Progress 01/01/04 to 12/31/04
Outputs
Fatty acids, starch content, total germination and time to 50% germination (T50) were determined for two lines of triploid watermelon seeds. Each line consisted of a high and a low germinating lot. A diploid line was also examined. The major fatty acids found were palmitic, stearic, oleic and linoleic acids. Major increases in the relative amounts of linoleic acid were noted with corresponding decreases in the other fatty acids, and the fatty acid content was particularly high in triploids with low germination. Starch content was lower in triploids than in the diploid and there was a high correlation between starch content and T50. Therefore, the high linoleic acid found in triploids (especially low germination lots) and low starch content observed in triploid seeds in comparison to diploids could be physiologically relevant to poor germination and vigor of triploid watermelon seeds.
Impacts
This is the first work to identify specific biochemical differences between triploid and diploid watermelon seeds. Future work would allow for the dissection of biochemical pathways and lead to a better understanding of seed germination and improvement of seedless watermelon seed germination.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
This project has evolved to examine the germination of seedless watermelon. Work on sweet corn was discouraged because it is not considered a major crop in Texas. The major problems in triploid watermelon production are involved to several morphological, physiological and environmental factors, such as hard thick seed coats, small and weak embryos, dense endotesta layers, large seed cavities, moisture control, planting depth and temperature control, instead of confining to a single factor. According to the conventional wisdom, better germination and vigor of watermelon seeds would be expected from seeds in which the cotyledons have higher storage reserves. In triploid seeds, though higher levels of fatty acid content and composition (especially linoleic acids) has been detected in triploids with low germination, it appears that excessive linoleic acids might cause the inhibition of isocitrate lyase activity, and thus triploids with low germination could not
efficiently utilize lipid reserves during the first few days of germination. Surprisingly, a more diverse starch content found in triploids within the same cultivar had no significant differences in T50 (time 50% germination), which could be indicative of seed vigor. This may suggest that starch content must be reached to some critical level to speed up the germination rate of triploid watermelon seeds in comparison to diploids. Ultrastructural differences examined in the seed cotyledon cells can clearly identify the storage of lipid reserves in triploids, and partly account for less availability of starch reserves during physiological and biochemical events in triploid watermelon seeds. The changes of respiration during seed germination provide further evidence that more oxygen is needed for lipid breakdown in triploids with low germination within the same cultivar of triploids since lipids are more reduced than starch in triploids during respiration. Therefore, chemical factors,
such as starch deficiency and fatty acid excess during the early stage of germination, seem to illuminate that failure to utilize seed reserves as an energy source contributes to poor germination in triploids with low germination within the same cultivar of triploids.
Impacts
This is the first work to identify specific biochemical differences between triploid and diploid watermelon seeds. Future work would allow for the dissection of biochemical pathways and lead to a better understanding of seed germination and improvement of seedless watermelon seed germination.
Publications
- Grange, S.L., Leskovar, D.I., Pike, L.M. and B.G. Cobb. (2003) Seedcoat Structure and Oxygen-enhanced Environments Affect Germination of Triploid Watermelon. J. Amer. Soc. Hort 128(2)253-259.
- Wang, T. Cobb, B.G., Sittertz-Bhatkar, H., and D.I. Leskovar. (2004) An Ultrastructural Study of Seed Reserves in Triploid Watermelons. Acta Hort. 631 71-77.
- Grange, S.L., Leskovar, D.I., Pike, L.M. and B.G. Cobb. (2000) Excess Moisture and Seedcoat Nicking Influence Germination of Triploid Watermelon. HortScience. 35:1355-1356.
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Progress 07/07/97 to 07/06/03
Outputs
Triploid watermelon seeds were examined to determine the physiological basis for their poor germiation. We found that respiration rates were lower and RQs suggested more lipid breakdown in triploids that occured in diploids. Starch was also found to be lower in triploids than in a diploid line.
Impacts
This is the first work to identify specific biochemical differences between triploid and diploid watermelon seeds. Future work would allow for the dissection of biochemical pathways and lead to a better understanding of seed germination and improvement of seedless watermelon seed germination.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
In the past we have examined the germination of sweet corn seeds. Seeds contain substantial quantities of at least two major food reserves, such as lipids and starch, in the form of complex polymers. These food reserves must be either hydrolyzed or degraded to their simpler monomers which are eventually catabolized enzymatically for the production of energy (ATP) and other essential metabolites for seed development and plant growth. This is the case for maize and the fact that lower starch contents do control germination is well known. We have extended this work to examine the germination of triploid seeds of watermelon. Major differences were found in starch content between the diploid and triploid cultivars. The lower starch content in triploids could have an influence on reserve mobilization during germination. That is, the low starch content relates to a longer T50 (time to 50% germination), which would be concordant with expected reduction in total rate of energy
utilization, and therefore resulted in low seed vigor contributed to poor germination of triploid watermelon seeds. Further research is needed to determine the practical levels of starch content for improving the germination in triploid seeds.
Impacts
The work that we are now engaged in is directed at identifying the reasons that seeds of some crops do not germinate well. The results will allow us to improve the germination of these seeds and thus improve their performance in the field.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
This research as expanded to other species that have germiation problems including triploid watermelon seeds. We have found that triploid watermelon seeds have altered starch content and lipid profiles compared to those of diploids. This combined with the other factors that may affect triploid seeds may help explain their poor germination performance.
Impacts
Understanding the mechanisms that control seed germiation will point to methods to enhance seed germination of major crop species.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
We have extended our work to examining lipid metabolism in diploid and triploid watermelon seeds. Analysis of the fatty acid composition of the total seed lipid from different polyploid watermelon seeds indicated that there were marked changes in the composition of the major saturated and unsaturated fatty acids. Changes in fatty acid composition were determined by analysis of FAMEs using gas chromatography. The analysis showed that six methyl ester peaks were found in the fatty acid profile of watermelon samples: myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0, oleic (C18:1), and linoleic (C18:2). The unsaturated fatty acids of triploid seeds, linoleic acid, contributed markedly to the total percentage of fatty acids while lower levels of other fatty acids were also present. The fatty acid compositions of the seed lipids from various samples of triploid watermelon were strikingly similar. The results suggest that in triploid watermelon seeds
there was an `overflow' of linoleic acid into all lipid classes. Apparently, the fatty acid composition of polar lipids was appropriate for their normal function in triploid watermelon seeds. It is interesting to note that the content of linoleic acid was low (35.1%) in diploid seeds, but rose rapidly to 54% or more in triploid seeds.
Impacts
These results point to a significant difference in the lipid content of triploid watermelon seeds suggesting that lipid metabolism may be involved in the poor performance of the triploid seeds.
Publications
- Grange, S.L., Pike, L.M., Cobb, B.G. 2000. Excess Moisture and Seedcoat Nicking Influence Germination of Triploid Watermelon. Hort Sci. 35:1335-1336.
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Progress 01/01/99 to 12/31/99
Outputs
Progress has been made in identifying factors that control germination of a variety of species. Two particular, sweet corn and triploid melon seeds have been investigated. For both it appears as though altered development controls, at least partially, germination. This year we have devoted most of the project to triploid seeds. They are characterized by thicker seed coats, lower carbohydrates and modified lipid reserves. SEM of the seed coats reveal many layers that are thicker than the diplid varities. Germination can be improved by removing or splitting the seed coat. However, germination in high oxygen concentrations does not improve germiation suggesting that the seed coat may act as a physical barrier than a barrier to gas transfer.
Impacts
This reseach addresses problems with horticultural species that are characterized by poor seed germination. Understanding the structural and physiological aspects that control germination may allow us to develop new treatments to improve seed performance.
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
We have extended our research to include triploid seeds. Triploid seeds are highly susceptible to excess moisture during germination. For example, when 8 commercial triploids (sources from four seed companies) and 7 diploids were incubated at optimum and submerged conditions at 25?C, germination was slightly reduced in diploids (98 vs. 90%; optimum vs. submerged), but was highly suppressed in triploids (89% vs. 10%). These results suggested that oxygen could be limited in triploids due to a thicker or impermeable seed coat. To test this the seeds were nicked using three triploid seed sources from Novartis (cvs. 121, 1614, and 1616) and one diploid. This resulted in an increase in germination from 46% to 76% in 121, 59% to 77% in 1614, and 65% to 71% in 1616. However, this is still lower than diploid controls (98 to 100%) and suggest that other factor(s) are involved. The susceptibility to excess moisture could also indicate a lack of ability of the triploids to
undergo anaerobic respiration, a process necessary for germination in conditions of excess moisture.
Impacts
(N/A)
Publications
- D. Andrews, Crystal Kelley, B. G. Cobb and James West. 1999. Ethanol attenuates lactate production in hypoxic postnatal day 3 rat cerebella. In press (Alcohol).
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Progress 01/01/97 to 12/31/97
Outputs
We have been improving germination methods because increased vigor will allow for the introduction of this crop into Texas. Importantly, we have identified seed treatments that can be utilized now to improve existing sh2 varieties and we now have in hand molecular strategies that we can utilize to develop new varieties of superior sweet corn for production in Texas. Our first goal is to improve the seed germination of existing sh2 sweet corn varieties using low-oxygen pre-treatments. We can increase germination from 40% to 90% using this method. Our second goal is to use RNA antisense technology to develop superior varieties that have high sugar during the market stage but high starch at maturity.
Impacts
(N/A)
Publications
- Paek.N, B. Lee, D. G. Bai, B. G. Cobb, C. W. Magill and J.D. Smith 1997 Regulatory Roles of Abscisic Acid for Anthocyanin Synthesis in Maize Kernels. (accepted, Maydica)
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Progress 01/01/96 to 12/30/96
Outputs
The priming of seed is a process in which the seeds are placed in an osmotic solution for a determined period of time, dried down to original moisture content, then germinated. Sweet corn 'Even Sweeter' is a mutant of the wild type Zea mays L. at the shrunken-2 loci. Seeds were primed with or without a 400 mM glucose solution (-1.0 and 0.0 MPa respectively) while no aeration (stagnant) or 0, 4, 21 or 40% (v/v) oxygen for 1 and 2 days. Primed seeds germinated faster and had a higher final percent germination than controls. Significant differences were found within the treatments, and especially between the treatments and the unprimed control. The effectiveness of the priming treatment depended on the osmotic potential of the solution, the percent of oxygen used for aeration, and the duration of priming. In general, seeds germinated better when primed at lower oxygen concentrations.
Impacts
(N/A)
Publications
- Giroux MJ, Shaw JBG, Cobb BG, Greene T, Okita T, Hannah LC. 1996. A single gene mutation that increases maize seed weight. PNAS 93(12):5824-5829.
- Yoo B, Lang H. Cobb BG. 1996. Priming with several salts increase germination of pansy seed at high temperatures. HortSci (in press).
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Progress 01/01/95 to 12/30/95
Outputs
We have found that maize seeds will survive an extended period of anaerobisis ifthey are first made anaerobic before imbibition. Maize seeds treated in this way will survive at least 10 days of anaerobisis. While they do no germinate during the anaerobic treatment they will germinate when returned to air. The ability to survive anaerobiosis is quickly lost if the seeds are germinated in air before the anaerobic treatment. Only 52% survived anaerobiosis if first germinated for 1 day and only 6% survived if germinated for 3 days before exposed to an anaerobic environment. This ability of seeds to survive anaerobisis is correlated with the presence of transcripts and enzymatic activity for the anaerobic pathways. Experiments are now underway to determine if seeds of other species show a similar survival mechanism.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
We have been examining how maize roots and seeds respond to oxygen deficit. Imbibed maize seeds will survive at least 20 days of anoxia. Imbibed seeds were placed in an anaerobic workbench for 2-20 days. While they do not germinate in anoxia, they do germinate when they are transferred back into a normal oxygen environment. However, if the seeds are allowed to germinate for greater than 2 days before they are exposed to anoxia, they do not survive even a limited exposure to anoxia. The switch from `anaerobic tolerant' to `anaerobic sensitive' must therefore occur during the first 48 hours of germination and is now the focus of our research.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
We are continuing our examination of the induction of anoxia tolerance in maize roots by pretreating the roots with low oxygen (hypoxia). We have found that adh1 nulls survive exposure to anoxia longer when they are pretreated with hypoxia than when they are transferred directly to anoxia. This occurs even though the ADH activity in the nulls is only 5% of that of the induced wild type seedlings. This residual activity is presumed to be ADH2 activity. These findings suggests that the high levels of ADH activity found in induced wild type seedlings is greater than that needed for survival, or alternatively, the induction of ADH2 activity that is know to occur during anoxia is necessary for survival. We will test this hypothesis my examining hypoxic induction of anoxia tolerance in mutants of Adh2. We are also characterizing the role of pyruvate decarboxylase (PDC) in the induction of tolerance. Preliminary, in wild type roots there is a transient induction of PDC
transcripts during the hypoxic pretreatment whereas PDC transcripts remain elevated in adh1 mutants throughout the pretreatment.
Impacts
(N/A)
Publications
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Progress 01/01/92 to 12/30/92
Outputs
Restricting oxygen during priming of maize sweet corn seed results in a significant improvement in low-temperature germination. Seeds of the sweet corn variety `Even Sweeter' were primed for 24 or 48h in solutions sparged with several concentrations of oxygen followed by re-drying the seeds for 4 days and then germinating at 18C. Seeds treated with high oxygen did not germinate as well as untreated controls. Seeds treated with lower than ambient concentrations of oxygen germinated significantly better than controls. The biochemical basis for these findings are unknown but data suggests that 2 different processes are occurring: 1) high oxygen during the first day of germination is detrimental to seed germination and 2) treating seeds with low oxygen may acclimate seeds to conditions that are normally encountered during germination. These results are similar to those we have observed with acclimation of maize roots tips to low oxygen.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
We have instituted work with a goal of elucidating the biological mechanisms that are invoked during the priming of seeds using maize as a model system. In priming seeds are placed in osmotically adjusted solutions that allow some metabolic events to occur without actual seed germination. We have recently directed our work to examining the role of anaerobic metabolism in the priming process. Priming is not as effective in mutants that lack the enzyme alcohol dehydrogenase, ADH1, suggesting the induction of anaerobic metabolism during priming. Interestingly, the addition of 40% oxygen to the priming solutions of wild type or ADH1 mutants does not enhance seed performance as compared to treatments with 4% oxygen. These data suggest that the induction of anareobic metabolism is necessary for enhancement of seed germination by seed priming. We have also extended the priming work to include priming by growers on site. We now are working with a grower who is priming seeds as
needed. We are developing the basic priming treatments in the lab which he has successfully used in a commercial setting. In the near future, we will produce solutions that can be used by growers to prime their seeds at the point of use.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
The research on salt stress has been divided into 2 areas: 1. Seed priming. Seeds are incubated in salt solutions then removed and germinated. These seeds germinate faster than untreated controls. Our goal is to understand physiological changes that occur during this "priming" treatment. We have found that enzymes are activated and that considerable respiration occurs during priming. Interestingly, we have found that seeds deficient in the enzyme alcohol dehydrogenase do not respond to priming as well as wild type seeds. This suggests that anaerobic metabolism is a requirement for effective seed priming. Measurements of RQ (respiratory quotient) in primed seeds confirms this. 2. Growth of cucumber seedlings. We have been examining the physiological mechanisms that roots use to cope with exposure to salt. A basic problem with this research is how to introduce the salt stress. In the past this has been done either by placing the plants directly in the salt
solution (shock) or by incrementally raising the salt concentration to avoid shock (mini-shock). To determine if there is differences in response to these treatments cucumber plants have been exposed to 80 mM NaCl as a shock (0 -80 mM NaCl), a mini-shock (0-80 mM NaCl in 20 mM increments) or gradient (0-80 mM in a continuous gradient over a 2 day period). Two high molecular weight protein bands seen in the control (0 mM) decrease in intensity in the gradient treatment, but disappear in the mini-shock treatment.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
There are 4 areas of research that we are involved in that deal with water or salt stress: 1) induction of invertase by salt stress 2) ABA regulation of gene action 3) anaerobic stress 4) vitrification. 1) We have found that invertase is induced in the roots of cucumber plants exposed to salt. Invertase levels increase within 1 hour of exposure to salt and remain high throughout the duration of the salt treatment. It is possible that invertase is converting sucrose to fructose and glucose to act as osmotica. 2) By using in vitro kernel culture we have shown that ABA controls the expression of anthocyanins in developing maize kernels. Kernels are grown in media supplemented with ABA, fluridone (an ABA inhibitor), or ABA plus fluridone. Kernels grown in the presence of ABA develop anthocyanin but kernels grown with fluridone do not indicating that ABA controls anthocyanin production. 3) Maize roots that are exposed to hypoxia (HPT) before an anoxic treatment are able to
survive anoxia longer than roots that are directly exposed to anoxia (NHPT). There are qualitative and quantitative differences in proteins produced, in energy change, and in respiratory pathways used between NHPT and HPT. We are now isolating mRNA to produce libraries from NHPT and HPT root tips. 4) Vitrification is a physiological disorder which can occur to plants asexually propagated in vitro.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
We have been examining seed development and germination as a system to study stress effects on plant growth. Seed germination has been examined by using seed priming techniques to ascertain the effect of salts on seed germination. Detectable seed respiration occurs during seed priming as does protein synthesis and carbon metabolism. It appears that anaerobic metabolism, as measured by glycolytic and fermentation markers, is preferentially activated during priming. By using in vitro kernel development we have simulated low water potentials by incorporation of PEG into the growth media and restricted carbohydrate availability by varying the sucrose concentrations in the growth medium. Kernels grown in the presence of PEG or with low sucrose concentrations do not develop well. Increasing available sucrose does result in increased kernel weight but the effect of water stress cannot be overcome.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
We have been investigating the role of salts in controlling the priming process in pepper. Pepper seeds have been primed in PEG, NaCl and CaCl(2). The activity of the major metabolic pathways (Kreb's, pentose phosphate, glycolysis, glycolate, anaerobic) were then assayed to determine which, if any, pathways are activated in primed seeds. Only the glycolate pathway, as measured by the activity of isocitrate lyase, was activated during priming. Seeds primed with PEG did show significant levels of anaerobic metabolism as measured as ADH activity. To date, we have found no detectable changes in starch or sugars in the primed seeds. We are now examining protein metabolism in the primed seeds. Preliminary results show that the concentration of amino acids increase in primed seeds.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
Two projects dealing with salt stress have been initiated in my lab. 1) Seed Germination. Pepper and cucumber seeds have been germinated in 10 different salts at concentrations of 25, 50, 100, 200 and 300 mM. The speed of germination is promoted at 25 mM but germination is totally inhibited at concentrations greater than 200 mM. After 15 days the salts are removed, the seeds washed, then germinated in distilled water. Whereas the cucumber seeds do not germinate after the salt treatments the pepper seeds are "primed to germinate" by the treatment and germinate much faster than controls. We are now examining the interaction of hormones and salts. By treating seeds with salts and hormones we can increase the speed of germination from 50% at 7 days to 80% after 1 day. We are concentrating on determining the physiological changes that occur during the priming process. 2) Salt Tolerance in Cucumber. We have been examining the effects of NaCl stress on cucumber growth
using a nutrient flow system in which salts can be added gradually to slowly increase the salt concentration. Leaves of salt stressed plants do appear to have a different carbohydrate pattern than controls. We have tentatively identified raffinose as one sugar that increases in the stressed plants. We are now examing the enzymes involved in raffinose metabolism to determine if they are affected by salt stress.
Impacts
(N/A)
Publications
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