Date August 21, 1991

Title of Project Production of Virus-Free Alstroemeria

Institution where work is being conducted Cornell University

Amount of Endowment Grant $ 5, 000
Covering Period 1/90 to 12/90

Anticipated Date of Project Completion/Final Report With submission of 1991 proposal; 12/91

Individual(s) Conducting Project:

(List Project Leader First)

Dr. Robert Langhans, Cornell - Title Professor of Floriculture

Telephone Number (607) 255-5113

Dr. R. Kenneth Horst, Cornell - Title Prof. of Plant Pathology

Dr. Mark Bridgen, University of Connecticut - Title Prof. of Horticulture

Dr. Richard Craig, Penn State - Prof. of Plant Breeding

Production of Virus-Free Alstroemeria

Robert Langhans, R. Kenneth Horst, Mark Bridgen, and Richard Craig

Cornell University

Progress Report to the American Floral Endowment, 8/21/91

A. Project Objectives:

1) to identify the virus or viruses present in Alstroemeria ‘Endowment Series’;

2) to determine a method to eliminate the viruses;

3) to assay Alstroemeria produced from the ‘Endowment Series’ to guarantee they are virus-

free; and

4) to produce pathogen-free stock plants in vitro and

in vivo and grow in a manner to prevent reinfection.

B. Summary of Work Conducted:

Alstroemeria cvs. developed from the

1985-88 breeding project which produced the ‘Endowment Series’

exhibited virus-like symptoms when ready for public introduction

in 1989. Our efforts shifted to virus detection, identification

and eradication techniques. Elisa testing to the 3 major virus

groups POTY, POTEX and carlavirus was performed. Antisera to 2

common Alstroemeria viruses (a carlavirus and Alstroemeria mosaic

virus) were obtained from A.A. Brunt in the United Kingdom and

tested using ELISA and EM scanning for reaction to our virus

particles. virus purification procedures to eliminate impurities

possibly interfering with virus transfer into indicator plants

included differential centrifugation, cesium chloride and sucrose

density fractionations.

Alstroemeria tissue was scanned under EM for particle shape

and size. Chrysanthemum B virus and colloidal gold were tested

for particle adherence. Virus inclusion bodies were looked for

using thin sectioning under the EM and orange and green staining

with the light microscope. A double stranded RNA gel test was

run with diseased and healthy tissue to look for virus-associated

bands to further substantiate virus presence. The buffer

formulation for plant bioassay inoculation was modified and

tested on both POTY and POTEX virus indicator plants, including

Datura, Chenopodium, Gomphrena and several Nicotiana species.

Heat therapy for virus elimination has been continued, along with

tissue culture procedures using very low (.1 mg/ml) phytohormone

levels and cytokinin/auxin combinations with activated charcoal.

Short day photoperiod for rhizome development has been trailed.

Embryo and ovule rescue have also been attempted.

C. Results to Date:

No reactions occurred using ELISA or EM

to the antisera for the 3 major virus groups or to Chrysanthemum

B virus or to Brunt’s Alstroemeria antisera, indicating the

presence of a unique virus. EM scanning showed flexuous rod

particles of 780 nm in length, categorizing the virus as a member

of the potyvirus group. Thin sectioning under the EM revealed

laminated aggregate inclusion bodies characteristic of potyvirus.

Staining techniques substantiated this finding under the light

microscope. The double stranded RNA gel test produced a virus-

associated RNA band in the diseased tissue only. Virus

purification techniques revealed a high ratio of protein present

when scanned with a spectrophotometer, indicating a high level of

impurity still remaining.

Inoculation of indicator plants with the modified buffer

increased concentration of virus particles under EM scanning as

compared to Alstroemeria tissue alone. In vitro rooting

attempts have improved using cytokinin/auxin combinations and

auxin alone directly on meristem shoot tips, along with an 8 hr.

photoperiod which appears to enhance rhizome and subsequent

rooting formation. Initial attempts at embryo and ovule rescue

have not produced new plants to date.

D. Future Plans Covered by the Endowment Grant:

The new Alstroemeria virus has now been

determined to be a member of the potyvirus group and purification

procedures developed for this virus group will be pursued in

depth, focusing on modifications in differential centrifugation,

cesium chloride and sucrose density fractionations. N.

clevelandii may function as a source for virus purification due

to deleterious compounds in Alstroemeria which are disruptive to

the virus. After purification, the virus will be injected into

rabbits for antibody production and the products screened for

effectiveness in an ELISA system. Further refinement of in vitro

techniques will produce plants that can be quickly screened by

ELISA to determine if heat therapy has produced virus-free

plants. Existing plants can also be tested for virus presence.

E. Anticipated Benefits for Floral Industry:

Plant vigor,

yield and quality of Alstroemeria flowers would be improved if

the virus can be eliminated. Prevalence of this virus in the

industry is speculative at present, but indications are that it

is a major problem. Once this technique is developed, it will be

possible to quickly and reliably screen Alstroemeria plants to

test for and produce pathogen-free stock plants. The improved

quality would be a competitive edge for U.S. growers using the

virus-free material.

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