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Colchicine-doubling of germinating seedlings of interspecific wildrye hybrids

T.A. Jones, D.C. Nielson and H. Jaussi
USDA-ARS and Utah State University, Logan, Utah 84322-6300, US
Lethbridge, Alberta, Canada X Acc:641 beardless wildrye of Colchicine has usually been applied to sterile clones of interspecific Jamieson, Oregon, USA), its reciprocal (L4PX-3R), L4PX-5 grass hybrids to restore fertility. However, when hybrids are partially (‘Trailhead’ Basin wildrye of Roundup, Montana, USA X Acc:641 fertile, colchicine can be applied to germinating seedlings. Four beardless wildrye), and its reciprocal (L4PX-5R), using a modified populations of Basin wildrye (Leymus cinereus) X beardless wildrye ryegrass (Lolium spp.) procedure (Arnold J.P. vanWijk of D.J. van (L. triticoides) were treated with a 0.2% colchicine solution to double der Have B.V. [Rilland, The Netherlands], pers. comm.). Seedlings chromosome number from 4x=28 to 8x=56. Doubling percentage with radicles just emerging were placed into a 5-cm petri dish lined was 28, 33, 42, and 44% of all root-tips examined for the four with a single disk of Whatman #2 filter paper soaked with 0.3 ml of populations. When plants without doubled sectors were discarded, 0.2% colchicine solution. Seedlings were arranged to ensure radicle doubling percentage increased to 56, 68, 78, and 78%. Plants with contact with the filter paper. After 2 h in the dark at 30°C, seedlings doubled sectors have been placed in crossing blocks to generate were gently rinsed twice with water and plated on moist blotter paper octoploid progeny. Octoploids identified through root-tip chromosome counts will be intercrossed to generate 8x populations.
Three root-tips were harvested from surviving seedlings at least 3 KEYWORDS
months old for determination of somatic chromosome number. Root- Chromosome-doubling, colchicine, interspecific hybridization, tips were placed on Whatman #2 filter paper soaked in colchicine solution (50 ml deionized water, 50 mg colchicine, 25 mghydroquinoline 10 ml dimelthylsulfoxide) and kept in the dark at a INTRODUCTION
room temperature for 3 h. Root-tips were transferred to a vial Basin wildrye (Leymus cinereus (Scribn. & Merr.) A. Löve) is a large- containing aceto-orcein (100 ml 45% acetic acid, 2 g certified orcein statured, grazing-susceptible bunchgrass that occurs at the tetraploid stain), incubated for 2 d at 4°C, and squashed for chromosome counts.
(2n=28) and octoploid (2n=56) levels. Beardless wildrye (L.
(Buckl.) Pilger) is a medium-statured, grazing tolerant, RESULTS AND DISCUSSION
rhizomatous grass that occurs only at the tetraploid (2n=28) level.
High rates of chromosome doubling were achieved for each of the Combining the good seed production and low seed dormancy of Basin four populations (Table 1). Averaged across the four populations, wildrye with the grazing tolerance, rhizomatous habit, and salinity over one-half of the plants had at least one of three examined root- tolerance of beardless wildrye is the goal of our breeding effort.
tips doubled from 28 to 56 chromosomes. Nearly one-quarter of the Hybrids occur naturally where the ranges of the two species overlap.
plants had all of three examined root-tips doubled. After discarding Dewey (1970) reported that this hybrid exhibited 13.86 of a possible undoubled plants, percentage of doubled root-tips for the four 14 bivalents and more than 10% stainable pollen, but no seed was populations ranged from 56 to 78% (mean=70%).
set. Using different parent populations, we have produced sufficienthybrid seed for large-scale colchicine treatment.
This method (Fig. 1) is an adoption of Lawrence's et al., (1990)approach for partially fertile interspecific hybrids instead of fertile Colchicine treatment is the most widely used method for chromosome material of nonhybrid origin. While treatment of germinating seeds doubling in crop plants. When sterile interspecific hybrid genotypes produces chimeras, as does the treatment of clonal tillers and buds, must be doubled, clonal tillers or buds are treated. Fertile doubled the proportion of the plant that is doubled is increased. Furthermore, sectors may be distinguished and separated from sterile undoubled because of the high frequency of success, nurseries can be established material. For production of autoploids from fertile populations, to produce sectorless seeds, many of which are chromosome doubled.
treatment of germinating seed in colchicine solution is the most Because the seed treatment procedure requires quantities of seed, it widely used method because of its simplicity (Speckmann, 1975).
is not feasible for use with sterile interspecific hybrids. However, While this method has been used in a variety of forage crops, where sterility is only partial, seed treatment may be more successful 'Tetrascan' Russian wildrye (Psathrostachys junceus (Fisch.) Nevski) than treating vegetative material because doubled and undoubled is the only perennial Triticeae cultivar developed using this method sectors are difficult to distinguish and laborious to separate.
(Lawrence et al., 1990). Nitrous oxide has also been used to doublechromosome number in Russian wildrye (Berdahl and Barker, 1991).
Our doubled hybrid Leymus populations may be used for four The principal advantage of nitrous oxide is reduced chimera frequency. However, because the treatment is performed in a Use directly as octoploid breeding populations. Fertility may pressurized tank, the number of plants which can be treated is limited.
be increased if cryptic structural hybridity (Stebbins, 1971) is This discourages generation of large populations desirable for plant responsible for partial sterility of the tetraploid interspecific breeding (Speckmann, 1975). Our objective was to chromosomally double interspecific Leymus populations by exposing germinating Hybridization with naturally occurring octoploid Leymus seeds to colchicine. This approach may be useful in developing large populations to introduce new traits.
populations of chromosome-doubled material from semi-fertile Hybridization with naturally occurring octoploid Leymus hybrid populations without making controlled pollinations and populations to assess relationships between genomes of tetraploid and octoploid Leymus populations as proposed byDewey (1970).
Hybridization with chromosome-doubled Elymus populations We applied colchicine to double chromosome number in two pairs for de novo synthesis of octoploid Pascopyrum germplasm.
of reciprocal populations, L4PX-3 (Acc:636 Basin wildrye of Dewey (1975) suggested an alternative approach of making Session 4 - Conventional and Novel Methodologies for Plant Improvement tetraploid Leymus X Elymus hybrids and producing fertile Lawrence, T., A.E. Slinkard, C.D. Ratzlaff, N.W. Holt and P.G.
Jefferson. 1990. Tetracan, Russian wild ryegrass. Can. J. Plant Sci.
70: 311-313.
Berdahl, J.D. and R.E. Barker
. 1991. Characterization of
Speckmann, G.J. 1975. Methods for chromosome doubling in
autotetraploid Russian wildrye produced with nitrous oxide. Crop fodder crops. Pp. 90-95. In: B. N˜uesch (ed.) Ploidy in fodder plants.
Sci. 31: 1153-1155.
EUCARPIA-European Association for Research on Plant Breeding,Zurich, Switzerland.
Dewey, D.R. 1970. Genome relations among diploid Elymus junceus
and certain tetraploid.
Stebbins, G.L. 1971. Chromosomal evolution in higher plants. E.
Arnold, London, U.K.
Dewey, D.R. 1975. The origin of Agropyron smithii. Amer. J. Bot.
62: 524-530.
Table 1
Percentage (number) of seedlings of four Leymus interspecific hybrid populations with 0, 1, 2, or
3 root-tips doubled in chromosome number by colchicine treatment.
Population No. 2n=56 root-tips (maximum=3) Figure 1
The procedure being used to chromosome-double L4PX populations.
Check chromosome number of remaining plants, discarding undoubled plants Establish doubled (may be chimeric) plants in a field block Check chromosome number of plants, discarding undoubled plants Establish doubled (now sectorless) first-generation progeny in a field block Harvest seed as a completely chromosome-doubled population Session 4 - Conventional and Novel Methodologies for Plant Improvement



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