JIRCAS]ournal No.4: 51-62 (1997) 51
Technical Advances in Wheat Haploid Production Using Ultra-wide Crosses
Masanori INAGAKI
Biological Resources Division, Japan Jnteniational Research Center for Agricultural Sciences (]JRCAS) (Tsukuba, Jbaraki, 305 japan)
Received November 12, 1996
Abstract
A technique for producing wheat haploids through ultra-wide crosses followed by embryo rescue was improved by successfully selecting pollen donors from different subfamilial species and applying a plant growth regulator. Efficient crossing procedures were developed by using stored pollen and detached-tiller culture. Production of wheat haploids from diverse genotypes is efficient enough to obtain two haploids per wheat spike. This technique could be useful as a strategy for reducing the time required to obtain homozygous breeding lines and improving selection efficiency of wheat breeding programs. Procedures for doubled haploid production are described.
Additional key words : Triticum aestivum, chromosome elimination, embryo rescue, breeding method
Introduction for developing homozygous breeding lines. Doubled haploids derived from heterozygous Practical breeding programs for self materials show complete uniformity when used as pollinating crops, such as wheat (Triticum recombinant inbred lines in selection procedures. aestivum L., 2n=6x=42), must include a process of An efficient technique for producing doubled genetic fixation for uniformity of agronomic traits haploids thus complements conventional breeding after genetic recombination to increase variation. programs. Repeated selection of heterozygous materials can Two major methods for producing wheat increase uniformity, but many generation cycles haploids (polyhaploids, 2n=3.x=21), one using are required to reach homozygosity in loci microspores (pollen) and one using megaspores associated with agronomic traits. Of great interest (egg cells), have been examined over the last two to plant breeders, haploid production followed by decades. The method for producing haploids from chromosome doubling offers the quickest method cultured pollen has developed as an anther- or 52 JJRCAS ]. No.4, 1997
pollen-culture technique, and is still constrained by Both Japanese and Chinese wheat varieties, and in the difference in the responses of wheat particular, local varieties, are highly crossable with 24, 51) 9, 10, 34) Wh . h genotypes . On the other hand, the method H. bu lb osum . eat genotypes carrymg t e using megaspores in ultra-wide crosses with dominant Kr gene(s) are not crossable with H. Panicoides subfamilial species followed by embryo bulbosum and cannot produce haploid embryos. 16) rescue has recently been developes . The lack of hybridization of wheat genotypes with This paper reports on the progress achieved in H. bulbosum is due to the failure of the pollen tube 44 developing a method for wheat haploid production to penetrate the embryo sac l. Application of plant through ultra-wide crosses since the publication of growth regulator 2,4-dichlorophenoxyacetic acid 12 t h e prev10us. review• ) on t h e pro d uction. of wh eat (2,4-D) promotes seed setting and embryo haploids through the bulbosum technique. The formation when crossable genotypes are used for achievements of the last decade can be attributed crosses, but cannot break the barrier of cross to the collaborative research projects of JIRCAS incompatibilit/0l. Since the efficiency of wheat with the International Center for Agricultural haploid production is greatly influenced by the Research in the Dry Areas (I CARDA), Syria, and crossability of H. bulbosum onto wheat, the method with the International Maize and Wheat using H. bulbosum crosses is restricted to Improvement Center (CIMMYf), Mexico. crossable wheat genotypes. Ultra-wide crosses of wheat with members of Pollen source the Panicoides subfamily have been attempted in 0 alien genetic transfe/ l. Maize (Zea mays L.) Wide crosses of wheat with a bulbous wild pollen can successfully hybridize with wheat egg 25 barley (Hordeum bulbosum L.) result in the cells and produce hybrid zygotes J, irrespective of 26) production of immature haploid embryos of wheat the presence of Kr gene(s) . The maize after preferential elimination of H. bulbosum chromosomes are rapidly eliminated from hybrid 3 chromosomes from hybrid zygotes J. However, the zygotes, requiring artificial rescue of proembryos 27 28 5) crossability of wheat with H. bulbosum is at early developmental stage · · . 2,4-D treatment genetically controlled by the genes Krl and Kr2 after pollination is critical to the enhancement of 43 . h . 46) located on chromosomes 5B and 5A, respectively · emb ryo deve 1opment m w eat x maize crosses . 1 42 · ). According to the pedigrees of wheat varieties, Maize pollination and subsequent 2,4-D treatment crossable genotypes can be traced to the variety onto wheat results in the production of immature 6 Chinese Spring or to materials of Asian origin l. wheat embryos capable of regenerating haploid
Table 1. Effect of 2,4-D treatment on embryo formation frequencies (%) in crosses of wheat with H. bulbosum and maize
Pollen 2,4-D Wheat variety
donor treatment Chinese Spring Norin 61 Mexipak 65 Highbury
None 0.0 0.0 0.0 0.0 None + 0.0 0.0 0.0 0.0
H. bulbosum 16.9 23.6 0.0 0.0 H. bulbosum + 25.0 38.5 0.0 0.0
Maize 0.0 0.0 0.0 0.0 Maize + 21.1 17.5 18.9 8.3
Source: Inagaki & Tahir (1990) 131 Masanori INAGAKI: Technical Advances in Wheat Haploid Production Using Ultra-wide Crosses 53
plants, even for wheat varieties that are cross require viable pollen at the time of crossing, that is, 13 incompatible with H. bulbosum (Table 1) ). Wheat flowering of wheat and pollen donors must be haploid production through maize crosses has synchronized. This method can only be applied in 13 33 been confirmed using diverse wheat varieties · · seasons and places where both wheat and pollen 40 ) . some species. re 1ate d to maize,. sueh as teosmte. donors grow. In addition, the environmental 49 (Zea mays L. spp. mexicana) ) and eastern conditions where wheat plants will develop after 40 gamagrass (Tripsacum dactyloides (L.) L.) , are crossing are critical to the efficiency of wheat alternative pollen donors for wheat haploid haploid production. Adequate techniques for long production. term pollen storage and for culturing wheat tillers Cytological evidence indicates successful with spikes under controlled conditions are helpful fertilization and elimination of paternal for peforming ultra-wide crosses without having to chromosomes from hybrid zygotes in sorghum synchronize flowering times of both parents. (Sorghum bicolor (L.) Moench) and pearl millet Maize and pearl millet pollen have been (Pennisetum glaucum (L.) R.Br.) crosses, which successfully preserved at ultra-low temperatures 4 suggests that sorghum and pearl millet are for long periods · SJ. Maize pollen dried to optimum potential pollen sources for wheat haploid water content range and stored at -SO'C produced . 29, 31, 1, 5) Wh h 1 "d b . d pro d uct10n . eat ap 01 s were o tame wheat haploid embryos, but the frequencies 39 at high frequencies from sorghum ) and pearl decreased to half in the case of fresh pollenl7). 18 millet ) crosses followed by 2,4-D treatment after Pearl millet is an alternative pollen source not only pollination. However, sorghum crosses expressed for haploid production but also for long-term pollen a strong genotypic barrier of wheat to embryo storage. The process of storing pollen involves 19 formation (Table 2) ). Therefore, haploid both drying and freezing. Understanding the production through crosses with maize and pearl effects of drying and freezing on pollen viability is millet appears more stable than other methods essential for achieving successful long-term pollen because of its less pronounced genotypic effect on storage. Pearl millet pollen is relatively tolerant to haploid embryo formation. drying and freezing, in contrast to maize pollen, which has a narrow water content range for Pollen storage and detached-tiller culture maintaining viability during drying and freezing 17 21 (Fig. 1) · ). As a result, pollen storage at ultra-low Methodologies using ultra-wide crosses temperatures does not affect embryo formation
Table 2. Effect of pollen donors on embryo formation frequencies(%) in crosses of wheat with maize, pearl millet and sorghum
Pollen Wheat variety
donor Chinese Spring Norin 61 Siete Cerros
19 Source: Inagaki & Mujeeb-Kazi (1995) > 54 ]JRCAS ]. No.4, 1997
80 a) Maize 80 Pollen germination b) Pearl millet
Pollen germination 60 .c.,-----1..J... 60 >, 0 C Fig. 1. Water content and germination frequencies of maize (a) and pearl millet (b) pollen after drying and freezing. Maize; F 1 hybrid line (CML-246 x CML-242), pearl millet; inbred line NEC-7006. Source: Inagaki & Mujeeb-Kazi (1994) 17 >, (1996)21> frequency in wheat x pearl millet crosses, but Haploid production greatly reduces the frequency in wheat x maize 22 crosses ). Stored pearl millet pollen can be used Wheat haploid production through ultra-wide as an alternative medium for producing wheat crosses consists of two consecutive steps: haploids when fresh pollen is not available. formation of immature embryos by crossing and A technique for artificially culturing detached regeneration of haploid plants from rescued wheat tillers has been developed through embryos. Factors such as the development stage physiological research on immature seed of the wheat florets used for crosses and of the 23 verna1 1zation. . ) . MaJor . components of t h e cu1 ture embryos rescued for plant regeneration are critical solution are sucrose as a nutrient and sulfurous to h ap 101 .d pro duct10n . e ffi c1ency. 3o. 35. rnJ . I n genera1 , acid for preventing fungal contamination. crossing at an early development stage of wheat Supplementing the culture solution with 2,4-D is florets results in higher frequencies of embryo essential for developing haploid embryos from formation. Artificial rescue at the appropriate 49 wheat x maize crosses ). Detached-tiller culture embryo development stage is required to attain has been extensively applied in wheat haploid higher frequencies of plant regeneration. production using maize crosses at the National Cytological examination of regenerated plants 37 Agriculture Research Center (NARC), Japan ). indicates that most of them are euhaploids with 21 The effectiveness of detached-tiller culture in c h romosomes ~~~ . Retent10n . of po 11 en d onor combination with pollen storage in wheat haploid chromosomes was detected at the proembryo 22 . . h . d h 25, 29) d production has also been confirmed (Table 3) J. stage m crosses wit maize an sorg um , an Detached-tiller culture enables to collect spikes at the tillering stage of plants in crosses with pearl 1 from wheat plants growing in distant sites and millet and maize • SJ. This does not eliminate the handle them in a laboratory. possibility of translocating chromosome segments These techniques also enable to avoid having of these pollen donors to the wheat genome to synchronize flowering times of both parents and background through somatic associations. result in considerable savings in terms of labor and However, variations in agronomic traits of doubled 11 45 space required for growing parent plants. They haploids were negligible in H. bulbosum · ) and 2 also provide greater flexibility in when and where maize crosses3 . 47). No significant distortion of wheat haploid production through ultra-wide segregation ratios of genetic markers was found in crosses can be performed. doubled haploids produced from hybrid progenies Masanori INAGAKI: Technical Advances in TVheat Haploid Production Using Ultra-wide Crosses 55 Table 3. Effects of pollen storage and detached-tiller culture on haploid production frequencies (%) in crosses of wheat with maize and pearl millet Pollen Tiller Embryo Plant Haploid donor culture formation (a) regeneration (b) production (axb) Stored On plant 2.8 65.0 1.8 Detached 7.0 46.5 3.3 Stored On plant 20.4 44.3 9.0 Detached 27.7 54.5 15.0 Wheat; variety Norin 61, maize; F1 hybrid line (CML-246 x CML-242), pearl millet; inbred line NEC-7006. Source: Inagaki, Nagamine & Mujeeb-Kazi (1997)221 14.15) • 48) through H. bulbosum or maize crosses. through ultra-wide crosses are described including Efficiency of wheat haploid production over a the improved techniques. three-year period at CIMMYT is shown in Table 4. The production scheme is summarized in Table 5. 1) Plant materials Production of wheat haploids is efficient enough to Sowing times of seed materials are obtain two haploid plants per wheat spike (1.5 determined in order to synchronize flowering doubled haploids per wheat spike after times of wheat and pollen donors. A continuous chromosome doubling). The process takes supply of pollen from pollen parents sown at one or approximately nine months, from sowing of plant two week intervals should be ensured. materials to harvesting of doubled haploid grains. However, it must be noted that production 2) Crossing efficiency varies due to the use of environment At ear emergence, wheat spikes are controlled facilities. emasculated following the removal of the central In case of haploid production of durum wheat florets of each spikelet and the apical and basal (T turgidum L. var. durum) through wide crosses spikelets (Fig. 2a). Emasculated spikes with 25 - with maize, frequency variation among durum 30 florets are enclosed in glassine bags. 40, 2, 20) w h eat genotypes h as b een reporte d . Polyethylene bags may be used for maintaining However, significant difference in fertilization humidity after emasculation. One day before 38) frequency was not found . These facts suggest predicted wheat anthesis, emasculated spikes are that the absence of the D genome in durum wheat pollinated with freshly collected donor pollen (Fig. causes differences in development of haploid 2b). On two consecutive days after pollination, the embryos from hybrid zygotes, resulting in uppermost internodes of wheat culms with frequency variation of haploid production. pollinated spikes are needle-injected with a 100 mg/1 2,4-D solution (Fig. 2c). Most pollinated Procedures for doubled haploid production florets produce plump seeds, but the seeds do not always contain embryos. Four embryos per wheat Steps in doubled haploid production of wheat spike on average can be obtained. 56 JJRCAS ]. No.4, 1997 Table 4. Efficiency of wheat haploid production using maize crosses Year Wheat No. spikes No. embryos No. plants materials pollinated obtained regenerated 1993 F 1 plants 284 1449 810 1994 F3 plants 456 1219 898 1995 F 1 plants 501 1894 1267 Total 1241 4562 2975 Table 5. Scheme of doubled haploid production in wheat using ultra-wide crosses Month Procedure Efficiency/day 0 Sow wheat materials 2.5 Cross wheat with pollen donors 10 spikes 3.0 Embryo rescue 40 embryos 4.0 Transfer haploid plants to soil 25 plants 5.0 Treat plants with colchicine 25 plants 7.5 Cover ears with glassine bags 25 plants 9.0 Harvest doubled haploid grains 15 plants 3) Pollen storage wide crosses. At ear emergence wheat tillers are Stored pollen can be used for crossing on cut off at the base and cultured in a flask containing wheat when fresh pollen is not available. Pollen is tap water. Wheat spikes are emasculated as collected from donors at anthesis and screened described above. After pollination, tillers are through a sieve to remove anthers. Ten grams of cultured in a solution containing 40 g/1 sucrose, 8 pollen are spread on a paper tray and dried with ml/1 sulfurous acid (6% S02) and 100 mg/1 2,4-D gentle ventilation at 35°C and 35 - 40% relative for two days. They are then transferred to a humidity. Pollen water content is determined from solution containing only sucrose and sulfurous a 0.5 g pollen sample dried at 95°C for five hours. acid, and cultured until embryo rescue. This Optimum water content for stored pollen is 10 - 12% process takes approximately 12 days (in maize for maize and 5 - 7% for pearl millet (Fig. 2d). The crosses) and 14 days (in pearl millet crosses) after dried pollen is distributed among cryopreservation pollination (Fig. 2e). The culture conditions are tubes. The sealed tubes are stored in liquid 22.5°C, 12-hr daylength and 60 - 70% relative nitrogen (-196°C) until use. After thawing sample humidity in a growth chamber. tubes containing pollen in a water-bath at 38°C for five minutes, pollen is checked for its germination 5) Embryo culture viability and used for crossing. The drying and Seed set in wheat crossed with pollen donors freezing process reduces pollen viability and may is somewhat smaller than in selfed seed, and seeds affect embryo formation frequency in wide crosses. are filled with an aqueous solution instead of solid endosperm as found in selfed seed (Fig. 2±). After 4) Detached-tiller culture 14 (in maize crosses) or 18 days (in pearl millet In the case of wheat plants growing in distant crosses) of spike growth on wheat plants, wheat sites, detaching tillers with spikes is feasible for seeds are removed from spikes and sterilized in a Masanori INAGAKI: Technical Advances in Wheat Haploid Production Using Ultra-wide Crosses 57 I ' Fig . 2a - I. (See captions p.58) 58 J/RCAS]. No.4, 1997 Fig. 2a - q . Stages in production of wheat doubled haploids through ultra-wide crosses. a) Wheat spikes at ear emergence (left), emasculation (center) and seed setting (right). b) Germination of maize pollen on wheat stigma. Bar 0.1 mm. C) Injection of 2,4-D solution into wheat culm using a syringe. d) Pearl millet pollen at collection (upper) and after being dried for two hours (lower). Bar 0.1 mm. e) Detached-tiller culture of wheat at emasculation (left), pollination (center) and seed setting (right). f) Wheat seeds obtained from self-pollination, and from crosses with maize, pearl millet and sorghum (from left to right). Bar 2 mm. g) Apparatus used for embryo rescue on a laminar flow bench. h) Wheat embryos obtained from self-pollination, and from crosses with maize pearl millet and sorghum (from left to right), showing differences in size and shape among crosses. Bar 2 mm. i) Plants developing from cultured embryos (from. left to right; 0, l , 2 and 3 weeks after incubation). j) Culture room accommodating test tubes and plastic dishes. k) Wheat haploid plants transferred to potted soil for further development. I) Somatic chromosomes (2n=3x=2 l) of a wheat haploid plant. Bar 1Oµm. m) Colchicine treatment of wheat haploid plants at tillering. n) Wheat plants transferred to potted so il after colchicine treatment. O) Wheat spikes of colchicine-treated plants, covered with glassine bag (leji), and producing doubled haploid grains (right). p) Doubled haploid plants grown for seed multiplication. q) Doubled haploid-tines grown in the field, showing uniformity within lines solution (1 - 2% sodium hypochloride of CLOROX further (Fig. 2k). The chromosome number can be and few drops/I Tween-20) for 10 minutes (Fig. examined in squashed preparations of root-tips 2g). After rinsing with sterilized water, embryos stained with acetocarmine or aceto-orcein (Fig. 21) . (approximately 1.0 mm in diameter) are aseptically excised (Fig. 2h) and transferred onto half strength 7) Chromosome doubling 6 Murashige & Skoog3 l medium supplemented with At tillering, roots are cut to about 2 cm below 20 g/1 sucrose and 6 g/1 agarose in test tubes or the crown. Plants are then immersed in colchicine plastic dishes (Fig. 2i). Embryos are incubated at solution (0.05 - 0.10% colchicine, 2% dimethyl 25 °C, using 16-hr daylength and ca. 5000 lux light sulfoxide (DMSO) and 15 drops/I Tween-20) in a intensity (Fig.2j). Plants are regenerated from flask for five hours at room temperature (Fig. 2m). embryos at frequencies of 50 - 70%. Treated plants are potted following thorough washing with tap water (Fig. 2n). Potted plants are 6) Plant regeneration grown under favorable, tillering-enhancing Cultured plants with fully developed roots and conditions. Wheat spikes from colchicine-treated leaves are transplanted to potted soil and grown plants are enclosed in glassine bags to prevent Masanori INAGAKI: Technical Advances in Wheat Haploid Production Using Ultra-wide Crosses 59 outcrossing. Wheat grains are harvested at a 60 - haploid production in wheat (Triticum 70% success rate (Fig. 2o). aestivum L.) by chromosome elimination. After multiplying harvested grains for one Nature (London) 256: 410-411. generation cycle (Fig. 2p), doubled haploid lines 4) Barnabas, B. & Rajki, E. (1981). Fertility of are planted in the field for evaluating genetic deep-frozen maize (Zea mays L.) pollen. Ann. variation (Fig. 2q). Bot. 48: 861-864. 5) Comeau, A et al. (1992). Media for the in Conclusion ovulo culture of proembryos of wheat and wheat-derived interspecific hybrids or A technique for producing wheat haploids haploids. Plant Sci. 81: 117-125. using ultra-wide crosses followed by embryo 6) Falk, D.E. & Kasha, K.J. (1981). Comparison of rescue has been developed over the last two the crossability of rye (Secale cereale) and decades. Significant technical advances have been Hordeum bulbosum onto wheat (Triticum achieved by using pollen selected from different aestivum). Can.]. Genet. Cytol. 23: 81-88. subfamilial species and applying plant growth 7) Falk, D.E. & Kasha, K.J. (1983). Genetic regulators. 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