Production Program for Virus-Free Alstroemeria Progress Report –March 1991
Date March 1, 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 Prof. 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 – Title Prof. of Plant Breeding
Production of Virus-Free Alstroemeria
Robert Langhans, R. Kenneth Horst, Mark Bridgen, and Richard Craig
Cornell University
- A. Project Objectives:
- 1) to identify the virus or viruses present in Alstroemeria ‘Endowment Series’;
- 2) to determine how the virus(es) are transmitted;
- 3) to determine a method to eliminate the viruses;
- 4) to assay Alstroemeria produced from the ‘Endowment Series’ to guarantee they are virus-free; and
- 5) to produce pathogen-free stock plants in vitro and in vivo and grow in a manner to preventreinfection.
- B. Summary of Work Conducted:
- Alstroemeria cvs. developed from the 1985-88 breeding project whichproduced the ‘Endowment Series’ exhibited virus-like symptoms when ready for public introduction in 1989.
Our efforts shifted to virus detection, identification and eradication techniques, for which a limited amount
of funding was received in 1990 and a part-time effort has been devoted toward this goal. Besides
maintenance of a large population of stock plants in the greenhouse, initial work included ELISA tests with
antisera. from AGDIA to the most common Alstroemeria virus, Alstroemeria mosaic virus. Electron
microscope scans of tissue samples from seedlings and mature plants for virus presence and particle size
were performed several times. Five species of bioassay indicator plants were inoculated, first with
phosphate buffer, then with the addition of mercapto ethanol at various dilution rates. Inoculations were
with tissue directly from Alstroemeria plants and from previously inoculated bioassay plants. Development
of heat therapy techniques have been continued.
- A major effort has been directed toward developing a tissue culture technique for meristem shoottips aimed at virus eradication, rather than rhizome buds. Rooting of shoots produced in vitro have been
attempted in greenhouse conditions using mist, fog and plastic-covered boxes. Cuttings and meristems
directly from the greenhouse without heat therapy were used for comparison. Rooting techniques produced in vitro
included the absence of hormones as well as factorial experiments of from 1 to 10 mg/l of the auxins NAA
and IBA in the medium. In addition, a 2 to 5 second quick dip in the hormones, an aquatic herbicide to
enhance rooting, both removal and nonremoval of callus, half and full strength sugar levels, several
Murashige & Skoog salt concentrations, activated charcoal and gelrite vs. agar were all trialed.
- C. Results to Date:
- After developing successful breeding techniques, new seedlings exhibitedvirus-like symptoms suggesting seed transmission of the disease. ELISA testing ruled out Alstroemeria
mosaic virus and pointed toward the Potex virus group with 470-580 nm sized particles, previously
unknown in Alstroemeria. Original bioassays with young plant material showed no reactions. However,
the use of order plants with a higher virus titer showed reactions on Datura and Chenopodium sp., but not
on 2 species of Tobacco and Gomphrena. A new buffer system containing mercapto ethanol and
phosphate at a pH of 7.6 was used. Reactions were obtained directly from both Alstoemeria tissue and
previously inoculated Datura and Chenopodium tissue. Heat therapy (95′F for 5 wks.) tissue has not yet
been tested for virus presence.
- Meristem shoot development in tissue culture has been successful. However, rooting under mistor fog has not worked and limited success has been obtained using plastic boxes in growth chambers or
the greenhouse. Rooting experiments in vitro have ranged from no hormones to a factorial trial of from 1-
10 mg/l of the auxins NAA and IBA. Limited success in vitro has been obtained without hormones, with
low levels of auxins and quick dips at low levels. High levels proved detrimental. Gelrite appears to be a
more effective medium than agar. All experiments require from 1 to several months before results are
obtained. Trends are becoming apparent with refinement of successful techniques and elimination of
others.
- D. Future Plans Covered by the Endowment Grant:
- The goals include: determine if a virus is present, identify and characterize it:develop a virus indexing system; and develop a production system for growing and maintaining pathogen-
free stock plants. Further EM work will be performed and a plant bioassay system will be refined,
considering proper conditions for inoculation and age of tissue sampling. Tissue culture material will be
screened for virus presence. Successful tissue culture procedures will be pursued and refined, including
cytokinin and auxin concentrations for shoot and root production. In vivo rooting techniques will include
bottom heat and capillary mats. Other directions of research will include photoperiodic control in culture
for rhizome development and etiolation. Embryo rescue for virus freedom will also be considered.
- E. Anticipated Benefits for Floral Industry:
- Plant vigor, yield and quality of Alstroemeria flowerswould be improved if the virus can be eliminated. There would be no virus carry-over through vegetative
propagation of rhizomes from generation to generation. Development of the crop from seed would
become feasible (especially potential for pot plants). Prevalence of this virus in the industry is speculative
at present, but indications are that it is a major problem. Development of a virus-indexing and therapy
program would produce large numbers of pathogen-free stock plants. The improved quality would be a
competitive edge for U.S. growers using the virus-free material.
