Botanical Studies (2006) 47: 163-166.

*

Corresponding author: E-mail: CAChang@wufeng.tari.

gov.tw

Virus-tested Lycoris aurea plants from apical meristems

of adventitiously regenerated shoots

Li-Chun HUANG

1

, Wha-Shin HSU

2

, You-Ming CHANG

2

, Pan-Chi LIOU

2

, Bau-Lian HUANG

1

,

Chin-Chu CHEN

2

, and Chin-An CHANG

2,

*

1

Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, Taiwan

2

Division of Plant Pathology, Agricultural Research Institute, Taiwan. 189, Chung-Cheng Rd., Wufeng, Taichung 413,

Taiwan

(Received September 8, 2004; Accepted December 2, 2005)

ABSTRACT.

Adventitious shoots were first regenerated in vitro from twin-scale sections of virus-infected

Lycoris aurea Herb. cv. ¡¥Golden spider lily¡¦. Their apical meristems were then excised and cultured to

redevelop shoots. Explants were not larger than 0.1 mm and were limited to the dome and subjacent stem,

without leaf primordia. The redeveloped shoots were multiplied, rooted, hardened, and acclimatized, before

transfer to an insect-free greenhouse. ELISA assays were performed for three lycoris potyviruses (lycoris

potyvirus, LPV; lycoris mild mottle virus, LyMMV; and lycoris virus Taiwan, LVT) and for cucumber mosaic

virus (CMV). Assays after one year and re-assays in the subsequent three years revealed no LyMMV, LPV,

or CMV in the meristem-derived plants or in their clonal progeny. The virus-tested plants are now being

increased as foundation stock.

Keywords: Cucumber mosaic virus; Lycoris mild mottle virus; Lycoris potyvirus; Lycoris virus Taiwan;

Lycoris; Meristem culture; Virus tested plants.

INTRODUCTION

With clonally propagated crops, which include

the bulbs, viruses are readily spread through plant

propagation. The spread can be minimized by employing

virus-free propagules. Commercial plantings of the

indigenous Lycoris aurea Herb. cv. ¡¥Golden Spider¡¦ in

Taiwan are infected by three lycoris potyviruses¡Xlycoris

potyvirus (LPV), lycoris mild mottle virus (LyMMV), and

lycoris virus Taiwan (LVT)¡Xand by cucumber mosaic

virus (CMV) (Chang et al., 2002). LyMMV induces

mild symptoms. No symptoms have been observed in

the rare instances of infection by LPV alone. This virus

is usually found in mixed infections with LyMMV. The

combination causes severe necrosis that can devastate

plantings severely. CMV infection has also shown no

characteristic symptoms. Currently, the four viruses

do not cause a serious problem, but they are a potential

threat to the budding Lycoris flower industry. Therefore,

this investigation has pro-active intents: (1) to disclose a

reliable method for recovering virus-tested plants from

infected material and (2) to establish a foundation of

uninfected stocks that can serve as a source of propagules

to avoid the risk of an epidemic because the three lycoris

potyviruses are easily spread through propagating by bulbs

and are readily transmitted mechanically and by vector.

MATERIALS AND METHODS

Regenerating adventitious shoots from bulb

scale segments

Bulbs of four commercially grown plants that tested

positively for LyMMV were potted in a 1:1:1 mixture of

peat:perlite:vermiculite and cultivated in an insect-free

greenhouse. These served as our infected controls and

sources of adventitious shoots for meristem culture. Twin-

scale segments were excised from the bulbs and cultured

by the method described previously to obtain adventitious

shoots (Huang and Liu, 1989).

Culturing meristems of regenerated shoots

When regenerated adventitious shoots were 5 cm in

length, their apical meristems were excised. A dissecting

microscope was employed to ensure that final explants

were not longer than 0.1 mm and comprised only the dome

and a portion of its subjacent stem without leaf primordia

PHYSIOLOGY

164

Botanical Studies, Vol. 47, 2006

(Figure 1). They were placed in a medium supplemented

with 0.44 £gM of BA (N

6

-benzyladenine) and 0.54 £gM

NAA (£\-naphthaleneacetic acid) to stimulate shoot

emergence. As shoots developed up to 1 cm, they were

removed and multiplied in another medium containing

44 £gM BA and 16 £gM NAA. When multiplied shoots

were 5 cm tall, they were rooted, either individually or in

clusters, in a third medium supplemented solely with 16

£gM NAA. Bulbs also differentiated in this medium. Basal

constituents of the three media were the same as those of

the twin-scale culture medium. Rooted plants were grown

in pots containing a 1:1:1 peat:perlite:vermiculite mixture;

hardened and acclimatized in a growth chamber, the

relative humidity of which was lowered in 5% increments

at weekly intervals from 95 to 80%; then transferred to an

insect-free greenhouse.

Virus assays

Initial virus assays were performed a year after

meristem-derived plants were transferred to the

greenhouse. They were re-assayed another year later

and again after the third year. The re-assays included

their clonal progeny. LPV, LyMMV, LVT and CMV

contents were determined by ELISA, following the

procedure for the detection tuberose potyviruses (Chen

and Chang, 1998) using the specific rabbit antisera for the

corresponding viral coat proteins. Samples of ca. 0.2 g

leaf tips were ground in 0.05 M phosphate buffer (pH 7.5),

and a duplicate of 200-£gl aliquots of juice supernatants

were coated onto ELISA plates by incubating at 37oC

overnight. Plates were washed, and individual rabbit

antisera were applied, followed by alkaline phosphatase-

conjugated goat antibody. Nitrophenylphosphate substrate

was added and OD

405

measured. Positive virus controls

were conducted with 1 mg ml

-1

each of LPV and LyMMV

coat proteins (Chang et al., 2002). LVT and CMV were

determined by infected tissues.

RESULTS

Previous observations with the presence the symptom

in Table 1 are the results of ELISA assays performed the

plants in vitro as well as on plants transplanted to the

greenhouse and grown in the field. The sample of a healthy

clone, previously tested as being free of the four viruses,

is indicated by H. Only values above the double value

of H (2 H) were considered evidence of virus presence.

LyMMV, LPV, LVT, and CMV denote positive controls

from the coat proteins of diseased plants. The leaf samples

of 17 original bulbs were analyzed for four viruses. As in

Table 1 the rate of infected plants was detected by either

single infection or mixed infection with a reading above

2 H on OD

405

.

Readings of 96 plants from their meristem

cultures and established in vitro were monitored first in

2001 and then again in 2004. They proved to be free of

the four viruses, Subsequently, each time with random

selection from 320 greenhouse plants in 2002 and 45 field

grown plants each time in 2002 and 2005 were conducted

thrice as seen in Table 1 indicated no virus presence. Virus

exclusion was thus confirmed when re-assays with ELISA

in were performed in the subsequent five years, which

included examinations of clonal progeny.

DISCUSSION AND CONCLUSIONS

Lycoris aurea cv. ¡¥Golden spider lily¡¦ is a very precious

indigenous plant in Taiwan with a high economic value.

The recent identification of viruses in commercial

plantings of Lycoris aurea serves as a wake up call to

the still emerging and promising component of Taiwan¡¦s

flower industry. A combined infection by the lycoris

potyviruses LyMMV and LPV can devastate diseased

fields (Chang et al., 2002). Recently, another strain LVT

was discovered specifically in Taiwan field plants (Dr.

Chin-An Chang, personal communication). Epidemics are

possible since these viruses are spread through propagules,

by mechanical means, and insect vectors. Crop losses

can be minimized by employing virus-certified plants

as sources of propagules, as has been done successfully

for citrus, strawberry, potato, carnation, and a number of

other clonally propagated crops (Murashige, 1974). Their

virus-certified plants were obtained by culturing shoot

apical meristems or by in vitro grafting of shoot apices

excised directly from infected plants (Murashige, 1974).

Applicability of the meristem culture method, albeit

modified, has now been demonstrated for Lycoris viruses.

To maximize exclusion of infected cells, the isolated

tissue was not larger than 0.1 mm and comprised only

the meristem dome and its immediately subjacent stem

without leaf primordia. Also, the meristems were isolated

from adventitious shoots that developed in tissue cultures

and not directly from greenhouse or field grown plants.

Use of these adventitious shoots made disinfestation

unnecessary. It also provided a large supply of meristems,

whereas only one explant was obtainable per field-grown

shoot. More importantly, the in vitro adventitious shoots

Figure 1. Excised shoot apical meristem of L. aurea cv. ¡¥Golden

spider¡¦ Feulgen stained and viewed with dissecting microscope.

Note absence of leaf primordia. The 0.1 mm tall tissue is shown

at 500-fold magnification.

HUANG et al. ¡X Virus-tested

Lycoris

165

were perhaps less likely to harbor viruses than field grown

shoots, thus, increasing the probability of virus free

meristems.

Smith and Murashige (1970) demonstrated that isolated

shoot apical meristems, without leaf primordia, were

capable of developing into whole plants when provided

suitable nutrient media. Hence as expected, a small but

sufficient number of our tiny isolates re-developed into

shoots very slowly and eventually grew into plant clones

of all four bulbs. All clones tested free of the viruses that

are currently detected in commercial plantings (Chang,

Chen and Hsu, in press). They are being increased as

foundation stock in insect-free greenhouses (Figure 2).

Lycoris plants that test free of LyMMV, LPV, LVT,

and CMV exist among commercial plantings and can

also be employed for establishing foundation stocks, but

they may contain other unidentified viruses. By confining

plant initials to small numbers, as was done in our

meristem culture method, removal of all infecting viruses

is virtually assured. In all instances the virus elimination

must be verified by specific tests, as were performed in

this investigation. We are also continuing to monitor our

foundation stock for any evidence of somaclonal variants

that sometimes occur among plants recovered through

tissue culture.

Acknowledgements. The authors acknowledge research

grants from the Council of Agriculture of Taiwan and the

Academia Sinica that enabled this investigation. We thank

Professor Toshio Murashige for critically monitoring our

work and reviewing the manuscript.

LITERATURE CITED

C ha ng , C .A. , C .C . C he n , a nd H. T. Hs u . 200 2. P a rt ia l

characterization of two potyviruses associated with golden

spider lily severe mosaic disease. Acta Hort. 568: 127-134.

Che n, C.C. and C.A. Chang. 1998. Cha ra cte riza tion of a

Figure 2. Foundation stock of virus-tested L. aurea cv. ¡¥Golden

spider¡¦ plants initiated from apical meristems of adventitious

shoots that regenerated in bulb scale segments in vitro.

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Botanical Studies, Vol. 47, 2006

potyvirus causing mild mosaic on tuberose. Plant Dis. 82:

45-49.

Huang, L.C. a nd D.M. Liu. 1989. Clonal multiplication of

Lycoris aurea by tissue culture. Scientia Hort. 40: 145-152.

Murashige, T. 1974. Plant Cell and Organ Culture Methods in

the Establishment of Pathogen-Free Stock. A.W. Dimock

Lectures, Cornell University. 2, pp. 1-26.

Smith, R.H. and T. Murashige. 1970. In vitro development of

the isolated shoot apical meristems of angiosperms. Amer. J.

Bot. 57: 562-568.