Volume 17, No. 2, 1993

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(pp. 1-8)
In Vitro Cultur of Embryonic Axes From Arachis Species for Germplasm Recovery
K.B. Dunbar, R.N. Pittman,* and J.B. Morris2
Germination of seeds from Arachis species is low after 20 yr in storage. This study was  conducted to develop procedures to recover germplasm from deteriorated seeds.  Embryonic axes from deteriorated seed of Arachis species were cultured on a medium containing MS salts, Gamborg's 85 vitamins, 30 g/L sucrose, and solidified with 8 g/L agar. 

Six to 8-week-old plants regenerated from embryonic axes were transplanted to Jiffy pots in the greenhouse. Nineteen samples of deteriorated seed between 20 and 31 years old were evaluated. Shoots were recovered from 31% of the seeds by in vitro rescue of embryonic axes and from 2.4% by germination in the greenhouse. In the same experiment, plants were recovered from 15 to 31-yr-old deteriorated seed of A. burkartii, A. glabrata, A. hagenbeckii, A. monticola, A. pusilla, A. rigonii, A. villosa, and A. villosulicarpa by in vitro rescue of embryonic axes, while no plants were recovered from seed of the same 15 seed lots germinated in the greenhouse. The in vitro rescue of embryonic axes can significantly  increase the recovery of germplasm from deteriorated seed of Arachis species.
Additional index words: Germination, peanuts, seed longevity, seed viability.
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(pp. 9-21)
Storage Duration and Freeze-Thaw Effects on Germination and Emergence of cicer milkvetch (Astragalus cicer) Seeds1
S.N. Acharya, E.G. Kokko, and J. Fraser2
'Oxley' cicer milkvetch (Astragalus cicer L.) seed lots harvested from a Lethbridge, Alberta nursery and stored in an uncontrolled room were used to study the effect of storage duration on hard seed content, germination, and indoor and field emergence. Seeds harvested in 1977 and 1981 to 1991 were also used to study the effect of repeated freeze-thaw treatments on the above traits. Hard seed content fell significantly with increasing age of seed lot and was accompanied by an increase in seed germination and emergence.

Cicer milkvetch seeds maintained above 90% viability for 10 yr under uncontrolled storage conditions. Germination on day 7 was significantly correlated with the germination on subsequent days and emergence. Thus, a standard 7-d germination test is sufficient to separate seed lots with differential ability to establish a stand. Heavier seeds did not have a higher inherent germinability than the lighter seeds, but were easier to scarify. Freeze-thaw treatment reduced the hard seed content from 46 to 9% and significantly increased germination and emergence. Seed lots with more hard seeds needed more freeze-thaw cycles than seed lots with fewer hard seeds. Freeze-thaw treatments may be an improvement over mechanical scarification because there is no loss due to breakage, small seed lots can be used, all the seeds are exposed to the same level of treatment and there is less risk of damage to the embryo.The presence and location of the strophiole was verified using a new, rapid and non-destructive technique which can be used to predict hard seededness.
Additional index words: Hard seed, Scarification, Seedling vigor, Strophiole.
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Proceedings of the


held at the

83rd Annual Meeting of the Association of Official Seed Analysts
70th Annual Meeting of the Society of Commercial Seed Technologists

Fort Collins, Colorado June 13,1993
Loren E. Wiesner Coordinator
Loren E. Wiesner and Eric E. Roos Presiding

(pp. 24-40)
Early History of NSSL and Contributions of Edwin James and Louis N. Bass
Eric E. Roos1
The National Seed Storage Laboratory (NSSL) opened for business in September of 1958 with the long-term mission of preserving the seed germplasm of those crops important to U.S. agriculture. The NSSL concept was a direct result of World War II, when it became apparent that germplasm may no longer be available, particularly from many foreign countries.

Dr. Edwin James was named the first Head of the Laboratory and served until his retirement in 1970. His primary accomplishments included the administration of the new facility, generating publicity about the Laboratory, and initiating the acquisition and storage of seed samples. In addition, he conducted research on such topics as seed longevity and seed deterioration, including biochemical aspects of red cotyledon in lettuce and viability assessment using such tests as the glutamic acid decarboxlyase test. Dr. Louis N. Bass joined NSSL at its inception and was named Head in 1970, a post he held until his untimely death in 1986. Louis oversaw many dramatic changes in the Laboratory, such as the shift from storing seeds at 5°C to storage at -18"C, primarily as a result of his research on seed storage under various conditions of temperature, relative humidity and in different gases. Louis brought the Laboratory national and international recognition through his participation in the Association of Official Seed Analysts (AOSA) (President, Merit Award, editorial duties, numerous committees), the International Seed Testing Association (ISTA) (Associate Editor of Seed Science and Technology), the American Society of Agronomy (C-4 Chair and Board Representative, Fellow) and the International Board for Plant Genetic Resources. A bibliography of James' and Bass' primary publications while at NSSL is included.
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(pp. 41-53)
Predicting the Optimum Storage Conditions for Seeds Using Thermodynamic Principles
Christina W. Vertucci1
Determination of the optimum moisture content for seed storage is difficult because deterioration is very slow at conditions close to the optimum. We have used thermodynamic principles to relate water phase behavior with different mechanisms of seed deterioration. We conclude that the optimum moisture level for storage represents a compromise between slowing aging reactions and preventing lethal ice formation by drying, and retaining the structural integrity of cellular constituents by supplying sufficient structural water.

For desiccation sensitive tissues, there may not be a moisture content and temperature combination at which aging reactions are sufficiently slowed, and lethal freezing injury and desiccation damage are prevented. In this case, storage protocols require vitrification, a process by which the thermodynamically stable state is avoided by cooling very rapidly. The thermodynamic principles invoked in this paper may indicate the optimum conditions for seed storage, but they are not sufficient to describe the rate of aging under these or other conditions.
Additional index words: Seed storage, seed aging, seed longevity,  bound water, calorimetry, glass, vitrification, recalcitrant, freezing injury, desiccation damage, water content, ultradry
Abbreviations: dw, dry weight; NMR, nuclear magnetic resonance, IR, infrared; ESR, electron spin resonance; TSDC, thermally stimulated direct current; DSC, differential scanning calorimetry; DTA, differential thermal analysis; DMA, dynamic mechanical analysis
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(pp. 54-64)
Imaging Techniques to Enhance the Preservation and Utilization of Seed Germplasm
M. Scott Howarth and Phillip C. Stanwood1
Several image processing techniques are being developed at the National Seed Storage Laboratory to enhance the preservation and utilization of seed germplasm. Two projects investigate the potential of this technology in controlling and providing additional information in germination testing, seedling growth rate analysis and tetrazolium testing.

Another thrust of the research has been to develop an image-based database. Investigations in this study concentrate on the development of features which describe physical characteristics of seed. An object-oriented database was developed within the  Windows 3.1 operating environment. This database is unique because it is capable of displaying many types of information, for example seed images, graphs, or color pads. Machine vision and image analysis have shown promise and many new applications of this technology are being developed.
Additional index words: machine vision, seed vigor, tetrazolium testing seed characteristics, slant board.
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(pp. 65-78)
Plant Germplasm Viability: Biochemical Insights and Noninvasive Assessments1
Sharon Sowa2
Biochemical research has been conducted at the National Seed Storage Laboratory to  examine parameters important to plant germplasm viability. Emphasis was placed on the key respiratory enzyme cytochrome c oxidase. Studies using effector molecules to probe Phaseolus respiration on a variety of physiological levels (whole seed to isolated enzyme) showed a direct correlation between rate of respiration and vigor, and implicated oxidase in loss of vigor/viability.

Recalcitrant seed storage in anesthetic atmospheres was shown to increase longevity. Infrared (FTIR) spectroscopy, with a variety of sampling techniques to accommodate (intact) biological samples, is a powerful analytical tool to examine biochemical structure/function relationships to plant germplasm viability. FTIR experiments conducted on suspension cultured cells showed measurement of CO2 production to be a noninvasive viability indicator. In pollen, structural changes in membrane lipids were correlated with imbibitional chilling injury, and distinct changes in structure and function were observed in vivo during germination. FTIR-photoacoustic spectroscopy, which can detect CO2 production during minimal hydration of intact seed, holds promise as a new noninvasive viability assessment method.
Additional index words: Respiration, cytochrome c oxidase, vigor, storage, infrared spectroscopy, FTIR, seeds, pollen, suspension cultured cells
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(pp. 79-90)
Long-Term Preservation of Clonal Germplasm: Advances and Concerns
L.E. Towill1
Numerous crops are maintained as clones in field, greenhouse or in vitro plant culture. Long-term germplasm conservation for these species is needed and may be accomplished by cryogenic preservation of seeds, pollen, and shoot tips or buds. Seed and pollen can be used to preserve genetic diversity of the crop. These two propagules from many clonal species are desiccation tolerant and survive low temperature exposure. Thus, methods already exist to apply longterm storage to certain crops. Cryopreservation of shoot tips or buds is needed for long-term conservation of the clone.

Substantial progress has occurred in defining two-step coaling and vitrification protocols, such that application to certain plant collections is now possible and feasible. For example, long-term storage of apple has been initiated at the National Seed Storage Laboratory using dormant vegetative buds collected En winter from the National Clonal Germplasm Repository at Geneva, NY. Survival after cryogenic exposure was found An all lines tested to date, with cold-hardy lines exhibiting a greater percentage of viability. Cold-tender lines will require a more complex protocol. Vitrification (application of cryoprotectants followed by rapid cooling such that a glass forms within the cells) is an easier method to apply than two-step cooling. Survival was found with apices from many species. A practical project using vitrification to preserve mint is underway. Survival for some species, using either vitrification or two-step cooling methods, still appears genotype-specific. Will a single method be applicable to all genotypes within a collection? A strategy to deal with diversity is needed to minimize the effort required to achieve practical cryopreservation.
Additional index words: germplasm preservation, cryopreservation, pollen, shoot tip, vitrification, clones.
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Proceedings of the


held at the

83rd Annual Meeting of the Association of Official Seed Analysts
70th Annual Meeting of the Society of Commercial Seed Technologists

Fort Collins, Colorado June 13, 1993
Jan M. Ferguson Coordinator
Richard L. Sayers Presiding


(pp. 92-100)
The History of Seed Vigor Testing1
Miller B. McDonald2
Seed vigor testing has emerged as a routine method to test seeds for field performance capability. This development can be traced to deficiencies in the philosophy of the purpose of a standard germination test. Early terms included "driving force" and "germination energy" of seedlings to capture the concept ot seed vigor. Yet, no organized approach to defining seed vigor or developing seed vigor tests was given until the International Seed Testing Association (ISTA) formed the first Biochemical and Seedling Vigor Committee in 1950. The Association of Official Seed Analysts (AOSA) established a Vigor Test Committee in 1961. Both committees

provided the central leadership in vigor testing that culminated in the production of respective Handbooks. This review of the history of seed vigor testing focuses primarily on those events which occurred within AOSA since the inception of that first Committee in 1961 and documents the impetus and momentum that led to vigor testing as an accepted measure of seed quality.
Additional index words: seed germination, seed quality, seed deterioration,
seed technology, field emergence.
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(pp. 101-104)
AOSA Perspective of Seed Vigor Testing
J.M. Ferguson1
Seed vigor tests have become a routine quality assessment tool for much of the seed industry. The Association of Official Seed Analysts (AOSA) has been actively involved in developing procedures for many of the vigor tests used today. An earlier presentation discussed the history of vigor testing and the numerous contributions of the AOSA Seed Vigor Subcommittee. Other presentations will discuss procedures for certain tests and will relate the views of industry and international trade on vigor testing. It is my challenge to examine the AOSA perspective of vigor testing and to discuss not only the current trends in using these tests, but also to take a close look at why vigor tests are important, how they are used, and the future of vigor testing.
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(pp. 105-109)
The ISTA Perspective of Seed Vigor Testing
J.G. Hampton1
The 1950 International Seed Testing Association (ISTA) Congress in Washington D.C. saw vigorous debate and discussion about discrepancies in germination test results between European and American laboratories (the so-called "commercial" versus "agricultural" concepts of seed testing). It was also the meeting at which ISTA became officially involved
with seed vigor.

The Biochemical and Seedling Vigor Committee (later to become the Vigor Test Committee) was established to "define seedling vigor and standardize methods for its determination." The difficulty of this task is illustrated by the following:

It took twenty-seven years for the Vigor Test Committee to agree on a definition of seed vigor, i.e. "...the sum total of those properties of the seed which determine the level of activity and performance of the seed or seed lot during germination and seedling emergence." This broadly based definition was adopted by the 1977 ISTA Congress.

The ISTA Rules for Seed Testing do not yet include vigor testing methods, although suggested procedures have been published in the ISTA Handbook of Vigor Test Methods, the first edition of which appeared in 1981 and the second in 1987.

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(pp. 110-120)
Accelerated Aging Test
Dennis M. TeKrony1
The accelerated aging (AA) test utilizes the environmental factors commonly associated with seed deterioration, namely storage temperature and relative humidity. A single layer of seed is placed on a screen tray which is inserted into an inner chamber (plastic box) containing a small volume (40 ml) of water (Figure 1). The inner chamber is then placed into an accelerated aging (outer) chamber and aged at high temperatures (41 to 45°C) for a specific period of time (i.e., 72 h). During the aging period the seeds take up water from the humid environment within the inner chamber and are stressed at high temperatures and seed moisture. High vigor seed deteriorate slower than low vigor seed and seed lots can be separated into various vigor levels.
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(pp. 121-126)
Commercial Vigor Testing
Alan Galbreth1
This morning I'd like to direct my comments about commercial vigor testing to the realities of running a commercial vigor testing lab. Other speakers have addressed very well the specifics of performing particular vigor tests, but I would like to concentrate on the day-to-day operations of running a commercial lab and the task of interpreting the results we provide for seedsmen. I'll be discussing corn and soybeans

but this could fit any crop. I hope my comments are as applicable to company labs as they are to service labs.

lndiana Crop lmprovement Association (ICIA) began corn cold testing in 1957 on Foundation seed lots. We ran cold tests on soybeans for the first time in 1966. In the early 1970's we further expanded our vigor testing program. The program had three core tests: cold tests, accelerated aging, tetrazolium. Our program today is still centered on these three tests though some of the procedures have been modified over time. This past season we ran 15,000 cold tests, 2000 accelerated aging tests, and 1000 tetrazolium tests.

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(pp. 127-133)
Industry Perspective of Vigor Testing
Dennis A. Berkey1
A great deal of research has gone into investigating seed vigor and vigor test methods. The Association of Official Seed Analysts (AOSA), through the Seed Vigor Test Committee, published an excellent guideline, "Seed Vigor Testing Handbook (1992). This handbook has proved to be an excellent guide to understanding vigor. The handbook also is an excellent starting point for setting up vigor test methods and can be said to be a way to standardize testing methods.

Those individuals that have made major contributions to vigor testing are to be commended.

While initial publication of the Seed Vigor Testing Handbook in 1983 was being hailed as a success in the public sector, it was viewed as another potential set of regulations and labeling requirements to the seed industry. In response to publication of the Handbook, the American Seed Trade Association (ASTA) published a position paper recommending that vigor test results be used for in-house information only and not for any other purposes such as labeling or advertisement.

Seed companies use vigor tests, but the methods are not necessarily the same as described in the Handbook. This makes it difficult to compare results between public and private laboratories or even between private laboratories. There appears to be a desire on the part of some individuals to force the standardization of vigor testing methods and labeling of vigor results. This movement has led to some challenging situations. The challenge is more of a dilemma for the seed industry.

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