Margaret S. Gibson, Ph.D.
American Crystal Research Center
Many definitions have been proposed for the meaning of seed and seedling vigor. The working definition decided upon by the Association of Official Seed Analysts in 1979 stated "Seed vigor comprises those seed properties which determine the potential for rapid uniform emergence and development of normal seedlings under a wide range of field conditions!'.
Some methods used to measure vigor are the following (1):
Field Emergence of Seedlings
Seedling Growth Rates
No one method is reliable in assessing vigor. Therefore a number of tests known to complement each other in measuring different aspects of vigor are much more certain to yield a reliable conclusion. The tests chosen should be easy to perform, fast, inexpensive, objective, reproducible, and have high correlation with field performance.
Germination results reveal little information about seed vigor. However, if germination is combined with other vigor tests such as accelerated aging and the stress of low temperature, valid vigor ratings appear. Data illustrating this are reproduced in Table 1 (2).
Studies of seedling emergence through sand cover 11 inches in depth and at lower temperatures (10-15°C) were useful in predicting vigor in the field. If the additional parameter of artificial aging was included, a much better assessment could be made (2). This is shown in Table 2.
The germination test, with the aging and low temperature stresses, is a practical one, since large numbers of samples may be easily handled by technicians with little training or equipment. The sand box emergence technique also requires little equipment or training, but it does take weeks to get results, and much labor is required to prepare and plant the boxes. Therefore, other rapid, reliable methods of predicting vigor were studied.
Rapid germination (15°C) of unaged and artificially aged small seed samples yielded some useful results for vigor assessment, as shown in Table 3. These data allow one to group varieties A, B, and D in a good vigor category, and C, E, and F in a poor one. The field ratings (very low = 5; very high = 1)show these conclusions from early germination would be valid. Unfortunately it would not be possible to sort out the varieties in the good or weak categories from these early germination data. Possibly root lengths of the germinated seed and/or tetrazolium staining of the imbibed seed might aid in making the rapid germination method useful in ranking the varieties for vigor. However, tetrazolium readings are very subjective in nature, and both stain reading and root measurements are time consuming and tedious, where many samples must be measured. Therefore, other tests with small samples which could be performed in a time period comparable to rapid germination, were examined.
Respiration of seed/seedling homogenates and production of soluble fraction protein were studied during the first 4-5 days after placing the seeds to germinate.
The method employed was as follows:
50 medium size seeds were surface sterilized in 1-37 sodium hypochlorite solution, washed, and allowed to imbibe sterile distilled H2O, while soaking 2-3 hours. They were then aseptically planted in sterile petri dishes having #3 What man filter paper moistened with distilled H2O. The dishes were placed in the dark at 15°C for germination. Respiration of homogenates and studies of the proteins in solution were performed 2-5 days after planting the seeds. All results given below are for seed received in the lab in 1980, except variety A, which was in the lab 2 years.
The homogenates were prepared by washing 40 seeds/seedlings in cold 0.07 MPO4 buffer, before grinding them by mortar and pestle with sufficient buffer (pH 7.0) to yield 2.2 ml homogenate, after squeezing the suspension through 3 layers of cotton gauze. The respiration readings were made at 25°C by Model 53 YSI Oxygen Monitor with the micro plungers and stirring bars. Generally, the O2 uptake values at the 3rd and 4th days were most useful.
For studies of the proteins from the soluble fraction, the homogenates utilized in respiratory determinations were centrifuged at 25,000 r.c.f.1 for 20 minutes (5°C) to yield the solution between the sedimented particulates and debris and the surface lipid. The soluble fraction was filtered through 0.22~m Millipore filter before measuring the protein content by Bio-Rad Protein Assay (3). The method involves only a few minutes for Coomassie Blue G-250 reagent to complex with the protein. The soluble protein-dye complex absorbance was read at 595 nm by a Spectronic 21. The values were related to a standard bovine plasma albumin solution.
Table 4 shows the respiration results from the homogenates of various varieties. The highly vigorous and weak varieties produced correspondingly high and low O2 uptake values. Varieties A and B have high vigor and C and F have low vigor. The respiratory data alone would not have correctly predicated the vigor of varieties D and E. Use of the protein values from the soluble fraction gave somewhat better assessments of the seed vigor (Table 5), but the rating of variety F did not agree with field performance.
To obtain greater reliability in measuring vigor by respiratory and protein studies, the technique of artificially aging the seed was utilized. The seeds were aged by placing them in plastic dishes in a water bath at 41.5° + 0.5°C
1 Relative centrifugal force (approximately 10070 relative humidity) for 120 hours. Upon removal they were allowed to return to lab moisture content by leaving them in open petri dishes for a few days.
As seen by Table 6 and Table 7, when the aging parameter was included, the vigor assessment became more accurate. The respiration of the homogenates from aged seeds held up well for the vigorous seed varieties (A: very good; B good; D: slightly less good; Table 6), but fell off in those varieties with low vigor. This was most dramatically illustrated by variety E, which had high O2 uptake values for the unaged seed/seedlings. Examination of the proteins from the soluble fraction showed a similar effect. If the variety was vigorous, the protein values for the aged seed increased, as in the unaged controls (A = high, B = good, and D = slightly less good vigor; Table 7).If the varieties were low in vigor, the protein values increased much slower in the aged seed compared to the unaged sample. Thus, varieties C, E, and F were classified as low in vigor by these protein studies. This is also shown by the field vigor ratings. When the artificial aging technique was included in the protein studies, the vigor measurement of variety F became reliable.
In conclusion, O2 uptake values of homogenates and soluble fraction protein readings of seeds/seedlings in early germination are reliable ways to measure the vigor of a sugarbeet variety, if the artificial aging parameter is included. These tests can be performed in a few days. The period required for a measurement is minutes, but preparations, if by mortar and pestle, do require sometime and accuracy. Some good equipment (O2 electrode with constant temperature bath and small sample capability and a spectrophotometer) and a trained technician are necessary.
(2) Gibson, Margaret, Value of various tests for assessing vigor of sugarbeet seeds, 1979 Sugarbeet Research and Extension Reports 10: 204, 1980.
(3) Bio-Rad Protein Assay, Bulletin 1069; 1979. Bio-Rad Laboratories, 2200 Wright Avenue, Richmond, CA 94804.
1980 Sugarbeet Research and Extension Reports, Volume 11, Page 143 - 147