The development of transgenic crops creates new possibilities for the improvement of quality, yield, and disease resistance in grains. There are however potential dangers in releasing such crops. Gene flow from transgenic crops to other commercially produced crops are undesirable and should be avoided at all costs. In this study the crossability between various grains will be determined. Plants will be hybridized by emasculating and then manually pollinating flowers. The following plant material will be used: Triticale (US2007, AS2009), rye (US3010, Duiker) and common wheat (US1010, SST88).Control as well as reciprocal crosses will be performed. Embryo rescue will then be performed to determine whether the seed from the cross is viable. In previous studies it has been found that the crossability between triticale and rye is low. The crossability between wheat and triticale however depends on which is used as the pollen parent. A field study will also be performed to asses long distance pollination. Not many studies have done on long distance pollination, but it would seem that pollination decreases with increasing distance from the pollen source. The final outcome of the study will be to publish the results. This may then serve as a basis when assessing a transgenic cultivar for release.
The improvement of wheat is of great importance since is the third most produced cereal in the world. The creation of hybrids is thus of great importance as it has greater potential of increasing yield than traditional breeding methods. Traditional breeding methods often only increase yield by less than 1% and this is why the production of hybrids should be explored to increase yield as well as other advantageous traits (Mahajan et al. 1998). In the late 1970's a hybrid cultivar was produces that increased the yield by 10-15% but was not widely accepted due to some limitations (Mahajan et al. 1997). This however proves that high yielding hybrids can be produced and should therefore be explored.
Improvement of wheat also requires enlargement of the gene pool that is available for breeding (Heslop-Harrison et al 1982). This will enable the breeder to increase yield, disease resistance as well as other agronomic important qualities. This is can also be obtained through hybridization.
Heterosis, also known as hybrid vigor, describes increased vigor, resistance to disease, yield and other characteristics in hybrids caused by the manifestation of heterozygosity. The result is thus plants that are superior to their parents. Certain parental combinations do not lead to hybrid vigor, while others do not perform as well as either parent (Briggle et al 1963). Hybrid vigor is therefore only available with specific parent combinations (Briggle et al 1963). Simple heterosis has been demonstrated in winter wheat (Pickett et al 1993).
Inbreeding depression is the result of inbreeding related individuals. This is caused by more recessive deleterious traits manifesting in the offspring. Offspring have more homozygous deleterious genes; the more closely related the parents are. This leads to unfit plants. Thus introducing genes from a different gene pool may reverse inbreeding depression.
Probably one of the most famous wheat-rye hybrids is Triticale. Breeders wanted to include certain traits of wheat such as productivity, grain quality and disease resistance with the vigor and hardness of rye (Oelke et al 1989). Wheat was used as the female parent while rye was used as the male parent (Oelke et al 1989). Triticale hybrids are sterile, but treatment with colchicine makes it fertile and thus able to reproduce (Later et al 2009). Commercial triticale is almost always a second generation hybrid (cross between two types of triticale).
As seen above the production of hybrids is of great importance. There are however also risks associated with gene flow from new transgenic crops to more conventional crops.
In the modern breeding world transgenic plants are also becoming of great importance as it holds unique possibilities in improving certain characteristics. Transgenic plants posses single or multiple genes transferred from one species to another. Transgenic plants are produced by using recombinant DNA technology where DNA from one species can be integrated into another plants genome by natural processes.
These new transgenic lines however need to be evaluated before it can be released. A risk assessment needs to be carried out to determine the potential risks there are of these lines outcrossing with conventional cultivars. Gene flow to related species should also be taken into account. This is why the crossability between species are of such great importance. If the plants are able to cross it may cause undesirable effects in non target plants.
Not many transgenic plants become invasive, but for management purposes it is necessary to identify the modifications that may lead to invasiveness.
Crossability refers to the ability of different species or cultivars to cross with each other. There is however various barriers to crossability with the large diversity of plant alone testify to this fact (Solbrig et al 1970). Most of these barriers are only partial that depends on physical separation (time, distance, etc.) (Bates et al 1973). All of these can however be manipulated by man. Absolute barriers cannot be controlled by man; these include hybrid breakdown and incompatibility between gametes (Bates et al 1973).
There has also been work done on the existence of so-called crossability genes (Jalani et al 1980). There has been found that it affects the hybridization of wheat with rye (rye as the pollen parent) (Jalani et al 1980). These genes modify the amount of pollen tubes that successfully move through the transmitting tract (Jalani et al 1980). Low crossability is due to rapid and complete disturbance of pollen tube growth (Jalani et al 1980).
There are two types of incompatibility between gametes. The first and most studied of the two is self-incompatibility that prevents inbreeding (Bates et al 1973). The other is cross-incompatibility and is the opposite of self-incompatibility. It prevents hybridization and promotes specialization (Bates et al 1973). When fertilization takes place gametic incompatibility ceases (Bates et al 1973).
There are limitations in the direct pollination from wheat as it is more suited for self-pollination. Possibility for wind-pollination does exist due to variability in floral structure, but the rate of gene floe is usually less than 1% (Rieger et al 2002). Receptivity of stigmas, viability of pollen and the availability of pollen during the period when female organs are receptive, are all factors that will determine whether gene flow from wheat will be successful (Johnson et al 1968). These factors are also influenced by the environment and genotype (de Vries et al 1974). From previous research, cultivars with a higher gene flow rate tend to posses low male fertility (Hucl et al 2001). Another factor to take into account is the cytoplasmic background as well as floral structures (Mahajan et al 1998). Both of these factors influence seed set after cross-pollination of self-pollinating crops
In previous studies it was found that the crossability between wheat and triticale ranged from 1.6 - 18.2% when triticale was the female parent and wheat the male parent (Vishwakarma et al 1985). It was found that seed set was low, but germination was good (Khanna et al 1990). However, when triticale was used as the male parent and wheat as the female parent seed set was good, but none of the seed germinate (Khanna et al 1990, Jouve et al 1984). The low germination of the seeds was due to embryo-less seeds (Khanna et al 1990). The low crossability is due to poor germination of pollen and retarded growth of the pollen tube. In some cases pollen tube growth is completely inhibited (Khanna et al 1990).
In a recent study (2007), carried out by L.M. Hall and colleagues, the crossability between triticale with various grains (common wheat, durum wheat and rye) where determined. They found that that outcrossing was higher when triticale was the male parent (>73%) and wheat the female parent. When triticale was used as the female parent in crosses with wheat the outcrossing was less than 23%. The emergence of F1 seed from crosses between wheat (female) and triticale (male) was only 1%. Even though outcrossing between wheat and triticale was high, only a few seeds emerged and they were not viable. Viable seeds were however produced when triticale acted as the female parent (Hall et al 2007).
The outcrossing between triticale and rye seemed to be cultivars specific. However, outcrossing was low for crosses made in both directions. Therefore the potential for rye crossing with triticale is very low. Even if all factors are favorable for crossing to takes place, all the seeds that emerge will be infertile. Therefore if introgression should occur the F1 seeds are sterile and thus prevents gene flow (Hall et al 2007).
In general outcrossing between these grains was lower than outcrossing obtained from crossing various triticales with each other. It should however be kept in mind that genetic as well as environmental factors influence outcrossing (Hall et al 2007).
The genetic control of crossability between wheat and triticale has also been studied. There are two recessive genes that control high crossability, kr1 and kr2 (Vishwakarma et al 1985). The dominant gene Kr1 however reduces crossability to a larger extent than Kr2 (Riley et al 1966). These dominant alleles of these genes lower crossability by inhibition of pollen tube growth and thus preventing fertilization (Vishwakarma et al 1985). The genes, Kr1 and Kr2, have been located on chromosome 5B and 5A respectively (Riley et al 1966).
Previous research has not focused on long distance (>300m) gene flow. Studies have only been carried out for distances shorter than 48m (Matus-Cádiz et al 2004). Many studies have shown that seed set can occur 5-48m from a pollen source (Miller et al 1975). There are however reports that pollen can travel as far as 60m (Jensen et al 1968). The amount of pollen usually decreases with increasing distance from the pollinator. Pollen loads are highest within 3-8m from the pollinator (Jensen et al 1968). Gene flow rates of up to 0.003-0.009% have been reported between two fertile common wheat lines. This gene flow rate was found at a distance of 27m (Hucl et al 2001). Studies by Matus-Cádiz have indicated that gene flow does not take place beyond distances of 300m (Matus-Cádiz et al 2004).
Rationale and Motivation
Crossing of closely related relatives creates another means for improving genetic variability of a gene pool. Improvement of the gene pool relies on the provision of new genes from different, but in this case, related species (Heslop-Harrison et al 1982). Improvement through breeding is dependent on genetic variability and thus cannot take place without it (Heslop-Harrison et al 1982).
Gene flow however is not always desirable. In the case of novel bioproducts the flow of genes from one species to the other should be prevented, especially if we do not know what effect it could have on other commercially produced crops. Gene flow to wild species should also be avoided as it could have a negative effect on the environment.
Genetic modification of wheat and other grains are of great importance. It opens the door to great possibilities in the improvement of quality, yield, disease resistance and other economical important traits. There are however concerns to what effect it may have on the environment as well as other commercially cultivated crops if crosses should occur.
Therefore in this study the crossability between various grains will be determined. This will enable us to determine the chance of crossing and production of viable seeds between novel bioproducts and other closely related species.
Research Aims and Objectives:
In this study various crosses will be carried out in order to asses if stable hybrids are produced in an effort to determine if successful flow of genes takes place from the crossed lines. Crosses will be carried out between all the relevant cultivars as well as reciprocal crosses.
Various tissue culture techniques, such as embryo rescue (hybrid rescue), will be performed to determine whether the seed formed from the crosses are viable. Many crosses fail because the endosperm of the seeds degenerates. This will indicate whether the plants will be able to reproduce and if seeds are produced as a result of crossing. Hybrid sterility will therefore be positive result as this will prevent the plants from reproducing.
Larger field studies are also necessary to determine the extent of pollen-mediated gene flow. A large pollinator block surrounded by the recipient plants are used to determine the gene flow that exists within increasing distance from the pollinator block. Transgenic plants that contain marker genes are commonly used in such experimental field trails. In this project the wheat line Amethyst will be used as it has distinctive purple grains.
If no crossing takes place, because fertilization was unable or because the seed produced is not viable, gene flow will be restricted. If this is the case genetically modified plants pose no threat to the environment or other closely related cultivated species.
Materials and Methods:
The following cultivars will be used in this study: Triticale (US2007, AS2009), rye (US3010, Duiker) and common wheat (US1010, SST88). All of these lines were released by (Stellenbosch University Plant Breeding) with the exception of SST88.
The plants will be grown in a greenhouse in small pots in a peat soil mix. Plant will be planted each week for about 8 - 10 weeks. When plants are ready to be emasculated planting will cease temporarily and as soon as plants are thrown out planting will recommence. Plants will be thrown out after all data from the cross has been collected.
Initially six plants will be planted in each pot and once the plants are strong enough they will be reduced to only four. The temperature in the greenhouse is between 21 and 28°C. Natural light provides the plants with the appropriate photoperiod for growth and plants will be watered daily and fertilized with liquid fertilizer.
Emasculation of flowers will take place when plants are 8 weeks old or just prior to anthesis. This is done by clipping of the top of the floret and using forceps, remove the anthers. When emasculating flowers, the central florets are removed, as well as poorly developed apical and basal spikelets. Uncontrolled pollination will be prevented by covering the emasculated spike with a brown paper envelope.
After emasculation flowers are given 4-6 days to become receptive and the flowers begin to open. For flowers to become fully open, tension must be relieved by removing all scar tissue caused by emasculation. Pollination of each cultivars in the study will be carried out with fresh pollen (must be used within half an hour of collection). Reciprocal crosses of the same amount are also carried out. The controls will consist of emasculating the flowers of a specific line and then continue to pollinate it with pollen from the same line. All four plants will be used in the crosses and will serve as replications of each cross. The crosses that will be carried out are indicated in table 1.
Table 1: Crosses that will be carried out as well as reciprocal crosses
After pollination the spikes are again covered with the brown paper envelope. On the envelope must be written the cross that is made and also the date of the cross. Before embryo rescue will be performed the seed set per spike and percentage seed set will be determined by using the number of seeds and florets.
Thus: % seed set = (number of flowers pollinated per spike) / (number of seed set per spike)
After 18-21 days embryo rescue is performed. Seeds will be sterilized in 70% EtOH for 30 seconds and in 30% jik (containing a few drops of Tween) for 8-10 minutes. The embryo dissected under a stereomicroscope and placed on a growth medium (Dr. G. Daniel's Wheat Embryo Regeneration Medium). Bottles containing embryos are placed in the dark for a week and then in a growth cabinet at 23-25°C with a 14 hour light / 10 hour dark cycle. (Refer to addendum for contents of the growth medium).
Any possibility for crossing of these lines and the production of viable seed has to be considered. This is why embryo rescue is performed. If all factors and environmental conditions are perfect for the growth of the offspring of the crosses, embryo rescue will indicate whether growth of these seeds will be possible in the field. If the rescued embryos do grow to become strong plants it does not necessarily mean that this is the case in the field. This only gives an indication of the possibility of growth under optimal conditions. It should however be kept in mind that optimum conditions are rarely achieved.
It is important to note the amount of flowers pollinated, amount set seed, ratio green : white seed, seeds with embryos and amount of embryos that grow. After adequate growth plants may be planted in pots and placed in the green house.
A 400m x 400m field trail will be sown on Welgevallen farm. A central pollinator block (50m x 50m) will be planted which consisted of Amethyst wheat and is surrounded by () in all directions to a distance of 175m. Amethyst is a wheat line with anthocyanin pigmentation, giving it a distinctive purple color. This purple color will act as a phenotypic marker, making it possible to determine whether the cross was successful. The following data will be collected: height, days to heading and days of flowering. This data will consist of the average of data collected at five random positions within the block. Climate data will also need to be collected to determine if the weather plays a significant role during pollination.
When maturity is reached 0.5 - 4m strips will be harvested and bagged. Grain will be harvested at increasing distance from the center pollinating block, 0.2, 1, 5, 10, 20, 60, 80, 100, 120, 140 and 160m, and in 8 directions (N, E, S, W, NE, SE, SW, and NW). Figure 1 illustrates the manner in which the grain will be harvested.
Samples in which cross pollination occurs will be identified by the expression of the purple pigment in the F1 seed. Seeds that are suspected of containing the purple pigment are kept separate from the remaining seed. Discolored seeds can often be confused for purple seeds because of disease or withering. These seeds are then grown in the green house. When maturity is reached it will be possible to determine if these seeds do indeed contain the purple pigment.
Seed are then surface sterilized for 8min with a solution of 2.5% sodium hypoclorite and 0.1% Tween20. Seeds are then rinsed for 5min with water and then with 70% alcohol. Seeds are then allowed to air dry and placed in a petri dish. Germination takes place at 15°C in the dark and will take about 10 days. The pre-germinated seeds are then planted into pots and placed in a greenhouse.
The amount of putative seeds need to counted and the amount of putative seeds that can produce established plants also needs to be determined (as described above).
All of the data collected from the greenhouse and field studies will be processed and this will give an indication of the crossability between these different grains.
Research Outputs and Potential Outcomes
When the study is completed the main outcome will be to publish the results in order to make it available for other researchers in similar fields of study. In this way the results can be shared and be used in similar studies. It will therefore serve to widen the field of reference when further studies are carried out.
Ultimately it may be used as a basis when assessing transgenic crops for released. Before a transgenic crop may be released as a commercial line, it must first be determined whether gene flow can take place from the transgenic line. The results of this study will give a preliminary answer to this question and serve as a stepping stone for further tests.
Weet nog steeds nie regtig wat om hier te sê nie!!
Dr. G. Daniel's Wheat Embryo Regeneration Medium
FeNa2EDTA stock 10ml
Micro Stock I 10ml
Micro Stock II 1ml
Thiamine HCl Stock 0.1ml
pH (KOH/HCl) 5.8 - 5.9
Micro Stock I in 500ml dH2O
Micro Stock II in 500ml dH2O
Thiamine Stock in 20ml dH2O
Thaimine HCl 80mg
FeNa2EDTA Stock in 200ml dH2O
Bates, L.S., Deyoe, C.W., 1973. Wide Hybridization and Cereal Improvement. Economic Botany, Vol. 27, No. 4 pp. 401-412
Briggle, L.W., 1963. Heterosis in wheat - a review. Crop Sci., 3: 407-411
de Vries, A.P. 1974. Some aspects of cross pollination in wheat (Triticum aestivum L): 4. Seed set on male sterile plants as influenced by distance from pollen source, pollinator: Male sterile ratio, and width of the male sterile strip. Euphytica 23:601-622.
Hall, L.M., Hills, M.J., Eudes, F., Messenger, D.F., Graf, R.J., Beres, B.L., 2007. Evaluation of crossability between triticale (X Triticosecale Wittmack) and common wheat, durum wheat and rye. Environ. Biosafety Res. 6, 249-257
Heslop-Harrison, J., 1982. Pollen-Stigma Interaction and Cross-Incompatibility in the Grasses. Science, New Series, Vol. 215, No. 4538 pp. 1358-1364
Hucl, P., and M.A. Matus-Cádiz. 2001. Isolation distances for minimizing out-crossing in spring wheat. Crop Sci. 41:1348-1351.
Jalani, B.S., Moss, J.P., 1980. Euphytica 29, 571
Jensen, N.F., 1968. Results of a survey on isolation requirements for wheat. Wheat Newsletter 15:26-28.
Jouve, N., Bernardo, A., Soler, C., 1984. Hybrid 6X triticale x Triticum turgidum L. And the obtention of its F2 and BC1 progenies. Cer. Res. Comm. 12 : 223-228
Khanna, V.K., 1990. Germination, pollen fertility and crossability between triticale and wheat and reversion patterns in early segregating generations. Cereal Research Communications, Vol. 18, No. 4, p359-362
Larter, E.N., 2009, "Triticale". Agriculture. The Canadian Encyclopedia. http://www.thecanadianencyclopedia.com/index.cfm?PgNm=TCE&Params=A1ARTA0008131. Retrieved 2009-06-19.
Mahajan, V., Nagarajan, S., 1998. Oppertunities in hybrid wheat - a review. PINSA B64 No.1 pp. 55-58
Matus-Cadiz, M.A., Hucl, P., Horak, M.H., Blomquist, L.K., 2004. Gene Flow in Wheat at the Field Scale. Published in Crop Sci. 44:718-727
Oelke, E.A., Oplinger, E.S., Brinkman, M.A.,1989. Triticale. Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108.
Pickett, A.A., 1993. Hybrid wheat: results and problems. Adv. Plant Breed., Suppl. J. Plant Breed., 15: 1-259.
Rieger, M.A., Lamond, M., Preston, C., Powles, S.B., Roush R.T., 2002. Pollen-mediated movement of herbicide resistance between commercial canola fields. Science 296:2386-2388.
Solbrig, 0.T., 1970. Principles and Methods of Plant Biosystematics (The Macmillan Co., Collier-Macmillan Canada, Ltd., Toronto, Ontario), pp. 108-109. 69.
Vishwakarma, S.R., Mani, S.C., 1985. Crossability between Triticale x Wheat and reversion patterns in early segregating generations. Current Science, Vol.54, No.1, p42-43