K.J. Kasha, E. Simion, Q.A. Yao, R. Oro
Dept. of Crop Science, University of Guelph
GUELPH, Ontario, Canada, N1G 2W1
Introduction:
Isolated microspore culture of barley has reached the stage where it can be used extensively for breeding new cultivars and in research. This has become feasible due to the high induction of embryo formation across genotypes and the subsequent high frequency of plant regeneration. These features have been developed and tested across a range of genotypes at Guelph. A similar system has been used exclusively in commercial barley (winter and spring) breeding programs in Denmark for the past 3 years (personal communication). This one company (Pajbiergfonden) produces 100,000 DH per year from ca 150 crosses (objective is 800 DH cross) with good success. This success demonstrates that it is feasible to develop a system that can overcome the genotype effects of haploid production in cereals.
Results in Barley at Guelph
We have observed high induction across 26 barley genotypes at Guelph with the procedures that we have developed for isolated microspore culture. In a few genotypes we have counted the # of embryos induced and % germination while for others we have only observed the massive embryo induction and initial germination (Fig. 1, Click here to view). Data on genotypes where ELS and germination were counted is provided in Table 1. These data are based on plating about 500,000 microspores on a plate on top of a filter paper on solid media. Plating about 150,000 microspores as liquid drops directly on solidified media worked equally well. The filter paper system was developed for use when the microspores are targets for transformation by particle bombardment. Transformation has been achieved by this system but frequencies are low, ca 1 in 107 microspores cultured (Jähne et al., 1994; Yao et al., unpublished).
Table 1: Numbers of embryo-like structures produced and plants regenerated from isolated microspore
| Regeneration %2 | ||||
|---|---|---|---|---|
| Genotypes | #ELS1 | Frequency | Green | Albino |
| A. Winter | ||||
| GBC 777 | 14,133 | 89.3% | 5.5% | 94.5% |
| GBC 778 | 7,667 | 83.9% | 61.0% | 39.0% |
| Igri | 12,380 | 93.3% | 98.4% | 1.6% |
| B. Spring | ||||
| Manley | 11,360 | 74.1% | 79.1% | 20.9% |
| AC Oxbow | 10,810 | 58.2% | 85.2% | 14.8% |
Regeneration from samples of embryos induced ranged from 59 to 93% across genotypes (Table 1). Winter barley genotypes showed good induction and regeneration but some had a high proportion of albino plants. The best 2-row spring malting barley cultivars grown in Canada (Manley, AC Oxbow) showed excellent response and a low level of albinos. We have sampled a range of European cultivars which respond like our Canadian spring barleys with few or no albinos. Spontaneously doubled completely fertile DH's comprised about 70% of the green plants produced.
Procedures for microspore induction and isolation have also been streamlined. We collect and remove the spikes at the mid-to-late uninucleate microspore stage and pretreat the spikes in 0.3M mannitol for 4 days at 4deg.C. Table 2 compares different pretreatment responses. The spikes are then cut into pieces with scissors into a Warring Blender Cup and blended for 6-10 sec. The mash is filtered through cheesecloth and then microspores are collected on a 100 u nylon mesh filter, washed off into centrifuge tubes and the pellet of microspores is collected by low speed centrifugation. Microspores are further washed by suspension in 0.3 M mannitol and recentrifuged. The pellet is again collected, dispersed in culture media and then placed in culture, either by collection on filter paper or as drops on solid media. The induction and regeneration media are completely defined and thus repeatable. A manuscript is being prepared for publication that will provide procedure and media details.
Table 2: Effects of Pretreatments on Isolated Microspore Culture of Barley cv. Igri (values are averages over 3 reps.)
| Pretreatment | Stage After Pretreat | # Embryos (ELS) | % Regeneration |
|---|---|---|---|
| Spikes 4deg. for 28d | uninucleate | 13,136 | 95.4% |
| Spikes in 0.3M mann. 4deg. for 4d. | uninucleate | 12,380 | 97.2% |
| Anthers, 0.3 M mann. 25deg. for 4d. | binucleate or fused uninucleate | 13,504 | 88.5% |
One barley spike will give about 70-100,000 good quality microspores. If more than 50% are dead, the live microspores are rescued on a 20% maltose gradient centrifugation prior to culture. Since thousands of plants can be recovered per plate inoculated with 500,000 microspores, it takes only a small number of spikes (7-8) per plate. The mass of embryos formed are spread by spatula over plates of regeneration media. Regenerating green plants are transferred to Magenta boxes and later to soil.
It might be noted that haploid production at the Danish breeding Co. is carried out by 1 scientist and 1 technician. Plants produced are put directly into the field and undergo some selection.
Besides the genotypes listed in Table 1, we have tested our procedures on another 21 cultivars, including Trinity, Lina, Cooper, OAC Elmira, Bruce, Morex, Excel, Hana, Disa, and Harrington, with induction similar to Igri and Manley (Fig. 2). The European cultivars regenerated essentially 100% green plants while some of the Canadian winter barleys give higher albino frequencies.
References:
Jähne A., D. Becker, R. Brettschneider, H. Lorz, 1994. Generation of transgenic, microspore-derived, fertile barley. Theo. Appl. Genet 89:525-533.
Yao, Q.A., E. Simion, M. Williams, J. Krochko and K.J. Kasha. Biolistic transformation of haploid isolated microspores of barley (Hordeum vnlgare L.) (Submitted for publication).