Efficient nursery plant production of dwarf cogongrass (Imperata cylindrica L.) through mass propagation in liquid culture

O R I G I N A L A R T I C L E

Efficient nursery plant production of dwarf cogongrass

(

Imperata cylindrica

L.) through mass propagation in liquid

culture

Nafiatul Umami1,2,†_{, Takahiro Gondo}3,†_{, Hidenori Tanaka}4_{, Mohammad M. Rahman}5

and Ryo Akashi3

1 Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, Miyazaki, Japan 2 Faculty of Animal Science, Gadjah Mada University, Yogyakarta, Indonesia

3 Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan 4 Graduate School of Agriculture, University of Miyazaki, Miyazaki, Japan

5 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

Keywords

Amplified fragment length polymorphism; dwarf cogongrass; mass propagation; multiple-shoot clump; nursery plant.

Correspondence

Ryo Akashi, Frontier Science Research Center, University of Miyazaki, Miyazaki 889-2192, Japan.

Email: rakashi@cc.miyazaki-u.ac.jp

†These authors contributed equally to this work.

Received 8 March 2012; accepted 21 September 2012.

doi: 10.1111/grs.12001

Abstract

Dwarf cogongrass (Imperata cylindrica L.) was developed as a dwarf mutant through heavy-ion beam irradiation 7 years ago. The dwarf mutant could be expected to use as a new variety for a cover plant with low maintenance, although it has poor seed fertility. To establish an efficient nursery production system for dwarf cogongrass, we attempted mass propagation of it in liquid culture and investigated the genetic stability of its regenerants. Multiple-shoot clumps (MSCs) were initiated from apical meristems of dwarf cogongrass on Murashige and Skoog (MS) solid medium supplemented with 0.1 mg L 1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg L 1_{benzylaminopurine (BAP).}

Mass propagation conditions were established from MSCs cultured in MS liquid medium containing 0.05 mg L 1 2,4-D and 0.5 mg L 1 BAP. The fresh weight of the MSCs per flask increased by more than 16 times in 14 days of liquid culture. Two different sizes of MSCs were produced in liquid culture. When smaller MSCs (<2 mm in diameter) were transferred to half-strength hormone-free MS solid medium, plant regeneration occurred at high frequency (93.3%). These tissues showed high regenerative potential with approximately 350 green shoots recovered within 50 days from 60 regenerating clumps. Fur-thermore, root elongation was vigorous in the regenerants growing in the same medium. Regenerated plants were acclimatized in hydrated Jiffy-7 pellets for 30 days and then grown in soil as nursery plants. The plant height of regener-ants was almost the same as original dwarf cogongrass and significantly lower than the wild type plants (P<0.01). Analysis of amplified fragment length polymorphism (AFLP) banding patterns generated using 10 primer combina-tions showed no major genetic variacombina-tions among the regenerated plants and original dwarf cogongrass.

Introduction

Cogongrass (Imperata cylindrica L.) is a widely grown perennial grass in tropical and temperate regions (Falvey 1981; Brysonet al.2010). This grass performs well under severe drought conditions and spreads over wide areas

cultivars could solve these problems, which could facili-tate the maintenance of the cover grass that prevent soil erosion of river levees etc.

In an earlier study, we established a tissue culture sys-tem for regenerating cogongrass and developed a dwarf mutant through heavy-ion beam irradiation (Shigekiet al.

2009). Although the plant height of common cogongrass is 30–80 cm, the height of this mutant is 15–20 cm, which is a sufficiently low height to preclude the need for cutting. The dwarf mutant could be expected to use as a new variety for a cover plant with a low maintenance. However, it has poor seed fertility. In order for the dwarf grass to be widely used as a ground cover, it is necessary to devise an efficient propagation system.

Plant propagation requires a long time to prepare the nursery, intensive labor, and significant amounts of space, and is dependent on field conditions throughout the growing season. These limiting factors can be solved by using an in vitro tissue culture method.In vitro propaga-tion enables rapid multiplicapropaga-tion without depending on the season and allows healthy plant production with the added benefit of germplasm storage. There are some reports of cogongrass tissue culture (Akashi and Ikeda 1989; Shigeki et al. 2009); however, these methods are not suitable for mass propagation since the proliferation rates are low.

In vitro propagation using liquid culture is the pre-ferred method for mass propagation. Several monocotyle-donous plants have been propagated using liquid cultures including garlic (Allium sativum ‘Howaito-roppen’) (Na-gakubo et al. 1993), bananas ‘Grande Naine’ (Alvard

et al. 1993) and orchids (Doritaenopsis 9 Phalaenopsis) (Parket al.1996). These liquid cultures established a high level of performance for propagation and demonstrated a high potential for nursery production. Successful mass propagation of dwarf cogongrass requires establishing an efficient liquid culture system. Further, it is important to assure the genetic uniformity of regenerated plants as nursery plants production. Amplified fragment length polymorphism (AFLP) fingerprinting is widely used to certify genetic stability in plants regenerated from tissue culture (Cloutier and Landry 1994; Hashmi et al. 1997), because it has a higher reproducibility with the effective multiplex ratio over other molecular markers techniques (Powellet al.1996).

Here, we described an efficient nursery plant produc-tion system of dwarf cogongrass by mass propagaproduc-tion in liquid culture. This system consists of three steps: prolif-eration of multiple-shoot clumps by liquid culture, plant regeneration and rooting, and acclimatization. We have established efficient methods for each step, making it pos-sible to mass produce dwarf cogongrass nursery plants. Moreover, we performed AFLP analysis to confirm the

genetic stability of nursery plants through mass propaga-tion.

Materials and methods

Induction of multiple-shoot clumps

Dwarf cogongrass was developed as a dwarf mutant through heavy-ion beam irradiation in a previous study (Shigeki et al. 2009). The origin of this strain is derived from field-grown native cogongrass in the University of Miyazaki. High quality MSCs were obtained from apical meristems through the screening of a large number of MSCs. One MSCs line was used for heavy-ion beam irra-diation and produced multiple plantlets. When the mor-phological characteristics were evaluated, the dwarf mutant showed the lowest plant height during the first 2 years. This was multiplied by plant divisions in a green-house in the University of Miyazaki.

Shoot-tillers of dwarf cogongrass collected from a greenhouse were surface-sterilized in 70% (v/v) ethanol for 1 min and in 2% (v/v) sodium hypochloride for 15 min, followed by three washings with sterile water. Apical meristems were excised from shoot-tillers and cul-tured in a 90-mm Petri dish with 25 mL of Murashige Skoog (MS) medium (Murashige and Skoog 1962) con-taining 3% sucrose, 0.3% phytagel with 0.1 mg L 1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg L 1 6-benzylaminopurine (BAP) (MS-D0.1B2) at 27°C under fluorescent lights (3500 lux for 16 h). After 30 days of culture, primary MSCs were transferred to fresh MS-D0.1B2 medium. Single apical meristem-derived compact and dividing clumps were subcultured every 30 days onto the same medium. All media were adjusted to pH5.6–5.8 prior to autoclaving at 121°C for 15 min.

Proliferation in liquid culture

For mass propagation, MS-D0.1B2 solid medium was replaced with MS liquid media containing different com-binations of 2,4-D (0.0, 0.05 and 0.1 mg L 1) and BAP (0.5, 1.0 and 2.0 mg L 1). 0.3 g of fresh weight MSCs were transferred to 20 mL liquid media in a 100 mL Erlenmeyer flask (Asahi Glass, Shizuoka, Japan). Cultures were incubated on a 100 rpm rotary shaker (Gallenkamp MIR 220 RL; Sanyo Electric, Osaka, Japan) at 27°C under fluorescent lights (3500 lux for 16 h) and subcultured every 14 days.

Plant regeneration and rooting

combinations (0.5 mg L 1 BAP+0.05 mg L 1 2,4-D; 2.0 mg L 1 BAP+0.01 mg L 1 2,4-D and 1.0 mg L 1 GA3+1.0 mg L 1kinetin; hormone-free) and half-strength

hormone-free MS medium. After 50 days of culture, regener-ated and rooted plants (10–15 mm in size) were acclimatized in the growth chamber.

Acclimatization

Regenerated and rooted plants were removed from the Petri dish carefully and washed in water to remove adher-ing culture media. Subsequently, the regenerants were acclimatized in Jiffy-7 peat pellets (Jiffy Products Interna-tional AS, Grorud Norway), soil (Yamasou, Miyazaki, Japan) and soil plus vermiculite (Ashahi Kogyo, Okay-ama, Japan) (2:1 v/v) at 27°C under 70–80% of moisture. After 30 days of acclimatization, plants were transferred to 5-cm nursery pots containing soil and placed in a greenhouse with temperatures between 18 and 26°C. These grown plants were transplanted to the field in plots at a spacing of 20920 cm together with original dwarf plants and wild type cogongrass on 18 May 2011, and plant heights were measured on 7 November 2011.

The data were analyzed by Tukey’s Test with Excel sta-tistics software (Social Survey Research Information, Tokyo, Japan).

AFLP analysis

Amplified fragment length polymorphism analysis of in vitro regenerated dwarf cogongrass was performed in order to evaluate the genetic variations in this strain. Total genomic DNA was extracted from the leaves of a single plant using the DNeasy Plant Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s proto-col. Genomic DNA (250 ng) was digested with restriction enzymes (Xba I andMse I) (PstI andMse I) and ligated to double-stranded adaptors. The adapter-ligated DNA was preamplified using the following cycling parameters: 20 cycles of 30 s at 94°C, 60 s at 56°C and 60 s at 72°C. The pre-amplified DNA was diluted in a ratio of 1:5 and was used as a template for the selective amplification using four Xba I and Mse I primer combinations (CTA/M-CTG, CAG/M-CAA, CTG/M-CAT and X-CGA/M-CGC) and six PstI and Mse I primer combina-tions (P-GTA/M-CGT, P-GCA/M-CAC, P-GCG/M-CGA, P-GAC/M-CCG, P-GTG/M-CAG and P-GTT/M-CTC). The cycling parameters were: one cycle of 30 s at 94°C, 30 s at 65°C, and 60 s at 72°C. The annealing tempera-ture was lowered by 0.7°C per cycle during the first 12 cycles followed by 23 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 60 s. The PCR products were separated and detected using a QIAxcel system with QX DNA High

Resolution Cartridge (Qiagen). The DNA banding pat-terns were measured using BioCalculator version 3.0.02 (Qiagen).

Results and discussion

Culture of MSCs on solid medium

Apical meristems as initial explants were isolated from shoot tillers of dwarf cogongrass (Figure 1a) and were cul-tured on MS-D0.1B2 at 27°C under fluorescent lights. The MSCs were maintained on the same medium. Repeated subculture at 30-day intervals led to a higher frequency of shoot-tips formed and the formation of more compact shoot-tip clusters (Figure 1b,c). The culture of MSC has been reported for other graminaceous species (Zhong

et al.1992; Zhang et al.1996; Sharma et al.2004; Gondo

et al.2007; Ishigakiet al.2009) and was found to result in potentially higher plant regeneration for a longer time in culture without the appearance of somaclonal variation compared with embryogenic callus (Gondo et al. 2007). The lack of somaclonal variation is one of the most important factors for nursery production, and the MSCs are reliable tissues for mass propagation.

Mass propagation of MSCs in liquid culture

For mass propagation, the MSCs were transferred to liquid MS medium containing 2,4-D (0, 0.05 and 0.1 mg L 1) and BAP (0.5, 1.0 and 2.0 mg L 1) (Figure 2). Different hormone combinations affected the characteristics and degree of proliferation of MSCs. Anthocyanin pigment accumulated in the MSCs cultured on liquid MS media without 2,4-D (0.0 mg L 1 2,4-D) (Figure 2a). MS media in the presence of 0.1 mg L 1 2,4-D induced callus formation from MSCs (Figure 2c). Real proliferation of MSCs occurred in the presence of 0.05 mg L 12,4-D (Figure 2b). High levels of MSCs pro-liferation was found in the medium containing 0.05 mg L 12,4-D with 0.5 or 2.0 mg L 1BAP.

Liquid culture has a high potential for mass propaga-tion and can provide a continuous supply for nursery production (Alvardet al.1993; Ilanet al.1995; Kimet al.

continu-ously by producing secondary shoot-tips, and this culture method facilitated mass propagation.

Plant regeneration and rooting

Multiple-shoot clumps, cultured in liquid MS media with 0.05 mg L 1 2,4-D and 0.5 mg L 1 BAP (Figure 1d), were transferred to MS media containing several different hormone combinations to compare their ability to sup-port plant regeneration and rooting. Table 1 shows the results of this experiment after 50 days of culture. Plant regeneration occurred only in hormone-free MS and half-strength hormone-free MS solid media with frequencies of 62.5% and 70.9%, respectively. The highest frequency of regeneration occurred with half-strength hormone-free MS solid medium, and rooting was induced in the same medium.

After more than 40–50 days of liquid culture, the sus-pension was composed of large (2–5 mm) (Figure 1e) and small aggregates (<2 mm) (Figure 1f,g). The small clumps appeared to consist of tiny shoots divided from a

large clump (Figure 1g). We sieved the MSCs through stainless mesh (2 mm) and cultured them in half-strength hormone-free MS solid medium. The smaller clumps showed a significantly higher percentage of plant regener-ation (93.3%) with 6.2 regenerated plants per MSCs (Table 2, Figure 1h). Furthermore, the roots of regener-ated plants vigorously elongregener-ated (Figure 1i). Plant regen-eration and rooting usually require different media (Gondo et al.2007; Ishigaki et al.2009); however, it was possible to perform both culture steps at the same time with high frequency using this protocol in dwarf cogon-grass. The small clusters consisting of many tiny shoots gave a clump high potential for plant regeneration and rooting.

Acclimatization and nursery plant production

The regenerated plants were removed from their Petri dishes, washed thoroughly in running tap water and transferred to three different acclimatization media (Jiffy-7 pellets, soil, and soil plus vermiculate). After 30 days of

(a)

(e)

(h) (i) (j)

(f) (g)

(b) (c) (d)

acclimatization, 95–100% of the regenerants had grown in all media; however, the growth rate was different for the three media. Regenerants grown in the Jiffy-7 med-ium had the greatest plant height, and plant height was significantly different compared with that in the other two media (Figure 3). It is likely that the Jiffy-7 pellets provided sufficient moisture and a higher nitrogen con-tent compared with those in other media (Noraini et al.

2009). After 30 days of acclimatization, plants grown in Jiffy-7 pellets were transferred to 5-cm pots containing soil for nursery production (Figure 1j). Subsequently, these grown plants were transplanted to the field plots together with original dwarf plants and wild type of

co-gongrass. The plant height of regenerants ranged between 15.2 and 18.5 cm, with an average value of 16.7 cm. This score was almost the same as the original dwarf cogon-grass and significantly lower than the wild type with an average value of 60.0 cm (P<0.01). These results indi-cated that plant height of regenerated dwarf plants was stable through mass propagation in liquid culture.

AFLP analysis

Amplified fragment length polymorphism due to its high multiplex ratio (Powell et al. 1996) and reproducibility has proven to be a highly efficient tool for characterizing somaclonal variation (Carolan et al. 2002; Popescu et al.

2002). Twenty-four regenerated plants, which were propa-gated in liquid culture for 2 years, and five original dwarf cogongrass were analyzed. A total of 101 clear reproduc-ible fragments were detected by AFLP analysis among all the samples (Table 3). Only one fragment indicated poly-morphic bands by using X-CGA/M-CGC primer combi-nation (Figure 4), while the other fragments (100 fragments) appeared monomorphic among the original dwarf cogongrass and regenerated plants. The results of the present investigation demonstrated that no major genetic variation among the original plants and regener-ants was observed. In the present study, genetic unifor-mity of the regenerants revealed the importance of the present establishment method as supplying a nursery plant production system of dwarf cogongrass.

Conclusion

In conclusion, we have established an efficient nursery plant production system for dwarf cogongrass through mass propagation in liquid culture and analyzed the genetic stability of the regenerated plants by AFLP analy-sis. Liquid cultures displayed a high potential for mass propagation that made it possible for large scale nursery Figure 2 Effect of plant hormone concentrations on characteristics

and proliferation of multiple-shoot clumps (MSCs) in dwarf cogon-grass. A, B and C indicate characteristics of MSCs in liquid MS media supplemented with 0.0, 0.05 and 0.1 mg L 12,4-D. Dotted line indi-cates proliferation of MSCs on MS-D0.1B2 solid medium. Values are the means of three replications and standard deviations are repre-sented by vertical bars. Letters indicate a significant difference by Tukey’s test (P_<0.05).

Table 1 Effect of medium composition on plant regeneration and rooting from multiple-shoot clumps (MSCs) of dwarf cogongrass

Medium composition

No. inoculated MSCs

No. regenerated MSCs (%)

Total no. regenerated plants

No. plants/

regenerated MSC Rooting† Basal medium Hormone concentrations (mg L 1₎

MS BAP (0.5+2,4-D (0.05) 74 0.0 0.0 0.0

BAP (2.0)+2,4-D (0.01) 74 0.0 0.0 0.0

GA3 (1.0)+Kinetin (1.0) 74 0.0 0.0 0.0

74 46.3 (62.5±2.1)b _90.0

±4.9b _2.0

±0.2b

+

1/2 MS 74 52.5 (70.9±4.9)a _145.5

±5.6a _2.8

±0.2a

+++

The values are means±standard deviations of three replications, and rooting was evaluated objectively. Letters indicate a significant difference by Tukey’s test (P_<0.05).

production. Sieving the small clusters in liquid culture was an important step for efficient plant regeneration and rooting. Regenerated plants were acclimatized in Jiffy-7 media for 30 days and grown in pots containing soil as nursery plants. The plant height of regenerants was almost the same as original dwarf cogongrass and signifi-cantly lower than the wild type plants in the field. Fur-ther, no major genetic variation was detected with AFLP Table 2 Effect of multiple-shoot clump (MSC) size on plant regeneration and rooting in dwarf cogongrass

Size of MSC Experiment

No. inoculated MSCs

No. regenerated MSCs (%)

Total no. regenerated plants

No. plants/

regenerated MSC Rooting†

<2 mm 1 60 56 341 6.1 +++

2 56 330 5.9 +++

3 56 367 6.6 +++

Mean 56.0 (93.3±0.0)a _346.0

±19.0a _6.2

±0.3a

+++

2–5 mm 1 60 28 78 2.8 +

2 31 57 1.8 +

3 30 84 2.8 +

Mean 29.7 (49.4±2.5)b _73.0

±14.2b _2.5

±0.6b

+

Mean values are indicated mean±standard deviations of three replications, and rooting was evaluated objectively. Letters indicate a significant difference by Tukey’s test (P_<0.05).

†Root rating: none,+poor,++moderate, and+++good.

Figure 3 Effect of acclimatization medium on nursery plant growth of dwarf cogongrass. Values are the means of 30 replications and standard deviations are represented by vertical bars. Letters indicate a significant difference by Tukey’s test (P_<0.05).

Table 3 Amplified fragment length polymorphism (AFLP) analysis of original dwarf plants and regenerated dwarf plants of cogongrass

Primer combination

Total fragments

Fragment details

Polymorphic bands Original

plant

Regenerated plant

X-CTA/M-CTG 15 15 15 0

X-CAG/MCAA 13 13 13 0

X-CTG/M-CAT 15 15 15 0

X-CGA/M-CGC 5 4 5 1

P-GTA/M-CGT 9 9 9 0

P-GCA/M-CAC 8 8 8 0

P-GCG/M-CGA 7 7 7 0

P-GAC/M-CCG 10 10 10 0

P-GTG/M-CAG 10 10 10 0

P-GTT/M-CTC 9 9 9 0

Total 101 100 101 1

analysis among the original dwarf cogongrass and regen-erants. Plans to create a new variety using dwarf cogon-grass have been initiated and are now in progress. This efficient nursery plant production system will be useful for propagating and commercializing a new variety of dwarf cogongrass in the future.

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