SUMMARY

Potential gene flow between genetically engineered (glufosinate herbicide resistant) canola and related weed species (field mustard (Brassica rapa L.), wild mustard (B. kaber (DC) L.C. Wheeler), and black mustard (B. nigra (L) W.J.D. Koch)) was examined by considering gene flow by seed and pollen movement along with cross compatibility, under glasshouse and field condition. Canola seeds are small and can be transported large distances by machinery, animals and through normal transportation. Cross pollination occurred between plants separated by 30 m. Therefore canola pollen can move at least this distance. Rate of embryo and endosperm development in canola is very similar to field mustard but differed markedly from wild mustard and black mustard. Greenhouse crosses between canola x wild mustard and canola x black mustard had high abortion rates. Crosses between canola and field mustard had a high proportion of normally developing embryos. Field studies showed that canola and field mustard will hybridize at relatively high frequency and almost all hybrids found have produced viable seed. Under field conditions, no hybridization was found between wild mustard and canola and between black mustard and canola.

INTRODUCTION

Lack of knowledge about the potential risks that may arise from large-scale commercialization of transgenic crops has fuelled a high degree of controversy. Some researchers feel that the risk associated by the release of transgenic crops is no greater than exists from crops arising from traditional breeding. Others, however, believe that release of transgenic crops, especially those containing genes from unrelated organisms, could have hazardous effects on agriculture and the environment because undesirable viruses, bacteria and weedy plant types may arise and be difficult to control.

Most researchers do, however, agree that the release of transgenes pose greater or lesser environmental risk according to the transformed trait. The most common type of plant transformation is herbicide resistance. The reasons for this are that the physiological basis for herbicide resistance is well characterized, resistance often is controlled by a single dominant gene and the transformed gene itself can be used as a selectable marker in the transformation process. In addition, many researchers feel herbicide resistance can be a desirable feature in an agricultural crop.

Canola crops can suffer severe yield loss due to weed infestation and since Brassica species can be readily transformed, and specific genes have been identified for herbicide resistance, it is no surprise that genetic engineers have targeted herbicide resistance in canola.

A major concern of introducing transgenic herbicide resistant crops into agriculture is the spread of the engineered gene(s), particularly by pollen, to related weed species. In addition, herbicide resistant canola plants could flourish as volunteer weeds on neighboring farms. The risk of transgenic characters being transferred from crops to weedy species will be related to: (1) seed movement; (2) pollen movement; (3) compatibility between crop plants and weeds; and (4) reproductive fitness of successful hybrid cross combinations. This paper examines factors (1), (2) and (3), above.

MATERIALS AND METHODS

Transgenic canola used in this study had been genetically engineered to confer resistance to glufosinate herbicide. Weed species used in this investigation included field mustard, wild mustard, and black mustard. All three weed species are closely related to canola and are serious weeds throughout the Pacific Northwest region of the U.S.A.

Pollen movement was monitored in three studies (1993, 1994 and 1995). In 1993, an area (20 m x 20 m) of glufosinate resistant canola was surrounded by an 8 m border of susceptible canola. At harvest the susceptible border was sampled in four directions (north, south, east and west) every 1.5 m from the herbicide resistant center. The following two years a plot of transgenic glufosinate resistant canola (20 m x 20 m) was surrounded by a 30 m wide border of susceptible canola. At maturity, seeds were collected from the susceptible border every 1.5 m along sixteen 26 m long rays spaced 22.5 apart (i.e., a Nelder wheel design).

Samples from all years were screened for herbicide resistance. Four samples, each of 200 seeds, were sown from each sample position. When seedlings reached a 3-5 leaf stage they were sprayed with glufosinate at 0.42 kg ai/ha. Surviving plants were sprayed a second time to avoid errors caused by escapes. Plants surviving the second spray were counted and assumed to have resulted from cross pollination with plants from the herbicide resistant center area.

Potential hybridization between canola, field mustard, wild mustard and black mustard were examined in hybridization studies carried out in the glasshouse. Two transgenic herbicide resistant canola genotypes were crossed in a complete diallel design along with three related weed species (field mustard, wild mustard and black mustard), where each genotype is hybridized to all other genotypes in the design, including selfs making a total of 6 x 6 = 36 possible cross combinations. Parent plants were grown in a glasshouse with mercury lights used to supplement natural lighting and provide a 16 hour day-length. Daytime temperatures were around 21 ± 5C and night temperatures were 14 ± 5C.

Two days after pollination, 10 siliques were taken at random to examine pollen germination on the stigma and pollen tube development down the style towards the ovary. The method used was similar to that of Martin (1959). Pollinated styles were fixed in absolute alcohol:glacial acetic acid (3:1) 48 hours after pollination and stored at 4C until examined. The fixative was replaced with 1N sodium hydroxide (NaOH) and the styles were left at room temperature. Hydrolysis was completed with a change of 1N NaOH and a further half hour at 60C. After the hydrolysed styles were rinsed with water and stained with a methyl blue solution [0.2% methyl blue + 2.0% K₃PO₄ (w/v)] they were observed for fluorescence using wavelength of light in the range 350-400 nm. The styles were examined for pollen germination and pollen tube growth in the style and ovary.

After pollination, developing siliques were harvested at 4 day intervals between 4 and 32 days after pollination and fixed in absolute alcohol:glacial acetic acid (3:1) and stored at 4C until examination. The ovary was opened and any developing ovules were removed. Individual ovules were opened and a few drops of HCl-carmine (Snow, 1963) placed inside. After a few minutes, excess stain was rinsed away with 70% ethanol. The ethanol was removed using a piece of filter paper and replaced with Rattenburys' Fluid (45% acetic acid:glycerine, 10:1). The endosperm and embryo were teased from the ovular tissue, which was then discarded. The prepared material was examined using a Jenval transmitted light research microscope. The developmental stage of the embryo and endosperm and any abnormalities present were noted.

To examine hybridization of transgenic canola with related weeds under field conditions, glufosinate resistant canola was planted in a 1:1 mixture with the three weed species in 1m x 5m plots arranged as a randomized complete block design with four replications. Seeding rate of each species was adjusted prior to planting to account for any differences in germination. Weed plants were harvested and threshed separately from the canola mixtures. Weed seeds were grown in the greenhouse to the 3-5 leaf stage and sprayed as before to detect herbicide resistant plants.

RESULTS

Seed movement. Canola plants have small seed (approximately 200 seeds/g). During normal farm operations the seed will inevitably be lodged in farm machinery and transported around the farm and surrounding area. Seed also can be distributed by animals and birds, and seed can be lost while being transported for processing. In the Pacific Northwest region of the U.S.A., spring canola has only recently been grown commercially and already volunteer plants have been observed several kilometers from where they originated.

Pollen movement. In 1993, pollen movement, as detected by percentage cross pollination between herbicide resistant plants and the non-resistant border, decreased with increased distance from the herbicide resistant pollen source. When grown adjacent to each other, there was 6.3% cross pollination between plants. However, cross pollination was 0.5% (1:200 seeds) when plants were separated by 7.5 m.

The 1994 growing season was hotter and dryer than 1993 and this greatly reduced the time that plants remained in flower, pollen load per flower and the number of insects observed in the experimental plots. Fewer cross pollinations compared were observed compared to wetter and cooler conditions of the previous season. Again hybridization was shown to decrease rapidly with increasing distance from the transgenic canola. Beyond 5 m from the pollen source the frequency of cross pollination was about 1:1000. A small proportion of herbicide resistant plants were found at the furthest distance from the pollen source examined (i.e. up to 26 meters).

Seed collected from the 1995 study had not been fully tested at the time of writing. However, initial observations and analyses indicate that a similar pattern of cross pollination to that observed in 1994 is highly likely with the proportion of cross pollination at greater distance being higher. Greatest frequency of cross pollination occurred down wind of the pollen source.

Compatibility. In all hybrid combinations examined, pollen germinated on the stigma, penetrated the style and showed some attraction to the ovary.

Rate of embryo development against time for canola was not significantly different (0.65) from field mustard (Table 1). Canola embryo development was significantly slower (P < 0.01) than wild mustard and significantly faster (P < 0.01) compared to black mustard. Similarly, endosperm development of canola against time was not significantly different from field mustard or black mustard but significantly (P < 0.05) slower than wild mustard.

Percentage of developing embryos was always highest in the self crosses (Table 2). Selfed pollinated canola showed healthy 93% embryo development while field mustard, wild mustard and black mustard had 79%, 91% and 57%, respectively. Percentage embryo development in hybrid crosses was low in crosses between canola and either wild or black mustard. Embryo development occurred at high frequency (74% and 81%) in crosses between canola and field mustard. The majority of embryos failed to develop due to abortion rather than non-fertilization (Table 2). Canola x wild mustard crosses had an average of 74% abortion and between canola and black mustard an average of 59% embryos aborted. Crosses between canola and field mustard showed very little abortion, only slightly higher than canola selfed.

Seed collected from field weed plants grown in resistant canola/weed mixtures, were screened for herbicide resistance. Over both locations of testing in 1994, 53,560 field mustard seedings, 340,896 wild mustard seedlings, and 40,572 black mustard seedlings were screened for glufosinate resistance. No herbicide resistant hybrid seedlings were observed between wild mustard and canola or between black mustard and canola. However, at least 18 hybrid plants were obtained between canola and field mustard, approximately one hybrid for every 3000 seedlings examined. Each canola x field mustard hybrid produced some viable seed and a small proportion (approximately 2%) of hybrids produced seed quantities similar to, or exceeding the original weed parent. These hybrids, and their progeny, are presently being investigated in more detailed breeding and cytological studies.

CONCLUSION

Initial indications from these studies are: (1) canola seed can be readily transported throughout a region, and therefore, there is a risk that these crops will become volunteer weeds; (2) canola pollen can move at least 26 meters and movement is affected by wind direction; (3) canola and field mustard have similar embryo and endosperm development rates and hybrids occur between the two species with relatively high frequency, and (4) canola and at least one related weed will combine, under field conditions, to produce hybrid plants.

ACKNOWLEDGEMENTS

We would like to thank Plant Genetic Systems of Belgium for providing the herbicide resistant lines of canola and permitting their use in this research.

REFERENCES

Brown, J., D.C. Thill, A.P. Brown, T.A. Brammer and H. Nair. 1996. Gene transfer between canola (Brassica napus) and related weed species. In: Proceedings of the 8^th Symposium on Environmental Releases of Biotechnology Products: Risk Assessment Methods and Research Progress, Ottawa, Canada, June 1996. pp.

Martin F.W. 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Tech. 34:125-128.

Snow, R. 1963. Alcoholic hydrochloric acid-carmine as a stain for chromosomes in squash preparations. Stain Tech. 38:9-13.

Table 1. Rate of embryo and endosperm development against time for canola and three weedy species

	Canola	Field mustard	Wild mustard	Black mustard
Embryo	0.65	0.60	0.86	0.47
Endosperm	0.16	0.19	0.24	0.17

Table 2. Percentage embryos developed in crosses between canola and three weedy species. Percentage aborted embryos are presented in parenthesis.

	Canola	Field mustard	Wild mustard	Black mustard
Canola	93 (2)	81 (4)	0 (82)	5 (74)
Field mustard	74 (9)	79 (1)	0 (0)	22 (66)
Wild mustard	8 (65)	0 (50)	91 (4)	52 (14)
Black mustard	13 (44)	0 (0)	24 (27)	57 (0)