Journal of Integrative Plant Biology 2006, 48 (10): 1163−1167

Received 17 Jan. 2006 Accepted 15 May 2006

Supported by the State Key Basic Research and Development Plan of

China (G2000046805).

*Author for correspondence. Tel: +86 (0)451 8219 1783; Fax: +86 (0)451

8219 1795; E-mail: <lumin-fu@163.com>.

**, Present address. The Finnish Forest Research Institute, PL, 18, 01301

Vantaa, Finland.

© 2006 Institute of Botany, the Chinese Academy of Sciences

Formation of Mycorrhiza-like Structures in CulturedRoot/Callus of Cathaya argyrophylla Chun et Kuang

Infected with the Ectomycorrhizal FungusCenococcum geophilum Fr.

Xue Sun, Yu-Hua Li and Lu-Min Vaario*,**

(College of Life Sciences, Northeast Forestry University, Harbin 150040, China)

Abstract

An in vitro system was used for ectomycorrhizal synthesis of Cenococcum geophilum Fr. with Cathayaargyrophylla Chun et Kuang, an endangered species. Calli initiated from stem segments and adventitiousroots differentiated from young seedlings were removed and cocultured with Cenococcum geophilum ona modified Murashige-Skoog medium. Fungal hyphae were visible within intercellular spaces of the callus4 weeks after inoculation, but definite and well-developed Hartig net structures did not form in the calli 8weeks after inoculation. The typical ectomycorrhizal structures (i.e. hyphal mantle and intracortical Hartignet) were observed in root segments 8 weeks after inoculation. This is the first report of asepticectomycorrhizal-like formation/infection between root organ/callus of Cathaya argyrophylla and theectomycorrhizal fungus Cenococcum geophilum. This culture system is useful for further investigation ofmycorrhizal synthesis in Cathaya trees.

Key words: ectomycorrhiza; endangered pine; Hartig net; in vitro.

Sun X, Li YH, Vaario LM (2006). Formation of mycorrhiza-like structures in cultured root/callus of Cathaya argyrophylla Chunet Kuang infected with the ectomycorrhizal fungus Cenococcum geophilum Fr. J Integr Plant Biol 48(10), 1163−1167.

doi: 10.1111/j.1672-9072.2006.00334.x; available online at www.blackwell-synergy.com, www.jipb.net

Cathaya argyrophylla Chun et Kuang, belonging to the familyPinaceae, is a very ancient relic conifer. It is native to the sub-tropical mountain regions of the Northern Hemisphere in Chinaand has great ecological importance within natural forests.This tree is distributed only in Guangxi, Hunan, Sichuan andGuizhou provinces (Wang 1990). It is a monotypic genus inChina, the pollen of which has been discovered in Eurasiantertiary deposits (Wang 1990). Because it has peculiar mor-phological characteristics and is similar to Pinus in embryonicdevelopment, C. argyrophylla is considered to have an

important meaning in phylogenetic development, geoflora, andpaleogeography (Fu and Jin 1996).

Nevertheless, propagation of C. argyrophylla in natural for-ests is hindered by several problems, such as low geneticdiversity, reproductive barriers, inferior capability of seedgermination, and habitat loss (Ying et al. 1983; Xie and Chen1999). The population of this endangered specie decreasesyear by year. It has been reported that there are fewer than4 000 C. argyrophylla trees in China that are taller than 1 m (Xieand Chen 1999).

Cathaya argyrophylla growing in natural forests has a verypowerful root system, with well-developed lateral roots to en-able its growth in places covered with bare rocks (Wang 1990).Ectomycorrhizal (ECM) fungi are known to play a fundamentalrole in enhancing the hydromineral nutrition in temperate andboreal forest ecosystems to affect host plant growth (Smithand Read 1997). A previous study (Vaario et al. 2006) reportedthat C. argyrophylla was a typical ECM tree based on in situand in vitro evidence and established an in vitro mycorrhizationsystem for this tree. However, physiological and biochemical

1164 Journal of Integrative Plant Biology Vol. 48 No. 10 2006

changes that are associated with ECM development remainunknown. It should be pointed out here that plant materials aredifficult to prepare for experiments because of the limited avail-ability of seeds. In our recent studies on the propagation of C.argyrophylla, it was observed that calli and adventitious rootswere obtained easily in vitro (X Sun et al., unpubl. obs., 2006).It is of interest to determine whether the symbiosis of C.argyrophylla only occurs at the whole-plant level and whetherthe cultured tissues and organs of C. argyrophylla are usefulfor mycorrhizal studies of C. argyrophylla.

The aim of the present study was to establish a system forthe in vitro synthesis of ECM between cultured root and callusof C. argyrophylla and Cenococcum geophilum Fr., which maybe applicable to other mycorrhizal fungus-host associations.

Results

Mycorrhizal infection in calli

After incubation for 2 weeks, the margin of the colony of C.geophilum reached the callus. Then, the hyphae began colo-nizing on the surface of the callus in the 3rd week.

The hyphae grew on the callus actinomorphically (Figure 1A).After 3 weeks incubation, a light-brown callus occurred on thebasal parts, which touched the medium; however, 80% of cal-lus numbers remained fresh and green. Following clearing,bleaching, and staining, fungal hyphae became visible withinthe callus intercellular spaces (Figure 1B, C). Eight weeks after

inoculation, the hyphae almost colonized the entire callus, butdid not overgrow the callus. Within the callus, hyphal apicesunderwent more extensive branching, showing greater invagi-nations (Figure 1D). In the semi-section, branched hyphae wereable to be distinguished from callus cells (Figure 1E).

Mycorrhizal colonization in roots

After 2 weeks incubation, the fungus began to make contactwith the adventitious root segments. One week later, root tipsbecame swollen and turned dark. It was clear that a network ofblack hyphae had formed around the root segments.Subsequently, the fungal hyphae completely enveloped the rootsegments and formed dark-brown mycorrhiza-like roots (Figure2A). Four weeks after inoculation, a structure composed ofdistinctly multi-branched hyhae had developed (Figure 2B) anda few hyphae had invaded and colonized the epidermal andcortical intercellular spaces, appearing between the epidermalhost cells in semi-sections (Figure 2C). Eight weeks afterinoculation, the definite and well-developed Hartig netstructures, “palmettis”, began to develop from the mycelial re-ticulate (Figure 2D). This structure was very similar to the“palmettis” that was observed previously within ectom-ycorrhizas collected from forest and in vitro specimens of thistree (Vaario et al. 2006). In addition, in the same samples mantlehyphae gave rise to profuse intracortical hyphae, which in-vaded and colonized the epidermal and cortical intercellularspaces, appearing to separate and isolate host cells (Figure2E).

Figure 1. Callus of Cathaya argyrophylla colonization by Cenococcum geophilum in vitro.

(A) The hypha was colonized on the surface of the callus initiated from a stem segment 3 weeks after inoculation. Bar, 3 mm.(B) Early infection structures, namely a branched hyphal apex (arrowhead) colonizing the cortical intercellular spaces, were observed 4 weeksafter inoculation. Bar, 10 µm.(C) Light micrograph of a semi-thin section of a callus shown in (B). Hyphal cells (h) were seen between the callus cells (c). Bar, 10 µm.(D) Reticulate hyphal structures (arrowhead) developing within the callus 8 weeks after inoculation. Bar, 10 µm.(E) Light micrograph of a semi-thin section of a callus shown in (D). A Hartig net-like structure (arrowhead) was observed between callus cells(c). Bar, 10 µm.

Figure 2. Adventitious roots of Cathaya argyrophylla colonization by Cenococcum geophilum in vitro.

(A) Adventitious roots were colonized along their length by hyphae to form a sparse mantle (m), from which copious extraradicle hyphaeemanate (em), 4 weeks after inoculation. Bar, 750 µm.(B) A distinct multibranched hyhae structure (arrowhead) within an adventitious root was developed 4 weeks after inoculation. Bar, 10 µm.(C) Light micrograph of a semi-thin section of an adventitious root shown in (B) showing the distinctive thick cell wall of cortical cells and anumber of hyphal cells (double arrowhead) between the cortical cells, as well as near the surface of a root tip. Bar, 20 µm.(D) A well-developed Hartig net structure, the “palmettis” (arrowhead), began to develop from the mycelial reticulate 8 weeks after inoculation.Bar, 10 µm.(E) Light micrograph of a semi-section of an adventitious root shown in (D) showing a loose and thin mantle (double arrowhead) on the roots.The Hartig net (arrowhead) colonizes the epidermis and cortex with no thick cell wall. Bar, 50 µm.

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1166 Journal of Integrative Plant Biology Vol. 48 No. 10 2006

Discussion

Previous studies have reported that Cathaya argyrophylla is atypical ECM tree on the basis of both in situ and in vitro evi-dence (Vaario et al. 2006). Nevertheless, a serious shortageof C. argyrophylla for experiment material hinders studies onthe mechanism of mycorrhization, the nutrition metabolism ofmycorrhizas of the Cathaya tree etc. It has been demonstratedthat cultured organs and calli are able to form mycorrhiza-likestructures in spruce (Sirrenberg et al. 1995) and Abies firma(Vaario and Suzuki 2004), and they have positive reactionswith ECM fungi in Pinus sylvestris (Niemi et al. 1998). In vitrocultures are frequently used in the study of the symbiosis ofarbuscular mycorrhizal fungi (Fortin et al. 2002; Declerck et al.2004). In order to maintain the mycorrhiza-forming ability ofisolated mycelia to their host plant, a few reports have sug-gested restoring this ability by passage through a mycorrhizalstage (Thompsom et al. 1993). The culture of host tissue ororgan/ECM fungi may become a useful mass inoculum for themycorrhization of plants in vitro or ex vitro.

With co-cultures of root segment-hyphae of Cenococcumgeophilum, typical morphological characteristics of mycorrhizalroots, such as swollen root tips and a network of blackextraradicle hyphae formed around the root base, were ob-served after inoculation for 4 weeks. The distinct and multi-branched hyphae structure was observed using the clearing,bleaching, and staining method. However, the cell walls of thecortex were still very thick, which is similar to that ofuninoculated roots of young seedlings of Cathaya argyrophyllain vitro (Vaario et al. 2006). Moreover, a well-developed Hartignet structure, the “palmettis”, began to develop in root seg-ments 8 weeks after inoculation. This phenomenon was in ac-cord with in vitro mycorrhizas of young seedlings of C.argyrophylla; however, development of the Hartig net in cul-tured roots was much quicker than that in roots of an intactplant and the latter took at least 14 weeks to form a distinctHartig net structure (Vaario et al. 2006). Conversely, Wenkartet al. (2001) have reported a slower rate of mycorrhizal devel-opment in hair root cultures of Cistus species. We are investi-gating the differences in the findings in terms of culture media,host plants, and the age of the roots.

Cenococcum geophilum was able to infect cultured calli ofCathaya argyrophylla and form the reticulate hyphal structureswithin a callus. After 8 weeks inoculation, mycorrhiza-like struc-tures were observed less frequently in callus cultures, in whichthe well-developed Hartig net structure “palmettis” did not form.Mycorrhizal infection at an early stage of callus culture is simi-lar to the finding in Abies firma-Laccaria bicolor by Vaario andSuzuki (2004).

The use of root cultures is particularly useful as a simplifiedmodel system for the investigation of physiological aspects,such as signaling between the symbiotic partners and

metabolism of the fungus, because most parameters can bestrictly controlled. Under these conditions, detailed non-destruc-tive observations at the morphological and cellular levels canbe made (Fortin et al. 2002). Root/callus cultures for mycor-rhizal investigations provide enough plant material for studieson Cathaya argyrophylla, so that more information will be ob-tained to re-establish this critical endangered species.

In summary, we have demonstrated that Cenococcumgeophilum and root/callus segments of Cathaya argyrophyllaare able to grow together on modified Murashige-Skoog me-dium (MMS) and form typical ECM associations and ECM infec-tion at an early stage. However, whether the mycorrhiza-likestructures substantially fulfill symbiotic functions remainsunknown. It is necessary to undertake a longer-term study tofollow the development of the mycorrhiza-like structures.

Materials and Methods

Fungal culture and plant material

Cenococcum geophilum Fr. was kindly provided by ProfessorK. Suzuki (University of Tokyo, Tokyo, Japan). The isolate wasmaintained at (23 ± 2) °C in darkness on an modified Melin-Norkrand medium (MMN; Marx 1969) containing 50.46 mmol/Lglucose instead of sucrose and solidified with 1.5% agar.

Seeds of Cathaya argyrophylla Chun et Kuang were col-lected in Dayaoshan, Guangxi Province, China, in October 2002,dried in air, and then stored in a polyethylene bag in darknessat 4 °C until use. Seeds were washed with tap water for ap-proximately 5 h. Seeds were surface sterilized with 70% etha-nol for 30 s and then in 30% H2O2 for 30 min. After three rinsesin sterilized deionized water, seeds were dried on a sterilizedPetri dish on the cleaning bench for 5 min. Subsequently, seedswere placed on a 1.4% agar medium containing 25.23 mmol/Lglucose for 7 d and the seed capsule was then removed usinga knife and forceps. Germinated seedlings were transplantedinto MMS medium containing 0.89 µmol/L 6-benzylaminopurine(6-BAP), 0.11 µmol/L α-naphthaleneacetic acid (NAA), 58.43mmol/L sucrose, and 0.75% agar. The MMS has a one-quarterstrength of macro-elements, one-half strength of micro-elements, full strength of organic elements, and the Fe salt ofthe MS medium (Murashige and Skoog 1962).

Stem segments approximately 1 cm in length were cut fromyoung germinated seedlings and placed on MMS containing2.22 µmol/L 6-BAP and 9.05 µmol/L 2,4-dichlorophenoxy (2,4-D) in a Petri dish. After 6 weeks inoculation, the callus wasinduced from each stem segment. Adventitious roots, whichwere induced from germinated seedlings without radicals grow-ing on MMS medium containing 2.69 µmol/L NAA, were also cutinto 1 cm segments. The 30 explants obtained from each callusand adventitious root were used for the in vitro induction of

Mycorrhiza-like Structures in Cultured Tissue of Cathaya Tree 1167

ECM-like structures with 10 explants used as controls.

Aseptic synthesis of ectomycorrhizas

One mycelia plug, 6 mm in diameter and cut from the margin ofthe fungal colony, was placed in the center of the Petri dishwith MMS, to which 0.50 µmol/L glucose had been added in-stead of sucrose and solidified with 0.75% agar. After 4 weeksinoculation, two segments of the callus or four segments ofadventitious root were placed adjacent to the margin of thefungal colony on MMS medium. Callus and adventitious rootsegments were placed on the same MMS medium without place-ment of a mycelia plug for control.

All incubations were performed under fluorescent lights at90 µmol.m−2.s−1 with a 16 h photoperiod at (23 ± 2) °C.

Preparation for ECM structure

Both 10 inoculated/control roots and 10 inoculated/control calliwere removed for microscopic observation from culture platesafter inoculation for 4 and 8 weeks.

Clearing, bleaching, and staining of whole infectedcallus and infected rootA set of five calli (approximately 2 mm3) and five roots (5–10mm in length) of Cathaya argyrophylla, which were infectedwith Cenococcum geophilum, were immersed in 70% ethanolfor 2 h, cleared in 10% KOH at 90 °C for 90 min, bleached in10% H2O2 in 10% KOH for 10 min, acidified with 0.1 mol/L HClfor 10 min, and then stained for 90 min at 90 °C with Chlorazolblack E (Gill et al. 1999). After being destained overnight inglycerol, samples were mounted in glycerol under a cover slipand were examined with a light microscope (BX51; Olympus,Tokyo, Japan) fitted with standard bright field optics.

Semi-thin sections of infected calli and rootsAnother set of five segments each of root (1–2 mm in length)and callus tissue (approximately 2 mm3) was immersed over-night in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH6.8, at 4 °C. Segments were then rinsed three times in 0.1 mol/Lphosphate buffer, pH 6.8, post-fixed in 2% OsO4 (SPI-CHEMTM;SPI Supplies, West Chester, PA, USA) in the same buffer for 90min, dehydrated for 20 min at each concentration of an as-cending acetone series (from 20% to 100%) at 20% increments,and washed three times with 100% propylene oxide for 15 mineach time. Tissues were finally embedded in Epon 812 resin(Serva, Heidelberg, Germany). Semi-thin sections were cut witha diamond knife on an ultramicrotome (Ultracut-E, AO, USA)and gently heat-fixed to glass microscope slides. Sections werethen stained with 0.1% Toluidine blue in 1% acetate buffer for10 min, kept in tap water for 15 min, dried in air, mounted in

100% glycerin under a cover slip, and examined with a lightmicroscope (BX51; Olympus).

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(Managing editor: Ya-Qin Han)