| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Genetics and Molecular Biology |
2Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 USA; 3Plant Biotechnology Centre, Department of Primary Industries, La Trobe University, Victoria 3086 Australia
Received for publication August 14, 2003. Accepted for publication November 14, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Key Words: forage grass • pollen longevity • pollen viability • tall fescue • transgenic plant
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
; Sleper and West, 1996
). It forms the forage basis for beef cow and calf production and also is widely used as turf in lawns, parks, football fields, highway medians, and roadsides (Sleper and Buckner, 1995
). Like many other forage and turf grasses, tall fescue is a wind-pollinated, highly self-infertile species. Because of its agronomic importance, tall fescue has been a major target species for genetic and agronomic studies (Jauhar, 1993
; Spangenberg et al., 1998
). In recent years, it has been used as a target species for large-scale sequencing of expressed sequence tags (ESTs), gene discovery, and genetic engineering in forage grasses (Wang et al., 2001a
). Because wind-pollinated grass species have a high potential to pass their genes to adjacent plants, risk assessment regarding pollen-mediated gene flow has become an extremely important issue for any future release of value-added transgenic cultivars. Pollen viability and longevity is one of the critical factors to be considered in the assessment.
Pollen viability is generally considered to indicate the ability of the pollen grain to perform its function of delivering the sperm cells to the embryo sac following compatible pollination (Shivanna et al., 1991
). Assessment of pollen viability on the basis of its function is cumbersome, time-consuming, and not always feasible (Heslop-Harrison et al., 1984
). Many short-cut methods that reflect the competence of the pollen to perform its normal function have been devised (Shivanna et al., 1991
). Pollen viability has been evaluated by various staining techniques (e.g., tetrazolium salts to detect dehydrogenase activity, aniline blue to detect callose in pollen walls and pollen tubes, iodine to determine starch content, fluorescein diacetate to determine esterase activity and the intactness of the plasma membrane), by in vitro and in vivo germination tests, or by analyzing final seed set (Adhikari and Campbell, 1998
; Dafni and Firmage, 2000
). The choice of method depends on the crop or species (Dafni and Firmage, 2000
). There has been very little information about the utility of most of these methods for tall fescue and other major forage grasses. Pollen viability in meadow fescue and Russian wildrye was estimated using iodine/potassium iodide staining (Wang et al., 1993
, 2002
). Even though the method offered a quick way of detecting aborted and non-aborted fresh pollen, however, it could not distinguish viable from dead pollen if the pollen was stored for some time. In other monocot species, such as sorghum, maize, and rice, reliable methods for estimating pollen viability have been reported (Khatun and Flowers, 1995
; Tuinstra and Wedel, 2000
; Luna et al., 2001
).
Pollen management has been investigated as a method to limit transgene flow in maize (Luna et al., 2001
). Knowledge about the duration of pollen viability will be helpful in developing various methods to manage pollen flow. The only report on pollen longevity of tall fescue indicated that pollen of this grass species could survive in open air for more than 48 h, but was completely dead 72 h after opening of anthers (Pacini et al., 1997
). The pollen viability test in the study was based on a fluorochromatic reaction (FCR) that measures the activity of cytoplasmic esterase and the integrity of the pollen plasma membrane (Pacini et al., 1997
). To date, there have been no reports on pollen viability and longevity of transgenic pollen in tall fescue and other forage grasses.
This paper describes (1) the optimization of an in vitro germination protocol to effectively detect viable pollen in tall fescue, (2) evaluation of the influences of different variables (temperature, humidity, UV-B treatment) on pollen viability, (3) comparison of viability and longevity of transgenic and nontransgenic pollen, and (4) pollen viability and seed set in hand pollinations using unique materials derived from tissue culture and genetic transformation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
An efficient plant regeneration system was established based on the use of embryogenic suspension cultures (Wang et al., 1994
, 1995
). Sterilized seeds/ caryopses were used as explants to induce callus. Embryogenic calluses were transferred to liquid culture medium to establish cell suspension cultures (Wang et al., 1994
, 2003a
). Plants regenerated from cell suspensions, namely primary regenerants (R0), were used in the hand-pollination experiment.
Cell clusters from embryogenic cell suspensions were used as direct targets for biolistic transformation to generate transgenic plants (Wang et al., 2003a
). A chimeric hygromycin phosphotransferase (hph) gene, which renders transformed cells resistant to hygromycin, was used as the selectable marker gene (Bilang et al., 1991
). A chimeric ß-glucuronidase (gusA) gene was co-transformed with the hph gene (Wang et al., 2003a
). Transgenic tall fescue plants (T0) were obtained after microprojectile bombardment of suspension cells and the subsequent selection of hygromycin-resistant calluses (Wang et al., 2003a
). Transgenic progenies (T1 and T2 generation) were obtained after reciprocal crosses between transgenic and nontransformed control plants (Wang et al., 2003a
, b
). Transgenic nature of the T0, T1, and T2 plants was confirmed by PCR and Southern hybridization analyses; all the plants contained multiple copies of hph and gusA transgenes (Wang et al., 2003a
, b
).
Primary transgenics, primary regenerants, and seed-derived plants were grown in the field during 2000–2001 in a randomized complete block with three replications. Progenies of primary transgenics, progenies of primary regenerants, and seed-derived plants were grown in the field during 2001– 2003. Additional seed-derived plants were available in a nearby field. Agronomic performance of the primary regenerants and transgenics were previously shown to be inferior to the seed-derived plants, probably from carryover effects of tissue culture, while the progenies of transgenics, regenerants, or seed-derived plants had no major agronomic differences (Wang et al., 2003c
). Thus pollen from transgenic progenies (T1 and T2) was compared with pollen from nontransformed seed-derived plants. Pollen from primary transgenics (T0) had various levels of viability. These plants were used in the hand- pollination experiment.
The experimental area was located in Ardmore, Oklahoma, USA, with mean annual temperature and rainfall of 17.7°C and 912 mm, respectively. The field trials with transgenic material were carried out under USDA regulations with a strict field protocol during the entire experimental period to fulfill the performance standard for field trials with transgenic grasses (Wang et al., 2003c
).
Tests for pollen viability by staining
A number of staining methods were assessed for fresh pollen and dead pollen (80°C for 2h) from seed-derived plants. (1) For the X-gal test for the presence of ß–galactosidase (Singh et al., 1985
; Trognitz, 1991
), the test solution consisted of 1 mg X-gal (5-bromo- 4-chloro-3-indoyle-ß-galactoside) dissolved in 50 µL N,N-dimethyl formamide and 1 mL acetate buffer (50 mmol, pH 4.8). The pollen grain was considered viable if it turned blue (Rodriguez-Riano and Dafni, 2000
). (2) For testing the presence of dehydrogenase, the test solution consisted of a 1% concentration of the substrate 2,3,5-triphenyl tetrazolium chloride (TTC) or 2,5-diphenyl monotetrazolium bromide (MTT) in 5% sucrose (Norton, 1966
; Khatun and Flowers, 1995
; Rodriguez-Riano and Dafni, 2000
). The pollen grain was considered viable if it turned deep pink. (3) Aniline blue was used to detect callose in pollen walls and pollen tubes (Hauser and Morrison, 1964
; Khatun and Flowers, 1995
). The aniline blue–lactophenol staining solution was made by adding 5 mL of a 1% aqueous aniline blue to a medium of 20 mL phenol, 20 mL lactic acid (ca. 85%), 40 mL glycerine, and 20 mL H2O. (4) The Lugol solution (Sigma, St. Louis, Missouri, USA) for detecting starch content consisted of iodine and potassium iodide. Black-stained pollen was considered viable. (5) For the fluorochromatic reaction (FCR) test for esterase activity and intactness of cell membrane, fluorescein diacetate was dissolved in acetone (2 mg/mL) and used at 10–6 mol/L in 0.8 mol/L sucrose (Heslop- Harrison and Heslop-Harrison, 1970
; Khatun and Flowers, 1995
). Pollen was viewed under a fluorescence microscope.
Tests for pollen viability by in vitro germination
Pollen collected from nontransgenic tall fescue plants was used to optimize media composition. A factorial experimental design, similar to the one described for sorghum (Tuinstra and Wedel, 2000
), was used to evaluate the effects of sucrose, boric acid, and calcium nitrate on pollen germination in tall fescue. Sucrose, boric acid, and calcium nitrate have been shown to be key substrates for pollen germination in other species (Steer and Steer, 1989
; Tuinstra and Wedel, 2000
). Media containing 1% (m/v) agar and different concentrations of the three reagents in all combinations were tested. Sucrose was initially tested at 0.4, 0.6, 0.8, 1.1, and 1.3 mol/L; boric acid at 0.16, 0.64, and 2.56 mmol/L; and calcium nitrate at 0.21, 1.27, and 2.54 mmol/L. The experiment was blocked in time with three replications in a randomized complete block design.
Based on the results from the pilot factorial experiment, media composition was further optimized by testing a wider range of reagents. Individual reagent concentration was varied while others were held constant as indicated in the pilot experiment (1% agar, 0.8 mol/L sucrose, 0.64 mmol/L boric acid, 1.27 mmol/L calcium nitrate). Effects of sucrose on pollen germination was evaluated at 0, 0.4, 0.7, 0.8, 0.9, 1.1, 1.3 mol/L; boric acid at 0, 0.32, 0.64, 1.28, 2.56, 5.12 mmol/L; and calcium nitrate at 0, 0.21, 0.63, 1.27, 2.54, 5.08 mmol/ L. These experiments followed a randomized complete block design with six replications.
Pollen was collected from field-grown material. Bulk pollen was distributed onto germination media in petri dishes and incubated at 24°C in the dark for 4 h. Germination was quantified as the percentage of germinated pollen grains per 100 evaluated. Pollen grains were considered germinated when the pollen tube length was greater than the diameter of the pollen grain (Tuinstra and Wedel, 2000
).
Effect of different conditions on pollen germination
Pollen germination at different conditions was evaluated on optimal germination medium (1% agar, 0.8 mol/L sucrose, 1.28 mmol/L boric acid, and 1.27 mmol/L calcium nitrate) in petri dishes after 4 h incubation at 24°C. To test the effect of temperature, pollen was immediately dispensed onto medium or incubated for 10 and 20 min at 18°, 24°, 28°, 32°, 36°, and 40°C before being dispensed onto the medium. The effect of relative humidity (RH) was tested after storing pollen at 40, 60, 80 and 99% RH for 10 min before being dispensed onto germination medium. Effect of UV irradiation was performed by exposing pollen to UV-B light (15-W UV-B lamps; UVP Inc., Upland, California, USA) fixed on an exposure stand for 10 min before pollen grains were germinated in vitro on optimal medium at 24°C. The UV-B doses were 300, 600, 900, 1200, and 1500 µW/cm2, determined by UVX digital radiometer with UV-B specific sensor UVX-31 (UVP). The dosage used in the experiment was in the range that plants normally encounter under natural conditions.
Pollen longevity was evaluated under controlled conditions in a growth chamber (24°C, RH 54 ± 5%) at various time points (0, 1, 2, 3, ... 24 h). Pollen longevity under natural conditions was tested by exposing pollen to outdoor open air during mid-April to mid-May (nine non-rainy days) in both 2002 and 2003. Pollen viability was assessed for 0, 15, 30, 45, 60, and 90 min under sunny conditions; pollen viability was tested for 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, and 300 min under shady/cloudy conditions.
Ten fully fertile plants were randomly selected from each category (regenerants, transgenics, and seed-derived plants) and used for pollen collection. At least three replications were carried out for each experiment involving different treatments. Pollen was collected onto paper and quickly distributed into petri dishes for different treatments. Germination of pollen was tested at 24°C for 4 h in the dark. Pollen germination was quantified as previously described.
Hand pollination
The recipient inflorescence of control plants was emasculated and then bagged together with two panicles from the pollen donor plant; water was supplied to the donor panicles from a 50-mL conical tube fixed to a bamboo stake. This is a common and effective practice in making crosses for tall fescue because the donor panicles will shed pollen to the recipient florets continuously (Hovin, 1980
). Unlike some cereal crops (e.g., wheat), the spikelets in tall fescue are pedicelled in a branched inflorescence, and the suitable pollination time for florets in the same panicle may vary greatly; thus it is inefficient to collect pollen grains for pollination in tall fescue. The primary transgenics regenerated from tissue culture had plants with various levels of pollen viability. These unique materials provided a good opportunity to study the influence of pollen viability on seed setting in tall fescue.
Statistical analysis
Statistical analyses were carried out using the SAS statistical package, version 6 (SAS Institute, Cary, North Carolina, USA). Each experiment was conducted as a randomized complete block design. Block effects were considered random, and treatment main effects and interaction were evaluated as fixed effects. Differences were declared significant when P < 0.05. Standard errors are provided where appropriate.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Pollen viability tested by in vitro germination
Variance analysis of the initial factorial experiment indicated significant differences in pollen germination associated with the sucrose, boric acid, and calcium nitrate treatments (data not shown). Near-optimum concentrations of sucrose (0.8 mol/L), boric acid (0.64 mmol/L), and calcium nitrate (1.27 mmol/L) were identified. Based on the results from the pilot experiment, a more detailed study was designed to optimize the concentrations of individual components (Table 1). As expected, sucrose concentration had a large impact on tall fescue pollen germination. Pollen burst in medium without sucrose, and germination was reduced at high concentrations of sucrose (1.3 mol/ L). The highest frequency of pollen germination was observed on medium containing 0.8 mol/L sucrose, although no significant difference was observed between 0.4 and 1.1 mol/L.
|
The effect of calcium nitrate on pollen germination was less pronounced than that of sucrose and boric acid. Nevertheless, pollen germination was significantly lower when no calcium nitrate was present in the medium or there was an excessive amount of calcium nitrate (5.08 mmol/L) in the medium. Pollen germination was maximized when the medium contained 1.27 mmol/L calcium nitrate (Table 1). Based on the optimization experiments, tall fescue pollen germination should be evaluated on agar medium containing 0.8 mol/L sucrose, 1.28 mmol/L boric acid, and 1.27 mmol/L calcium nitrate. Most of the pollen germinated within 8 min in the medium. The optimized pollen germination medium allowed a reliable evaluation of pollen viability in tall fescue. No killed pollen was able to germinate on this medium.
The in vitro germination medium was also tested for assessment of pollen viability in perennial ryegrass (Lolium perenne L.), Italian ryegrass (Lolium multiflorum Lam.), and meadow fescue (Festuca pratensis Huds.). From these species 69–83% of fresh pollen germinated in the medium, indicating that the protocol can be used, directly or with slight modifications, for a range of widely grown forage and turf grass species. The development of the in vitro germination protocol allowed us to study a number of parameters affecting pollen viability and longevity.
Effects of temperature on pollen viability
Pollen germination in nontransgenic tall fescue was tested over a range of temperatures that might be encountered under field conditions (Fig. 1). When pollen was harvested and immediately distributed onto the germination medium, no significant difference in pollen viability was found at incubation temperatures of 18°, 24°, 32°, and 36°C, but germination was significantly reduced at 40°C (P < 0.05). When pollen was incubated at various temperatures for either 10 or 20 min before being distributed onto germination medium, the negative impact of high temperature on pollen viability was more obvious; pollen germination was greatly reduced at 36°C and 40°C, with less than 5% of the pollen still viable after incubation at 40°C for 20 min.
|
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
; Rodriguez-Riano and Dafni, 2000
). However, in this study, none of the staining methods tested (X-gal, aniline blue, TTC, MTT, Lugol solution, and fluorescein diacetate) could distinguish viable and dead pollen grains of tall fescue. These staining methods are thus not suitable for the study of pollen viability and longevity in tall fescue.
In vitro pollen germination is believed to provide the best estimate of pollen viability in vivo (Stone et al., 1995
; Tuinstra and Wedel, 2000
). In fact, in most of the studies on staining techniques, the effectiveness of staining methods was evaluated based on their correlation with pollen germination in vitro (Khatun and Flowers, 1995
; Dafni and Firmage, 2000
; Rodriguez-Riano and Dafni, 2000
). Therefore, we optimized conditions for pollen germination in tall fescue and established a simple medium that allowed effective testing of pollen viability. The medium is also suitable for testing pollen viability in some closely related grass species, e.g., ryegrasses.
Environmental factors can vary considerably during the tall fescue flowering period. Because pollen of tall fescue shed immediately after anthesis, short duration (10 or 20 min) treatments of pollen at different conditions were evaluated. When fresh pollen was germinated at different temperatures, germination was reduced only at 40°C, a result similar to that in sorghum (Tuinstra and Wedel, 2000
). When pollen was stored for 10 or 20 min, its viability was not significantly changed at 18–32°C, but viability was reduced when temperature was at or above 36°C. These results indicated that pollen viability was not influenced over the range of temperatures often encountered during the pollination season, while excessively high temperatures during this period may negatively influence pollen viability. Relative humidity is one of environmental factors that might affect pollen viability (Adhikari and Campbell, 1998
). Although low relative humidity is favorable for pollen storage in many species, pollen from graminaceous species requires high relative humidity (Adhikari and Campbell, 1998
). In the present study, exposure of tall fescue pollen to different humidities did not affect pollen viability, probably because the exposure period was relatively short (10 min). For pollen storage in buckwheat, a high relative humidity was required to retain the viability of pollen for more than an hour (Adhikari and Campbell, 1998
). Besides temperature and humidity, the effect of UV-B irradiation on pollen viability also was evaluated. Higher UV-B doses reduced pollen viability, indicating ultraviolet irradiation may have contributed to the reduced pollen longevity under direct sunlight. In addition, pollen-mediated gene flow could be affected in geographic regions with an increase in UV-B irradiation, due to the reduction of stratospheric ozone.
The potential of biotechnology to improved forage grasses has become evident in recent years (Spangenberg et al., 1998
; Wang et al., 2001a
), and there have been many reports on the generation of transgenic plants in tall fescue (Wang et al., 1992
, 2001b
, 2003a
; Dalton et al., 1995
; Spangenberg et al., 1995b
; Kuai et al., 1999
; Cho et al., 2000
; Bettany et al., 2003
; Chen et al., 2003
) and other important forage species, such as perennial ryegrass (Spangenberg et al., 1995b
; Dalton et al., 1998
, 1999
; Altpeter et al., 2000
), Italian ryegrass (Wang et al., 1997
; Ye et al., 1997
; Dalton et al., 1998
, 1999
; Bettany et al., 2003
), red fescue (Spangenberg et al., 1994
, 1995a
), orchardgrass (Horn et al., 1988
; Denchev et al., 1997
; Cho et al., 2001
), switchgrass (Richards et al., 2001
), and bahiagrass (Smith et al., 2002
). In spite of important progress made in the development of transformation techniques and the production of transgenic grasses, to date there have been no reports on detailed pollen viability and longevity assessments for any transgenic forage grass.
Evaluation of viability and longevity of transgenic pollen is an important aspect of risk assessment and biosafety study for transgenic plants. Pollen from T1 and T2 generations of transgenics and R1 and R2 generations of regenerants had similar viability when compared with that of seed-derived nontransgenic plants, although primary transgenic (T0) and regenerants (R0) had various levels of pollen viability among individual plants. The results complement and are also consistent with a field performance study, in which the primary transgenics and regenerants had lower productivity than seed-derived plants, whereas progenies of these transgenics and regenerants had agronomic performance similar to seed-derived plants (Wang et al., 2003c
). The lower seed yield in the primary transgenics might be related to lower pollen viability. Together with the field data reported by Wang et al. (2003c)
, the results indicate that once seeds are obtained from the primary transgenic plants, normal pollen viability and agronomic performance of the progenies can be expected.
Primary regenerants and transgenics with different pollen viability were used as unique material in a hand-pollination experiment. No seed was obtained with donor panicles having 5% or less pollen viability, indicating seed setting is closely related to pollen viability.
A rapid decline of pollen viability may greatly diminish effective pollen flow with pollen longevity thus being a crucial factor in pollination efficiency. Longevity of pollen from transgenic progenies of tall fescue was compared with seed-derived plants under different conditions. Under controlled conditions in the growth chamber, both transgenic and nontransgenic pollen could survive a relatively long time, with 
5% viability in 12 h and a complete loss of viability in 22 h. However, the situation was much different under ambient atmospheric conditions, where pollen viability was lost rapidly. It took only 30 min to reduce pollen viability to 5% under sunny conditions and to the same level in 150 min under cloudy conditions. Thus, the longevity of pollen could be influenced drastically by the weather conditions. Although sporadic germination could be observed for a relatively longer time, viability was completely lost after 90 min and 240 min under sunny and cloudy conditions, respectively. Pacini et al. (1997)
reported that tall fescue pollen could survive nearly 3 d in the open air. However, the results were based on FCR evaluation of pollen viability. As we showed here, this method is not suitable for longevity study in tall fescue because fluorescence could not distinguish viable pollen from the dead ones in the FCR test, a phenomenon also observed in some other species (Kapyla, 1991
). Short periods of pollen viability have also been reported in other grass species: viability was completely lost within 65–70 min in wheat and 110–120 min in triticale (Fritz and Lukaszewski, 1989
). In maize, no viable pollen was detected after 2 h atmospheric exposure (Luna et al., 2001
).
Under either growth chamber conditions or outside atmospheric conditions, pollen longevity from transgenic progenies had trends similar to those of seed-derived plants, further confirming that there was no difference between transgenic and control pollen.
Forages are critical to livestock industries throughout the world. They play a major role in providing high quality roughage for the economical production of meat, milk, and fiber products and are important in soil conservation and environmental protection. Most of the important forage grasses are outcrossing species and require wind pollination to set seed (Jensen et al., 1990
). Synthetic cultivars have been the main product of breeding in most of the outcrossing forage species. Therefore, transgenic grasses pose a great challenge for risk assessment and biosafety studies. Because human consumption is indirect, biosafety evaluation of transgenic grasses will likely focus on their environmental or ecological impacts, in which pollination biology plays a critical role. As an important step toward understanding one of the essential aspects of risk assessment, in the present research an effective protocol was developed for assessing pollen viability, the influences of different environmental factors on pollen viability were tested, viability of transgenic and nontransgenic pollen was compared, information on longevity of pollen under different conditions was gained, and the correlation of pollen viability and seed set in hand pollinations using unique materials derived from tissue culture and genetic transformation was assessed. This report is the first covering several aspects of pollination biology using transgenic and nontransgenic materials in an important forage species. The information obtained from this study could be useful for other closely related grass species.
|
FOOTNOTES |
|---|
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Altpeter F. J. P. Xu S. Ahmed 2000 Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage- and turf-type cultivars. Molecular Breeding 6: 519-528
Barnes R. F. 1990 Importance and problems of tall fescue. In M. J. Kasperbauer [ed.], Biotechnology in tall fescue improvement, 1–12. CRC Press, Boca Raton, Florida, USA
Bettany A. J. E. S. J. Dalton E. Timms B. Manderyck M. S. Dhanoa P. Morris 2003 Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam). Plant Cell Reports 21: 437-444[ISI][Medline]
Bilang R. S. Iida A. Peterhans I. Potrykus J. Paszkowski 1991 The 3'-terminal region of the hygromycin-B-resistance gene is important for its activity in Escherichia coli and Nicotiana tabacum. Gene 100: 247-250[CrossRef][ISI][Medline]
Chen L. C. Auh P. Dowling J. Bell F. Chen A. Hopkins R. A. Dixon Z. Y. Wang 2003 Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnology Journal 1: 437-449[CrossRef][ISI][Medline]
Cho M. J. H. W. Choi P. G. Lemaux 2001 Transformed T0 orchardgrass (Dactylis glomerata L.) plants produced from highly regenerative tissues derived from mature seeds. Plant Cell Reports 20: 318-324[CrossRef][ISI]
Cho M. J. C. D. Ha P. G. Lemaux 2000 Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed- derived highly regenerative tissues. Plant Cell Reports 19: 1084-1089[CrossRef][ISI]
Dafni A. D. Firmage 2000 Pollen viability and longevity: practical, ecological and evolutionary implications. Plant Systematics and Evolution 222: 113-132[CrossRef][ISI]
Dalton S. J. A. J. E. Bettany E. Timms P. Morris 1995 The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arundinacea Schreb). Plant Science 108: 63-70[CrossRef][ISI]
Dalton S. J. A. J. E. Bettany E. Timms P. Morris 1998 Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide fibre-mediated transformation of cell suspension cultures. Plant Science 132: 31-43[CrossRef][ISI]
Dalton S. J. A. J. E. Bettany E. Timms P. Morris 1999 Co- transformed, diploid Lolium perenne (Perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (Darnel) plants produced by microprojectile bombardment. Plant Cell Reports 18: 721-726[CrossRef][ISI]
Denchev P. D. D. D. Songstad J. K. McDaniel B. V. Conger 1997 Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojectile bombarded leaf cells. Plant Cell Reports 16: 813-819[CrossRef][ISI]
Fritz S. E. A. J. Lukaszewski 1989 Pollen longevity in wheat, rye and triticale. Plant Breeding 102: 31-34[CrossRef][ISI]
Hauser E. J. P. J. H. Morrison 1964 The cytochemical reduction of nitroblue tetrazolium as an index of pollen viability. American Journal of Botany 51: 748-752[CrossRef][ISI]
Heslop-Harrison J. Y. Heslop-Harrison 1970 Evaluation of pollen viability by enzymatically induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Technology 45: 115-120[ISI][Medline]
Heslop-Harrison J. Y. Heslop-Harrison K. R. Shivanna 1984 The evaluation of pollen quality and a further appraisal of the fluorochromatic (FCR) test procedure. Theoretical and Applied Genetics 67: 367-375[CrossRef][ISI]
Horn M. E. R. D. Shillito B. V. Conger C. T. Harms 1988 Transgenic plants of orchardgrass (Dactylis glomerata L.) from protoplasts. Plant Cell Reports 7: 469-472[CrossRef][ISI]
Hovin A. W. 1980 Cool-season grasses. In W. R. Fehr and H. H. Hadley [eds.], Hybridization of crop plants, 285–298. American Society of Agronomy and Crop Science Society of America, Madison, Wisconsin, USA
Jauhar P. P. 1993 Cytogenetics of the Festuca-Lolium complex: relevance to breeding. Springer-Verlag, Berlin, Germany
Jensen K. B. Y. F. Zhang D. R. Dewey 1990 Mode of pollination of perennial species of the Triticeae in relation to genomically defined genera. Canadian Journal of Plant Science 70: 215-225[ISI]
Kapyla M. 1991 Testing the age and viability of airborne pollen. Grana 29: 430-433
Khatun S. T. J. Flowers 1995 The estimation of pollen viability in rice. Journal of Experimental Botany 46: 151-154
Kuai B. S. J. Dalton A. J. E. Bettany P. Morris 1999 Regeneration of fertile transgenic tall fescue plants with a stable highly expressed foreign gene. Plant Cell Tissue and Organ Culture 58: 149-154[CrossRef][ISI]
Luna V. S. M. J. Figueroa M. B. Baltazar L. R. Gomez R. Townsend J. B. Schoper 2001 Maize pollen longevity and distance isolation requirements for effective pollen control. Crop Science 41: 1551-1557
Norton J. D. 1966 Testing of plum pollen viability with tetrazolium salts. Journal of the American Society for Horticultural Science 89: 132-134
Pacini E. G. G. Franchi M. Lisci M. Nepi 1997 Pollen viability related to type of pollination in six angiosperm species. Annals of Botany 80: 83-87
Richards H. A. V. A. Rudas H. Sun J. K. McDaniel Z. Tomaszewski B. V. Conger 2001 Construction of a GFP-BAR plasmid and its use for switchgrass transformation. Plant Cell Reports 20: 48-54[CrossRef][ISI]
Rodriguez-Riano T. A. Dafni 2000 A new procedure to assess pollen viability. Sexual Plant Reproduction 12: 241-244[CrossRef][ISI]
Shivanna K. R. H. F. Linskens M. Cresti 1991 Pollen viability and pollen vigor. Theoretical and Applied Genetics 81: 38-42[ISI]
Singh M. B. P. M. O'Neill R. B. Knox 1985 Initiation of postmeiotic beta-galactosidase synthesis during microsporogenesis in oilseed rape. Plant Physiology 77: 225-228
Sleper D. A. R. C. Buckner 1995 The fescues. In R. F. Barnes, D. A. Miller, C. J. Nelson, and M. E. Heath [eds.], Forages, 345–356. Iowa State University Press, Ames, Iowa, USA
Sleper D. A. C. P. West 1996 Tall fescue. In L. E. Moser, D. R. Buxton, and M. D. Casler [eds.], Cool-season forage grasses, 471–502. American Society of Agronomy; Crop Science Society of America; Soil Science Society of America, Madison, Wisconsin, USA
Smith R. L. M. F. Grando Y. Y. Li J. C. Seib R. G. Shatters 2002 Transformation of bahiagrass (Paspalum notatum Flugge). Plant Cell Reports 20: 1017-1021[CrossRef][ISI]
Spangenberg G. Z. Y. Wang J. Nagel I. Potrykus 1994 Protoplast culture and generation of transgenic plants in red fescue (Festuca rubra L). Plant Science 97: 83-94[CrossRef][ISI]
Spangenberg G. Z. Y. Wang I. Potrykus 1998 Biotechnology in forage and turf grass improvement. Springer-Verlag, Berlin, Germany
Spangenberg G. Z. Y. Wang X. L. Wu J. Nagel V. A. Iglesias I. Potrykus 1995a Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra) plants from microprojectile bombardment of embryogenic suspension cells. Journal of Plant Physiology 145: 693-701[ISI]
Spangenberg G. Z. Y. Wang X. L. Wu J. Nagel I. Potrykus 1995b Transgenic perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells. Plant Science 108: 209-217[CrossRef][ISI]
Steer M. W. J. M. Steer 1989 Pollen tube tip growth. New Phytologist 111: 323-358[CrossRef][ISI]
Stone J. L. J. D. Thomson A. S. J. Dent 1995 Assessment of pollen viability in hand-pollination experiments: a review. American Journal of Botany 82: 1186-1197[CrossRef][ISI]
Trognitz B. R. 1991 Comparison of different pollen viability assays to evaluate pollen fertility of potato dihaploids. Euphytica 56: 143-148
Tuinstra M. R. J. Wedel 2000 Estimation of pollen viability in grain sorghum. Crop Science 40: 968-970
Wang G. R. H. Binding U. K. Posselt 1997 Fertile transgenic plants from direct gene transfer to protoplasts of Lolium perenne L. and Lolium multiflorum Lam. Journal of Plant Physiology 151: 83-90[ISI]
Wang Z. Y. J. Bell Y. X. Ge D. Lehmann 2003a Inheritance of transgenes in transgenic tall fescue (Festuca arundinacea Schreb). In Vitro Cellular & Developmental Biology Plant 39: 277-282[CrossRef][ISI]
Wang Z. Y. J. Bell M. Scott 2003b A vernalization protocol for obtaining progenies from regenerated and transgenic tall fescue (Festuca arundinacea Schreb.) plants. Plant Breeding 122: 536-538[CrossRef][ISI]
Wang Z. Y. A. Hopkins R. Mian 2001a Forage and turf grass biotechnology. Critical Reviews in Plant Sciences 20: 573-619[CrossRef][ISI]
Wang Z. Y. G. Legris J. Nagel I. Potrykus G. Spangenberg 1994 Cryopreservation of embryogenic cell suspensions in Festuca and Lolium species. Plant Science 103: 93-106[CrossRef][ISI]
Wang Z. Y. G. Legris G. Spangenberg 1995 Establishment of and plant regeneration from embryogenic cell suspensions and their protoplasts in forage grasses. In I. Potrykus and G. Spangenberg [eds.], Gene transfer to plants, 295–304. Springer-Verlag, Berlin, Germany
Wang Z. Y. D. Lehmann J. Bell A. Hopkins 2002 Development of an efficient plant regeneration system for Russian wildrye (Psathyrostachys juncea). Plant Cell Reports 20: 797-801[CrossRef][ISI]
Wang Z. Y. M. Scott J. Bell A. Hopkins D. Lehmann 2003c Field performance of transgenic tall fescue (Festuca arundinacea Schreb.) plants and their progenies. Theoretical and Applied Genetics 107: 406-412[CrossRef][ISI][Medline]
Wang Z. Y. T. Takamizo V. A. Iglesias M. Osusky J. Nagel I. Potrykus G. Spangenberg 1992 Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts. Bio/Technology 10: 691-696[CrossRef][Medline]
Wang Z. Y. M. P. Valles P. Montavon I. Potrykus G. Spangenberg 1993 Fertile plant regeneration from protoplasts of meadow fescue (Festuca pratensis Huds). Plant Cell Reports 12: 95-100
Wang Z. Y. X. D. Ye J. Nagel I. Potrykus G. Spangenberg 2001b Expression of a sulphur-rich sunflower albumin gene in transgenic tall fescue (Festuca arundinacea Schreb.) plants. Plant Cell Reports 20: 213-219[CrossRef][ISI]
Ye X. Z. Y. Wang X. Wu I. Potrykus G. Spangenberg 1997 Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells. Plant Cell Reports 16: 379-384[ISI]
This article has been cited by other articles:
|
|
 |
|
  S. Koti, K. R. Reddy, V. R. Reddy, V. G. Kakani, and D. Zhao Interactive effects of carbon dioxide, temperature, and ultraviolet-B radiation on soybean (Glycine max L.) flower and pollen morphology, pollen production, germination, and tube lengths J. Exp. Bot., February 1, 2005; 56(412): 725 - 736. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
