edulis Sims [56], Alocasia [57], Dendranthema nankingense [58]. The stomatal parameter may be a powerful index of ploidy level change [59, 60]. In this study, the palisade tissue thickness, spongy tissue thickness and leaf tissue structure tightness (CTR%) of tetraploid B. papyrifera were significantly higher than those of diploid. The tissue structure of plant leaves is of great significance to the study of plant stress resistance [61, 62]. Previous studies have shown that the increase in palisade tissue thickness and sponge tissue thickness may help plant leaves store more water to improve their drought resistance [63, 64]. Therefore, the drought resistance of tetraploid B. papyrifera may be better than that of diploid. It has also been reported that polyploid plants are more drought tolerant than their corresponding diploids, such as Arabidopsis [65], and Rangpur lime [66]. Table 8 showed that the chlorophyll content, nitrogen content and net photosynthetic rate of tetraploid were significantly higher than those of diploids. Chlorophyll is a part of the photosynthetic system, which absorbs light energy and ultimately promotes CO2 assimilation. And the net photosynthetic rate of plants is an important index to measure plant health and photosynthesis, which affects the accumulation of carbohydrates in plants [67, 68]. These indicators of tetraploid B. papyrifera are higher than diploid, indicating that the photosynthetic capacity of tetraploid B. papyrifera may be better than diploid. Polyploids not only differ from diploids in morphological indicators, photosynthetic features, and leaf structure, but some reports also indicate that the nutritional content of polyploid plants is also different from that of diploids. For example, tetraploid Sorghum bicolor (L.) Mönch had higher protein content [69], and tetraploid Zea mays L. × Zea mays ssp.mexicana (Schrad.) Kuntze had higher soluble sugar content [70]. Therefore, the nutritional components of tetraploid B. papyrifera can be determined in the future to study its feasibility as a forage tree species.


This study shows that tetraploids can be obtained by treating leaves, calli and seeds of B. papyrifera with liquid and solid colchicine. This is the first report of the successful induction of tetraploid B. papyrifera and a detailed comparison of diploid and tetraploid B. papyrifera. The data indicate that treating leaves with 450 mg·L−1 liquid colchicine for 3 d is the optimal condition to obtain polyploid plants. Tetraploids showed differences from their corresponding diploids in morphology, stomata, leaf cell structure and photosynthetic characteristics, including larger leaves, thicker ground diameter, larger stomata, thicker leaves, thicker palisade tissue and sponge tissue, higher net photosynthetic rate and higher stomatal conductance. Overall, the results of this study provide an important technical basis for polyploid breeding of B. papyrifera, and make an important contribution to improving varieties and creating new germplasm.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


  1. Sattler MC, Carvalho CR, Clarindo WR. The polyploidy and its key role in plant breeding. Planta. 2016;243(2):281–96.

    Article  CAS  PubMed  Google Scholar 

  2. Ramsey J, Ramsey TS. Ecological studies of polyploidy in the 100 years following its discovery. Philos T R Soc B. 2014;369(1648):20130352.

    Article  Google Scholar 

  3. Van De Peer Y, Mizrachi E, Marchal K. The evolutionary significance of polyploidy. Nat Rev Genet. 2017;18(7):411–24.

    Article  PubMed  Google Scholar 

  4. Fawcett JA, Maere S, Van de Peer Y. Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. P Natl Acad Sci USA. 2009;106(14):5737–42.

    Article  CAS  Google Scholar 

  5. Levin DA, Soltis DE. Factors promoting polyploid persistence and diversification and limiting diploid speciation during the K-Pg interlude. Curr Opin Plant Biol. 2018;42:1–7.

    Article  PubMed  Google Scholar 

  6. Wood TE, Takebayashi N, Barker MS, Mayrose I, Greenspoon PB, Rieseberg LH. The frequency of polyploid speciation in vascular plants. P Natl Acad Sci USA. 2009;106(33):13875–9.

    Article  CAS  Google Scholar 

  7. Soltis PS, Liu XX, Marchant DB, Visger CJ, Soltis DE. Polyploidy and novelty: Gottlieb’s legacy. Philos T R Soc B. 2014;369(1648):20130351.

    Article  Google Scholar 

  8. Godfree RC, Marshall DJ, Young AG, Miller CH, Mathews S. Empirical evidence of fixed and homeostatic patterns of polyploid advantage in a keystone grass exposed to drought and heat stress. Roy Soc Open Sci. 2017;4(11):170934.

    Article  Google Scholar 

  9. Hollister JD. Polyploidy: adaptation to the genomic environment. New Phytol. 2015;205(3):1034–9.

    Article  PubMed  Google Scholar 

  10. Hahn MA, van Kleunen M, Muller-Scharer H. Increased phenotypic plasticity to climate may have boosted the invasion success of polyploid Centaurea stoebe. Plos One. 2012;7(11):e50284.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Majdi M, Karimzadeh G, Malboobi MA, Omidbaigi R, Mirzaghaderi G. nduction of tetraploidy to feverfew (Tanacetum parthenium Schulz-Bip.): Morphological, physiological, cytological, and phytochemical changes. Hortscience. 2010;45(1):16–21.

    Article  Google Scholar 

  12. Adams KL, Wendel JF. Novel patterns of gene expression in polyploid plants. Trends Genet. 2005;21(10):539–43.

    Article  CAS  PubMed  Google Scholar 

  13. Adaniya S, Shirai D. In vitro induction of tetraploid ginger ( Zingiber officinale Roscoe) and its pollen fertility and germinability. Sci Hortic-Amsterdam. 2001;88(4):277–87.

    Article  Google Scholar 

  14. Renny-Byfield S, Wendel JF. Doubling down on genomes: polyploidy and crop plants. Am J Bot. 2014;101(10):1711–25.

    Article  PubMed  Google Scholar 

  15. Xi XJ, Jiang XB, Li D, Guo LQ, Zhang JF, Wei ZZ, Li BL. Induction of 2n pollen by colchicine in Populus X popularis and its triploids breeding. Silvae Genet. 2011;60(3–4):155–60.

    Article  Google Scholar 

  16. Hu YL, Sun DQ, Hu HG, Zuo XD, Xia T, Xie JH. A comparative study on morphological and fruit quality traits of diploid and polyploid carambola (Averrhoa carambola L.) genotypes. Sci Hortic-Amsterdam. 2021;277:109843.

    Article  CAS  Google Scholar 

  17. Mo L, Chen JH, Chen F, Xu QW, Tong ZK, Huang HH, Dong RH, Lou XZ, Lin EP. Induction and characterization of polyploids from seeds of Rhododendron fortunei Lindl. J Integr Agr. 2020;19(8):2016–26.

    Article  CAS  Google Scholar 

  18. Hias N, Svara A, Keulemans JW. Effect of polyploidisation on the response of apple (Malus x domestica Borkh.) to Venturia inaequalis infection. Eur J Plant Pathol. 2018;151(2):515–26.

    Article  CAS  Google Scholar 

  19. Cheng ZM, Korban SS. In vitro ploidy manipulation in the genomics era. Plant Cell Tiss Org. 2011;104(3):281–2.

    Article  Google Scholar 

  20. Kaensaksiri T, Soontornchainaksaeng P, Soonthornchareonnon N, Prathanturarug S. In vitro induction of polyploidy in Centella asiatica (L.)  Urban Plant Cell Tiss Org. 2011;107(2):187–94.

    Article  Google Scholar 

  21. Tsuro M, Kondo N, Noda M, Ota K, Nakao Y, Asada S. In vitro induction of autotetraploid of Roman chamomile (Chamaemelum nobile L.) by colchicine treatment and essential oil productivity of its capitulum. In Vitro Cell Dev Pl. 2016;52(5):479–83.

    Article  Google Scholar 

  22. Shao JZ, Chen CL, Deng XX. In vitro induction of tetraploid in pomegranate (Punica granatum). Plant Cell Tiss Org. 2003;75(3):241–6.

    Article  CAS  Google Scholar 

  23. Ramanna MS, Jacobsen E. Relevance of sexual polyploidization for crop improvement - a review. Euphytica. 2003;133(1):3–18.

    Article  Google Scholar 

  24. Lu Y, Chen JJ, Xiao M, Li W, Miller DD. An overview of tubulin inhibitors that interact with the colchicine binding site. Pharm Res-Dordr. 2012;29(11):2943–71.

    Article  CAS  Google Scholar 

  25. Chung HH, Shi SK, Huang B, Chen JT. Enhanced agronomic traits and medicinal constituents of autotetraploids in Anoectochilus formosanus Hayata, a Top-Grade Medicinal Orchid. Molecules. 2017;22(11):1907.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Zhou J, Guo FL, Fu JP, Xiao Y, Wu JP. In vitro polyploid induction using colchicine for Zingiber Officinale Roscoe cv. “Fengtou” ginger. Plant Cell Tiss Org. 2020;142(1):87–94.

    Article  CAS  Google Scholar 

  27. Madani H, Hosseini B, Dehghan E, Rezaei-chiyaneh E: Enhanced production of scopolamine in induced autotetraploid plants of Hyoscyamus reticulatus L. Acta Physiol. Plant 2015, 37(3).

  28. Beck SL, Dunlop RW, Fossey A. Evaluation of induced polyploidy in Acacia mearnsii through stomatal counts and guard cell measurements. S Afr J Bot. 2003;69(4):563–7.

    Article  Google Scholar 

  29. Penailillo J, Olivares G, Moncada X, Payacan C, Chang CS, Chung KF, Matthews PJ, Seelenfreund A, Seelenfreund D. Sex distribution of Paper Mulberry (Broussonetia papyrifera) in the Pacific. Plos One. 2016;11(8):e0161148.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Peng XJ, Teng LH, Yan XQ, Zhao ML, Shen SH. The cold responsive mechanism of the paper mulberry: decreased photosynthesis capacity and increased starch accumulation. BMC Genomics. 2015;16:898.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Chen RM, Hu LH, An TY, Li J, Shen Q. Natural PTP1B inhibitors from Broussonetia papyrifera. Bioorg Med Chem Lett. 2002;12(23):3387–90.

    Article  CAS  PubMed  Google Scholar 

  32. Guo FJ, Feng L, Huang C, Ding HX, Zhang XT, Wang ZY, Li YM. Prenylflavone derivatives from Broussonetia papyrifera, inhibit the growth of breast cancer cells in vitro and in vivo. Phytochem Lett. 2013;6(3):331–6.

    Article  CAS  Google Scholar 

  33. Park MH, Jung S, Yuk HJ, Jang HJ, Kim WJ, Kim DY, Lim G, Lee J, Oh SR, Lee SU, et al. Rapid identification of isoprenylated flavonoids constituents with inhibitory activity on bacterial neuraminidase from root barks of paper mulberry (Broussonetia papyrifera). Int J Biol Macromol. 2021;174:61–8.

    Article  CAS  PubMed  Google Scholar 

  34. Ryu HW, Lee BW, Curtis-Long MJ, Jung S, Ryu YB, Lee WS, Park KH. Polyphenols from Broussonetia papyrifera Displaying Potent alpha-Glucosidase Inhibition. J Agr Food Chem. 2010;58(1):202–8.

    Article  CAS  Google Scholar 

  35. Su YY, Chen GS, Cai Y, Gao BL, Zhi XJ, Chang FJ. Effects of Broussonetia papyrifera-fermented feed on the growth performance and muscle quality of Hu sheep. Can J Anim Sci. 2020;100(4):771–80.

    Article  CAS  Google Scholar 

  36. Si BW, Tao H, Zhang XL, Guo JP, Cui K, Tu Y, Diao QY. Effect of Broussonetia papyrifera L. (paper mulberry) silage on dry matter intake, milk composition, antioxidant capacity and milk fatty acid profile in dairy cows. Asian Austral J Anim. 2018;31(8):1259–66.

    Article  CAS  Google Scholar 

  37. Huang HM, Zhao YL, Xu ZG, Zhang W, Jiang KK. Physiological responses of Broussonetia papyrifera to manganese stress, a candidate plant for phytoremediation. Ecotox Environ Safe. 2019;181:18–25.

    Article  CAS  Google Scholar 

  38. Wu YY, Liu CQ, Li PP, Wang JZ, Xing D, Wang BL. Photosynthetic characteristics involved in adaptability to Karst soil and alien invasion of paper mulberry (Broussonetia papyrifera (L.) Vent.) in comparison with mulberry (Morus alba L.). Photosynthetica. 2009;47(1):155–60.

    Article  CAS  Google Scholar 

  39. Nagpal UMK, Bankar AV, Pawar NJ, Kapadnis BP, Zinjarde SS. Equilibrium and kinetic studies on biosorption of heavy metals by leaf powder of paper mulberry (Broussonetia papyrifera). Water Air Soil Poll. 2011;215(1–4):177–88.

    Article  CAS  Google Scholar 

  40. Chen HL, Lu ZW, Wang J, Chen T, Gao JM, Zheng JL, Zhang SQ, Xi JG, Huang X, Guo AP, et al.  Induction of new tetraploid genotypes and heat tolerance assessment in Asparagus officinalis L. Sci Hortic-Amsterdam. 2020;264:109168.

    Article  CAS  Google Scholar 

  41. Wang XL, Zhou JX, Yu MD, Li ZG, Jin XY, Li QY. Highly efficient plant regeneration and in vitro polyploid induction using hypocotyl explants from diploid mulberry (Morus multicaulis Poir.). In Vitro Cell Dev Pl. 2011;47(3):434–40.

    Article  Google Scholar 

  42. Xie X, Agüero CB, Wang Y, Walker MA. In vitro induction of tetraploids in Vitis×Muscadinia hybrids. Plant Cell Tiss Org. 2015;122(3):675–83.

    Article  CAS  Google Scholar 

  43. Sarathum S, Hegele M, Tantiviwat S, Nanakorn M. Effect of concentration and duration of colchicine treatment on polyploidy induction in Dendrobium scabrilingue L. Eur J Hortic Sci. 2010;75(3):123–7.

    CAS  Google Scholar 

  44. Huang HP, Gao SL, Chen LL, Wei KH. In vitro tetraploid induction and generation of tetraploids from mixoploids in Dioscorea zingiberensis. Pharmacogn Mag. 2010;6(21):51–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gantait S, Mandal N, Bhattacharyya S, Das PK. Induction and identification of tetraploids using in vitro colchicine treatment of Gerbera jamesonii Bolus cv. Sciella Plant Cell Tiss Org. 2011;106(3):485–93.

    Article  CAS  Google Scholar 

  46. Viehmannova I, Cusimamani EF, Bechyne M, Vyvadilova M, Greplova M. In vitro induction of polyploidy in yacon (Smallanthus sonchifolius). Plant Cell Tiss Org. 2009;97(1):21–5.

    Article  Google Scholar 

  47. Widoretno W. In vitro induction and characterization of tetraploid Patchouli (Pogostemon cablin Benth.) plant. Plant Cell Tiss Org. 2016;125(2):261–7.

    Article  CAS  Google Scholar 

  48. Caperta AD, Delgado M, Ressurreicao F, Meister A, Jones RN, Viegas W, Houben A. Colchicine-induced polyploidization depends on tubulin polymerization in c-metaphase cells. Protoplasma. 2006;227(2–4):147–53.

    Article  CAS  PubMed  Google Scholar 

  49. Ravandi EG, Rezanejad F, Zolala J, Dehghan E. The effects of chromosome-doubling on selected morphological and phytochemical characteristics of Cichorium intybus L. J Hortic Sci Biotech. 2013;88(6):701–9.

    Article  Google Scholar 

  50. Nilanthi D, Chen XL, Zhao FC, Yang YS, Wu H. Induction of tetraploids from petiole explants through colchicine treatments in Echinacea purpurea L. J Biomed Biotechnol. 2009;2009:343485.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Rauf S, Khan IA, Khan FA. Colchicine-induced tetraploidy and changes in allele frequencies in colchicine-treated populations of diploids assessed with RAPD markers in Gossypium arboreum L. Turk J Biol. 2006;30(2):93–100.

    CAS  Google Scholar 

  52. Omidbaigi R, Mirzaee M, Hassani ME, Moghadam MS. Moghadam MS: Induction and identification of polyploidy in basil (Ocimum basilicum L.) medicinal plant by colchicine treatment. Int J Plant Prod. 2010;4(2):87–98.

    Google Scholar 

  53. Sun QR, Sun HS, Li LG, Bell RL. In vitro colchicine-induced polyploid plantlet production and regeneration from leaf explants of the diploid pear (Pyrus communis L.) cultivar, “Fertility.” J Hortic Sci Biotech. 2009;84(5):548–52.

    Article  CAS  Google Scholar 

  54. Hessen DO, Jeyasingh PD, Neiman M, Weider LJ. Genome streamlining and the elemental costs of growth. Trends Ecol Evol. 2010;25(2):75–80.

    Article  PubMed  Google Scholar 

  55. Ishigaki G, Gondo T, Suenaga K, Akashi R. Induction of tetraploid ruzigrass (Brachiaria ruziziensis) plants by colchicine treatment of in vitro multiple-shoot clumps and seedlings. Grassland Sci. 2010;55(3):164–70.

    Article  Google Scholar 

  56. Rego MM, Rego ER, Bruckner CH, Finger FL, Otoni WC. In vitro induction of autotetraploids from diploid yellow passion fruit mediated by colchicine and oryzalin. Plant Cell Tiss Org. 2011;107(3):451–9.

    Article  CAS  Google Scholar 

  57. Thao NTP, Ureshino K, Miyajima I, Ozaki Y, Okubo H. Induction of tetraploids in ornamental Alocasia through colchicine and oryzalin treatments. Plant Cell Tiss Org. 2003;72(1):19–25.

    Article  CAS  Google Scholar 

  58. Liu SY, Chen SM, Chen Y, Guan ZY, Yin DM, Chen FD. In vitro induced tetraploid of Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress tolerance. Sci Hortic-Amsterdam. 2011;127(3):411–9.

    Article  CAS  Google Scholar 

  59. Kadota M, Niimi Y. In vitro induction of tetraploid plants from a diploid Japanese pear cultivar (Pyrus pyrifolia N. cv. Hosui). Plant Cell Rep. 2002;21(3):282–6.

    Article  CAS  Google Scholar 

  60. Masterson J. Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperms. Science. 1994;264(5157):421–4.

    Article  CAS  PubMed  Google Scholar 

  61. Jiang CD, Wang X, Gao HY, Shi L, Chow WS. Systemic regulation of leaf anatomical structure, photosynthetic performance, and high-light tolerance in sorghum. Plant Physiol. 2011;155(3):1416–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Lauterbach M, van der Merwe PD, Kessler L, Pirie MD, Bellstedt DU, Kadereit G. Evolution of leaf anatomy in arid environments - a case study in southern African Tetraena and Roepera (Zygophyllaceae). Mol Phylogenet Evol. 2016;97:129–44.

    Article  PubMed  Google Scholar 

  63. Bacelar EA, Correia CM, Moutinho-Pereira JM, Goncalves BC, Lopes JI, Torres-Pereira JMG. Sclerophylly and leaf anatomical traits of five field-grown olive cultivars growing under drought conditions. Tree Physiol. 2004;24(2):233–9.

    Article  PubMed  Google Scholar 

  64. Guerfel M, Baccouri O, Boujnah D, Chaibi W, Zarrouk M. Impacts of water stress on gas exchange, water relations, chlorophyll content and leaf structure in the two main Tunisian olive (Olea europaea L.) cultivars. Sci Hortic-Amsterdam. 2009;119(3):257–63.

    Article  CAS  Google Scholar 

  65. del Pozo JC, Ramirez-Parra E. Deciphering the molecular bases for drought tolerance in Arabidopsis autotetraploids. Plant Cell Environ. 2014;37(12):2722–37.

    Article  PubMed  Google Scholar 

  66. Allario T, Brumos J, Colmenero-Flores JM, Iglesias DJ, Pina JA, Navarro L, Talon M, Ollitrault P, Morillon R. Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production. Plant Cell Environ. 2013;36(4):856–68.

    Article  CAS  PubMed  Google Scholar 

  67. Lindsey AJ, Barker DJ, Metzger JD, Mullen RW, Thomison PR. Physiological and morphological response of a drought-tolerant maize hybrid to agronomic management. Agron J. 2018;110(4):1354–62.

    Article  CAS  Google Scholar 

  68. Li YL, Gao XM, Li T, Jin HF, Zhu H, Wu QX, Lu BL, Xiong QX. Estimation of the net photosynthetic rate for waterlogged winter wheat based on digital image technology. Agron J. 2023;115(1):230–41.

    Article  Google Scholar 

  69. Ardabili GS, Zakaria RA, Zare N. In vitro induction of polyploidy in Sorghum bicolor L. Cytologia. 2015;80(4):495–503.

    Article  CAS  Google Scholar 

  70. Niazi IAK, Rauf S, da Silva JAT, Iqbal Z, Munir H. Induced polyploidy in inter-subspecific maize hybrids to reduce heterosis breakdown and restore reproductive fertility. Grass Forage Sci. 2015;70(4):682–94.

    Article  CAS  Google Scholar 

Download references


Not applicable.


This work was supported by the Young Innovative Talents Project of Guangdong Province (2020KQNCX005); the Forestry Technology Innovation Program, the Department of Forestry of Guangdong Province (2018KJCX001). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Author information

Authors and Affiliations



All authors contributed to the study conception and design. J.L. and B.Z. conceived and designed research. J.L., B.Z., J.Z. and H.Y. conducted experiments. J.L., J.Z., Z.L. and P.Z. analyzed the data. J.L. and J.Z. wrote the manuscript. X.C. and W.Z. supervised the overall work and critically analyzed all the results. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Xiaoyang Chen or Wei Zhou.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1:

Table S1. Variance analysis of leaf explants induced by solid colchicine medium. Table S2. Variance analysis of callus explants induced by solid colchicine medium. Table S3. Variance analysis of leaf explants induced by liquid colchicine medium. Table S4. Variance analysis of callus explants induced by liquid colchicine medium. Table S5. Variance analysis of seeds induced by liquid colchicine medium.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lin, J., Zhang, B., Zou, J. et al. Induction of tetraploids in Paper Mulberry (Broussonetia papyrifera (L.) L’Hér. ex Vent.) by colchicine. BMC Plant Biol 23, 574 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: