Open Life Sci. 2017; 12: 34–41

AbbreviationsMS Murashige and Skoog mediumNAA Naphthalene acetic acid6-BA 6-benzylaminopurine2, 4-D 2, 4-dichlorophenoxyacetic acid

1 IntroductionAmorphophallus konjac (Araceae) belongs to a group of perennial plants characterized by tubers and pinnately  compound leaves. This species is widely distributed throughout Southwest Asia and Africa [1,2], and has been used for a long time as a medicinal plant. A. konjac is also utilized for pharmaceutical, chemical industries as well as in agriculture and food source because its tuber contains a high level of water-soluble glucomannan [3]. Although A. konjac demonstrates a great potential for commercial application and offers favorable economic benefits, the poor disease resistance and low-efficiency propagation of A. konjac are hindering its production.

Plant tissue culture technology has been applied to produce healthy seeds and seedlings for many crops, such as potato, corn, and wheat. Apparently, the tissue culture technique could be an important and efficient method to solve the problem in the A. konjac industry. Many studies on A. konjac tissue culture have been reported. These studies used different explants from A. konjac, such as shoot tip and bud [4], anther, corm, leaf, root [5] side-bud, subcutaneous tissue, main-bud, basilar-tissue, budlet [6], petiole [7], and unpollinated ovaries [8]. In addition, the A. konjac plant regeneration of these studies was almost developed from organogenesis instead of somatic embryogenesis. Plant regeneration during somatic embryo genesis offers certain advantages over organogenesis, given that embryos contain both root and shoot meristems. Somatic embryos are single cells and clonal in origin. Somatic embryogenesis is a powerful tool for genetic improvement of any plant species [9].

DOI 10.1515/biol-2017-0004Received September 10, 2016; accepted November 30, 2016

Abstract: Amorphophallus konjac (Araceae) a perennial herb, it has high medicinal and industrial value. In this study, a simple and efficient system for direct somatic embryogenesis and plantlet regeneration of Amorphophallus konjac was developed. The floral axis was used as the experimental material. The primary callus, developed from the floral axis grown on Murashige and Skoog (MS) medium supplemented with different hormone combination at different concentrations. The highest rate of embryogenic callus formation was observed on the MS medium containing 9.04 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 5.37 µM naphthalene acetic acid (NAA). The maximum induction rate was 79.8%, and the embryogenic calli were able to subculture on a medium containing similar hormone combination for over 1 year. The calli were also placed on different media for regeneration and it produced complete plants with shoots and root systems simultaneously. The highest differentiation rate of the embryogenic calli grown on differentiation medium supplemented with 8.88 µM 6-benzylaminopurine (6-BA) and 5.37 µM NAA was 95.6%. Flow cytometry analysis showed no ploidy variation in all the regenerate plantlets.

Keywords: Amorphophallus konjac, Embryogenic callus, High reproduction rate, In vitro production, Tissue culture

© 2017 L. Zhong et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.

*Corresponding author: Zhongli Hu, State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, 430072, Wuhan, Hubei, PR China, E-mail: huzhongli@whu.edu.cn Lin Zhong, State Key Laboratory of Hybrid Rice, College of Life Scien-ces, Wuhan University, 430072, Wuhan, Hubei, PR ChinaErxi Liu, Chaozhu Yang, Institute of Konjac, Enshi Academy of Agri-cultural Sciences, Enshi, P. R. China; Surong Jin, Institute of Chemical and Life Science, Wuhan University of Technology, Wuhan, P. R. China; Ying Diao, Lotus Engineering Research Center of Hubei Province, College of Life Sciences, Wuhan University, 430072, Wuhan, Hubei, PR China

Lin Zhong, Erxi Liu, Chaozhu Yang, Surong Jin, Ying Diao, Zhongli Hu*

High embryogenic ability and regeneration from floral axis of Amorphophallus konjac (Araceae)

Research Article Open Access

High embryogenic ability and regeneration from floral axis of Amorphophallus konjac (Araceae)    35

and 50 μmol m−2s−1 irradiance) and then maintained at 28 ± 2°C and 60% relative humidity.

Induction of somatic embryogenesis and plantlet regeneration

The explants were transferred into a fresh induction medium every 2 weeks until the somatic embryos were induced. The embryogenic calli were then detached from the explants. Some of embryogenic calli were subcultured on the primary medium to keep multiplying and maintained under the same conditions. The remaining embryogenic calli were transferred to the regeneration medium (Table 2). Each treatment was replicated thrice.

2.2 Histological analysis

The calli were obtained for histological examination. The samples were fixed in FAA (formaldehyde/acetic acid/ethanol=10:5:85 v/v/v) for 48 h, followed by 30 min of dehydration at increasing ethanol gradations [25, 50, 75, 95, 100% (v/v)]; dehydration was performed twice for each step. Ethanol was replaced by gradual dilution with trichloroethane solution. The specimens were infiltrated in a vessel that contained the paraffin wax and kept at 56°C for at least 24 h; finally, the samples were embedded in the paraffin wax. The samples were sectioned into 10 µm-thick slices using a rotary microtome (AO 820, Scientific Instruments) and then mounted on glass slides. The sections were dewaxed in xylene for 5-10 min, stained with Heindenhain’s hematoxylin, mounted using a drop of neutral balsam, and covered by the coverslips before examination under a Nikon ANTI-MOULD microscope (15×40).

2.3 DNA ploidy analysis

Flow cytometry analysis was performed to determine the ploidy level of the regenerated plants by using the leaves of randomly selected 21-month-old regenerated plantlets. The leaves of the mother plant were used as control. Flow measurement was performed as described by [13].

To determine the nuclear DNA content, flow cytometric analysis was performed using the protocol of Arumuganathan and Earle [14] at a laser wavelength of 488nm. Briefly, a sharp razor blade was used to manually scrape the young leaf tissues (typically 30 mg) on ice in 1.5 ml of nucleus extraction buffer containing 5.55 mg of KCl, 3.69 mg MgSO4, 1.8 mg of Hepes, 1.5 mg dithiothreitol, 36.3 µl of 10% Triton X-100 and a little of 5% polyphenol oxidase, which helps prevent polyphenol oxidation in the

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue [10]. In addition, in most cases the somatic embryos or the embryogenic cultures can be cryopreserved, which makes it possible to establish gene banks. Embryonic cultures are an attractive target for genetic modification. [11]. It is also an efficient method for clonal propagation of genetically stable regeneration in many species [12]. However, the induction of somatic embryogenesis in A. konjac is rarely reported worldwide. Hu [7] found that the Murashige and Skoog (MS) medium supplemented with high concentration of auxin alone with of 13.57 μM 2,4-dichlorophenoxyacetic acid (2, 4-D) and 8.88 μM 6-benzylaminopurine (BA) or 21.48 μM naphthalene acetic acid (NAA), and 6.66 μM 6-BA can be used for somatic embryo induction, but no plantlets were regenerated.

We have developed an efficient method for somatic embryogenesis from floral axis of A. konjac. This system will not only improve the production of healthy seed tubers but also contribute to transgenic and functional genome researches on A. konjac.

2 Methods

2.1 Plant materials and culture conditions

Amorphophallus konjac flower buds were gathered from the greenhouse of Life Science College of Wuhan University in China. The floral axes were cut off as the explants for the further treatment. The explants were cut into pieces, surface sterilized with 75% ethyl alcohol for 30 s followed by 0.1% (w/v) HgCl2 for 6-8 min with occasional shaking, and then rinsed thoroughly at least thrice with sterile distilled water. The floral axes were cut into small pieces (approximately 1cm  ×  1cm) by a sterile knife; 2-3 pieces were placed in 250 ml jars containing 50 ml of medium with various concentrations of plant hormones. These various combinations were used to test the callus induction (Table 1).

The explants cultured in the culture bottles with MS (Murashige and Skoog, 1962) medium supplemented with 3% sucrose, 0.2% polyvinyl pyrrolidone and 0.3% phytagel (Sigma) and containing various combinations of plant hormones. The pH of each medium was adjusted to 5.8 by using 0.1 M HCl and 0.1 M NaOH; each medium was then autoclaved at 121°C and 1.2 kg·cm−2 pressure for 20 min. The cultures were incubated in the dark before being put under cool-white fluorescent light (12 h photoperiod

36   L. Zhong, et al.

arranged cells of the callus, and each developmental process of the callus can be observed in the microscopic field (Figure 3). Transferring the compact calli to the differentiation medium resulted in morphogenetic events. Typical features of meristemoid (cluster of organgenic cells) were small in cell size with cytoplasmic density, minimal vacuoles and relatively large nuclei.

After approximately 25 days, some of the embryogenic calli began to differentiate, part of the calli turned green and small bud-like structure gradually formed (Figure 4a) on the differentiation culture medium containing various concentrations of NAA and 6-BA. Over the  subsequent  week, the buds could be observed

sample. The nuclear suspensions were filtered through a 30 µm mesh nylon cloth and then placed into a labeled test tube. Following filtration, supernatant was aspirated by centrifuged at 3000 rpm at 4°C for 15 min, and nuclei were resuspended in 450 µl MgSO4·buffer containing 5.55 mg KCl, 3.69 mg MgSO4, 1.8 mg of Hepes and 50 µl RNase A (50 µg/ml) was added to prevent staining of double-stranded RNA. The suspension was subsequently stained with 5 µl of propidium iodide and then incubated in the dark for 15 min at 37°C.

2.4 Statistical analysis

The experiments were set up in a completely randomized design with three replicates for each treatment and each replicate included 10 explants. The number of tissue clumps showing embryogenesis in each petri dish was counted, and percentage induction was calculated as the ratio of the number of clumps showing embryogenesis to the number of total clumps and then expressed as percentage. The data were analyzed using one-way analysis of variance (ANOVA) and the significance of the differences among treatments of each experiment was evaluated by Duncan’s multiple range test at P = 0.05 using SPSS (version 16.0).

3 Results During the first few days of culture, some small pieces of floral axes swelled and folded on the MS medium supplemented with different hormone combination of various concentrations. Approximately 4 weeks later, the embryogenic calli were obtained from the cut edges of most pieces (Figure 1), these calli formed directly from the explants without an intervening callus phase and can be easily distinguished from the explants’ surface. The calli were induced by all combinations of auxin and cytokinin, although induction percentage varied significantly depending on the hormone combinations. Nearly no callus was induced in hormone-free medium.

Induction percentage (%) in different concentrations is presented in Table 1. The optimum callus induction was observed on MS medium supplemented with 9.04 µM 2, 4-D and 5.37 µM NAA among all other auxins and cytokinins combinations and concentrations used. The corresponding highest induction rate was approximately 79.8%. The calluses on this type of medium were friable, semi-transparent and yellowish (Figure 2). In addition, histological examination showed the regularly

Fig. 1. Embryogenic calli induction from explants

Fig. 2. Embryogenic callus multiplication

High embryogenic ability and regeneration from floral axis of Amorphophallus konjac (Araceae)    37

almost all of them were dead ultimately. Alteration in the concentrations of the hormones keeping ratios constant remarkably decreased the percentage of differentiation. Few regenerated plantlets were observed in hormone free medium.

The flow cytometry results shown in Fig. 5 strongly indicate that the analyzed regenerated plants were diploid and all the samples analyzed have the same ploidy level. No polyploidy occurred when somatic embryos were germinated and converted to plantlets.

clearly, their numbers increased rapidly and their height increased was visible (Figure 4b). Approximately 40 days later, most of the calli developed into 3.0-5.0 cm long plantlets bearing well-formed roots (Figure 4c). The highest rate of differentiation is 95.6% (Table 2), and the medium supplemented with 8.88 µM 6-BA, 5.37 µM NAA was the most suitable  to support normal  development. In some differentiation medium, the calli differentiated into malformed buds or took roots only. These calli cannot develop into complete plantlets,

Table 1. Effect of different concentrations of 2, 4-D, 6-BA and NAA on embryogenic callus induction of A. konjac on MS medium

Growth regulators (µM) Frequency of explants with embryogenic callus produced (%)

2, 4-D 6-BA NAA

0 0 0 0

2.26 10.23 ± 1.63cd

4.52 2.43 ± 0.47ab

9.04 0

0 2.22 2.5 ± 0.42ab

2.26 2.22 30.20 ± 1.73gh

4.52 2.22 33.33 ± 1.93hi

9.04 2.22 35.57 ± 1.13i

0 4.44 11.37 ± 1.38d

2.26 4.44 33.33 ± 1.93hi

4.52 4.44 43.67 ± 2.23j

9.04 4.44 26.30 ± 0.40fg

0 8.88 5.60 ± 1.27bc

2.26 8.88 35.57 ± 1.13i

4.52 8.88 49.23 ± 2.05k

9.04 8.88 46.67 ± 1.93jk

0 2.69 3.47 ± 0.50ab

2.26 2.69 17.80 ± 1.10e

4.52 2.69 24.43 ± 1.13f

9.04 2.69 45.57 ± 1.13jk

0 5.37 7.50 ± 0.49bcd

2.26 5.37 48.87 ± 5.57k

4.52 5.37 71.10 ± 1.10m

9.04 5.37 79.80 ± 1.10n

0 10.74 11.70 ± 0.87d

2.26 10.74 45.17 ± 1.00jk

4.52 10.74 58.47 ± 0.61l

9.04 10.74 62.50 ± 0.96l

Values represent the mean of three replicates ± SE. Mean values in a column followed by the same superscript are not significantly different (P = 0.05) based on Duncan’s multiple range test (DMRT).

38   L. Zhong, et al.

Fig. 4. The differentiation of embryogenic calli. a Small bud-like structure formed from the calli. b Shoot proliferation from the calli gradu-ally. c Approximately 40 days later, the calli developed into plantlets bearing well-formed roots.

Fig. 3. Histochemical analysis during the initiation of the somatic embryogenesis in A. konjac. a Initial phase of proembryo formation with densely stained meristematic cells indicating intensive cell divisions. b Globular embryo (arrow) clearly discernible from the surrounding tissue c Heart-shaped embryo stage. d Further differentiation of heart-shaped resulted in the formation of torpedo stage.

Fig. 5. Flow cytometer analysis of DAPI stained nuclei of A. konjac. Each flow cytometric analysis of the somatic embryo-obtained plants along with the parent grown showed that they all had same peaks.

High embryogenic ability and regeneration from floral axis of Amorphophallus konjac (Araceae)    39

Table 2. Effect of auxins (6-BA and NAA) and their concentrations on differentiation of embryogenic callus

Growth regulators (µM) Differentiation rate of embryogenic callus (%)

6-BA NAA

0 0 00 5.37 42.57 ± 4.45e

0 10.74 16.93 ± 0.69c

4.44 0 8.87 ± 0.81b

4.44 5.37 58.10 ± 0.70f

4.44 10.74 59.77 ± 1.5f

8.88 0 23.73 ± 0.23d

8.88 5.37 95.63 ± 0.73g

8.88 10.74 47.13 ± 1.59e

Values represent the mean of three replicates ± SE. Mean values in a column followed by the same superscript are not significantly different (P = 0.05) based on Duncan’s multiple range test (DMRT).

exhibited the same ploidy level and that no polyploidy was observed in embryogenic callus tissue. The stability reported here is not described in other species, in which polyploidisation was found during somatic embryogenesis (23-27). Endemann [27] found that tetraploidy occurs 8% of the clones tested over a culture period of 7 years in Quercusrobur. In Q. canariensis, tetraploidy occurred in somatic embryogenesis after 14 months of continuous subculture [23]. Prolonged culture, any unfavorable condition or any substance that affects or blocks plant metabolism, growth or development may result in polyploidization [27].

Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. In some cases, somatic embryogenesis is favored over other methods of vegetative propagation because of the possibility to scale up the propagation by using bioreactors. In grapevine (Vitis vinifera L.) cultivars, the maximum rates of induction and germination were 99.5% and 68%, respectively [28]. Pavlović [29] reported that the highest frequencies of embryogenic calli induction in cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleraceavar. botrytis) were 83.3% and 87.5%, correspondingly. Moreover, the development of somatic embryos into plantlets was 56% in cabbage and 79% in cauliflower. Hence, the induction and differentiation rate in these species can satisfy the production needs. A study showed that unpollinated ovaries and petiole of A. konjac can be used as explants to induce development of embryogenic callus. But the embryogenic callus induced from the petiole cannot display normal development,

4 DiscussionSomatic embryogenesis is a complex method of plant regeneration which can be affected by numerous factors: the genetic background of plant material, the manipulation of primary explants initiating culture and culture conditions [12]. Some studies have reported that the culture medium exerts stronger effect on somatic embryogenesis than other factors [15]. The type of hormone used, as well as the corresponding concentration, also significantly influences callus induction. In some plant species, 2, 4-D is the most effective auxin for somatic embryogenesis [16,17], whereas high Cytokinin in concentrations are required to initiate somatic embryogenesis in another species [18]. Our results show that somatic embryogenesis slightly occurred in media containing NAA or 6-BA, whereas embryogenic callus was induced much faster and more frequently when 2, 4-D was used, suggesting that floral axis was much more sensitive to 2, 4-D than to NAA and 6-BA. However, higher concentrations of 2, 4-D could not increase the induction rate nor stimulate callus development. Instead, high concentrations of 2, 4-D can result in soft, slow-growing, and malformed callus cultures, which will eventually turn brown/black [19]. A similar trend was also observed in 6-BA and NAA. Some researchers have reported that most species exhibit a lower frequency of primary than secondary somatic embryos induction [20,21]. Similar results are obtained in the present study.

We also found that the protocol developed by Pinto [22] combined a simple methodology with a low-hormone method reducing the risk of somaclonal variation. Our results strongly indicated that all the samples analyzed

40   L. Zhong, et al.

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and no plantlets are regenerated except that some adventitious buds have formed [7]. When unpollinated ovaries were utilized as explants, the rate of embryogenic callus induction was 34%, and the highest plantlet formation efficiency was only 35.5%, both of which are too low to be applied in A. konjac production [8]. It is the only report to date that obtains somatic embryogenesis of A. konjac. Given that the reported frequency of somatic embryogenesis is extremely low [7], we developed an efficient and reproducible tissue culture method for A. konjac. This method successfully induced the development of A. konjac embryogenic callus, which developed into healthy virus-free plantlet sex habiting a high percentage of acclimatization and slight ploidy variation. The entire regeneration procedure, including the acclimation period, took approximately 5 months and the recurrent somatic embryogenesis of A. konjac retained high embryogenic potential after 1 year.

Our findings will provide guidance  for the practical and rapid propagation of A. konjac. It has proven that the floral axis is a better explant than the unpollinated ovaries and the petiole thus could be further applied in rapid propagation for production, with a comparatively high induction rate of 79.8% and differentiation rate of 95.6%, respectively. Moreover, the tuber of A. konjac is rich sources of water-soluble glucomannan, and widely used in  industry. However, the floral axis is hardly used as industrial material. In conclusion, the floral axis is an ideal explant for induction of somatic embryogenesis and planet regeneration in A. konjac. The present study described a high-efficiency method for repetitive somatic embryogenesis for A. konjac by using floral axis as explants which is the most efficient so far. The efficient method for induction of primary and plant regeneration described in present study may contribute to the improvement of the propagation rate of A. konjac, which is useful in production. Therefore, based on this achievement, genetic  improvement and application on breeding could be realized easily.

Acknowledgments: This study was supported by grants from the National Science and Technology Supporting Program (No. 2011BAD33B03), the Fundamental Research Funds for the Central Universities (No. 2042016kf1106)) and Philanthropic Project of Scientific Research of Hubei (No. 2012DBA11001).

Conflict of interest: Authors state no conflict of interest.

High embryogenic ability and regeneration from floral axis of Amorphophallus konjac (Araceae)    41

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