Culture of freshly isolated wheat (Triticum aestivum L.) microspores treated with inducer chemicals

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1 Plant Cell Rep (2001) 20: DOI /s CELL BIOLOGY AND MORPHOGENESIS M. Y. Zheng W. Liu Y. Weng E. Polle C. F. Konzak Culture of freshly isolated wheat (Triticum aestivum L.) microspores treated with inducer chemicals Received: 14 March 2001 / Revision received: 16 August 2001 / Accepted: 17 August 2001 / Published online: 11 October 2001 Springer-Verlag 2001 Abstract Microspores were isolated from wheat (Triticum aestivum L.) spikes by means of a micro-blender immediately following their removal from the donor plants. Isolated microspores were then subjected to a chemical treatment consisting of 0.18 mm 2-hydroxynicotinic acid, minerals and 9% maltose in the dark at 25 C for h. Following purification via filtration and gradient centrifugation on 21% maltose, the microspores were cultured in the presence of excised ovaries in liquid medium at 27 C. Embryoid yield, percentage of green plants, and the frequency of spontaneously doubled haploids ranged from 360 to 4,914 embryoids per spike, 15% to 95%, and 39% to 78%, respectively. Other compounds that were effective in maintaining viability and triggering microspore embryogenesis were benzotriazole-5-carboxylic acid and violuric acid monohydrate. This system has proven to be highly efficient for producing doubled haploids over a range of genotypes. Keywords Androgenesis Doubled haploid Embryoid 2-Hydroxynicotinic acid Wheat Abbreviations DH: Doubled haploid GP: Green plants 2-HNA: 2-Hydroxynicotinic acid NPB: Northwest Plant Breeding Company Introduction The production of haploid/doubled haploid plants from gametic cells i.e., microspores is useful in crop improvement, genetic manipulation, and in many areas of basic research in plant developmental biology (Reynolds 1997; Touraev et al. 1996a; Vicente et al. 1991). Since Communicated by J.M. Widholm M.Y. Zheng ( ) W. Liu Y. Weng E. Polle C.F. Konzak Northwest Plant Breeding Co, 2001 Country Club Rd, Pullman, WA 99163, USA mzheng63@yahoo.com Tel.: , Fax: homozygosity is achieved in one generation, breeders can eliminate the numerous cycles of inbreeding required by conventional systems as well as substantially reduce population sizes required for effective selection of superior trait combinations. Haploids/doubled haploids are also frequently used in plant genome mapping (Kasha et al. 1990). Assuming an efficient plant regeneration system is available, gametic cells are also the preferred targets for transformation and transgenic studies (Kasha et al. 1990). The chromosomes of the resulting haploid seedlings may be doubled spontaneously or by colchicine or anti-microtubule herbicides to achieve instant homozygosity. Historically, haploids/doubled haploids have been predominantly produced by anther cultures (Chuang et al. 1978; Guha and Maheshwari 1964; Hu and Yang 1986; Zheng and Konzak 1999; Zhou et al. 1991; Ziauddin et al. 1990). Only in recent years has substantial progress been made in wheat doubled haploid production through the culturing of isolated microspores (Gustafson et al. 1995; Hu et al. l995; Hu and Kasha 1997; Mejza et al. 1993; Touraev et al. 1996b; Tuvesson and Öhlund 1993). Isolated microspore culture offers several advantages over anther culture. First, this method opens avenues for analyzing the basic mechanisms of pollen embryogenesis at the molecular and biochemical levels (Raghavan 1986; Reynolds 1997). Second, it also provides opportunities to increase the frequency of induction and thereby doubled haploid yield by directly manipulating androgenic processes at the cellular level (Touraev et al. 1997). Thirdly, once freed from the barrier of anther walls, microspores become a favorable target for gene delivery. To realize these benefits, however, an efficient culture system for isolated microspores must first be established. Major factors affecting the efficiency of microspore culture include genotype, donor plant physiology, microspore developmental stage, pretreatment conditions, physical and chemical conditions for induction culture, and plant regeneration (Custers et al. 1994; Gustafson et al. 1995; Hu et al. 1995; Hu and Kasha 1997; Touraev et

2 686 al. 1997). It is generally agreed that microspores must be at an appropriate stage of development when placed into culture. The stages most responsive to androgenic induction seem to be either mid- to late-uninucleate or earlybinucleate (Raghavan 1986; Reynolds 1997). Some type of stress treatment is needed to initiate or enhance androgenesis, and this may include low or high temperature (Custers et al. 1994; Kasha et al. 1990; Touraev at al. 1996a, b, 1997), water deficiency (Imamura and Harada 1980), anaerobic conditions (Imamura and Harada 1981), and starvation (Kyo and Harada 1986; Touraev et al. 1997; Zarsky et al. 1992). Once induced for androgenesis, microspores are isolated and placed into culture. The concentrations and nature of the ingredients in the media used for induction culture and plant regeneration are also critical. Type and level of sugar, hormones, and additives, osmolarity of the media, and ovary co-culture can all affect the ultimate success (Bruins et al. 1996; Hu et al. 1995; Hu and Kasha 1997). Described herein is a novel system for haploid/doubled haploid production using freshly isolated microspores. Microspores were first isolated and then subjected for a brief period to treatment with an inducer chemical under the proper physical conditions. The inducer chemical helped to trigger microspore embryogenesis and to maintain microspore viability, both of which are prerequisites for successful plant regeneration. 2-Hydroxynicotinic acid and other compounds were selected for our experiments based upon preliminary testing in anther cultures conducted at NPB. Upon spraying or painting of these compounds over anther donor plants at the completion of meiosis, the anther culture response frequencies were increased. Following treatment with inducer compounds, this microspore culture system allows for the production of hundreds of green plants by microspores isolated from a single spike. This microspore culture system is effective for a wide spectrum of genotypes, amenable to the genetic transformation and production of transgenic plants for crop improvement (Konzak et al., unpublished). Materials and methods Growth of donor plants The genotypes used in all of the experiments described herein include Calorwa, Chris, Pavon 76, Waldron, WED 202 and WPB 926; these were in NPB's seed stock and can be obtained from USDA Agriculture Research Service (Aberdeen, Idaho). All of the genotypes were grown in the same greenhouse. One to three seeds were sown in each pot (20 25 cm) filled with premixed soil. Plants were grown in temperature-controlled greenhouses at 27 ±2 C (day) and 17 ±2 C (night) under a 17/7-h (day/night) photoperiod. Fertilizers (N, P, K) were premixed with soil at the time the seeds were sown, with subsequent applications through daily watering with water containing liquid forms of nitrogen, phosphorus, and potassium (20:20:20). In general, any standard conditions for growing wheat in a greenhouse are acceptable provided that healthy plants can be obtained. With regard to winter wheats, in order to ensure normal plant development and hence optimum culture, it is essential that the vernalization be complete. Collection of tillers Fresh tillers that contained microspores at the mid- to late-uninucleate stage were cut at the second node from the top of the tiller, and the base of each tiller was immersed immediately in a 250-ml flask containing ml distilled water. All of the leaves were removed by severing leaf blades at their bases. The morphological features of tillers containing microspores at the mid- to late-uninucleate stage can easily be established for each genotype via microscopic examination of microspores in acetocarmine stain or distilled water (Zheng and Konzak 1999). Isolation of microspores All foliage beneath the first node was removed, keeping only the boot encasing the spike. Boots were then disinfected through immersion in 1.5% sodium hypochlorite solution in a 100-ml graduated cylinder for 20 min, followed by three rinses with sterile distilled water over 3 min. The spikes were aseptically removed from each disinfected boot and placed on top of a sterile blender cup (Waring MC II) that had been autoclaved. Florets were cut from their bases with awns (if present) removed and allowed to drop into the open blender cup. Florets obtained from three to six spikes were used for each run of blending in 50 ml of autoclaved 0.3 M mannitol solution. After a sterile lid was placed and secured on top, the blender cup was assembled to the MCII Waring blender and run at 16,000 rpm for 20 s. To eliminate the larger debris, we passed the resulting slurry through a 100-µm stainless steel mesh filter. The blender cup was rinsed three times using 5 ml of 0.3 M mannitol each time, which was also poured through the filter. The filtrate was then poured onto a 38-µm mesh filter. This essentially trapped all of the viable microspores and some of the small debris and dead microspores. The microspores were then rinsed three times on the filter with 5 ml of solution A each time (in mg/l: KCl, 1,492; MgSO 4 7H 2 O, 246; CaCl 2.2H 2 O, 148; KH 2 PO 4, 136; H 3 BO 3, 3; KI, 0.5; MnSO 4.4H 2 O, 8.0; ZnSO 4.7H 2 O, 3.0; FeSO 4.7H 2 O, 56.0; maltose, 9,000; 2-HNA, 50), then washed off the filter into a petri dish ( mm) using 15 ml of solution A (with the ph adjusted to 6.5). Treatment with inducer chemical formulation Using mm-petri dishes, we adjusted the concentration of 2- HNA in solution A to 0.06, 0.12, 0.18, and 0.30 mm. The microspore density was held constant at approximately 10,000 per milliliter as determined with a hemocytometer, with a total volume of 4 ml per dish. All inducer chemicals were dissolved in solution A prior to being added to the pretreatment cultures. Once microspores and 2-HNA were gently mixed together, the petri dishes were sealed with Parafilm and incubated in the dark for 2 days at 25 C. At this temperature, however, the treatment time may vary from 38 h to 52 h depending on the genotype. Following the incubation, the microspores were recovered on a 38-µm stainless steel mesh filter and then rinsed once with 5 ml 0.3 M mannitol. Microspores trapped on the 38-µm mesh were rinsed off the filter with 2 ml of 0.3 M mannitol into 5 ml of 21% maltose in a 15-ml conical tube, which was then capped and centrifuged at 450 g for 3 min. Using a pipette, we then collected the embryogenic microspores from the band at the interface between 21% maltose and 0.3 M mannitol and transferred them to another 38-µm mesh filter. Microspores retained on the mesh were rinsed three times with 3 ml of culture medium each time [NPB-99 (Konzak et al. 1999) in mg/l: (NH 4 ) 2 SO 4, 232; KNO 3, 1,415; CaCl 2 2H 2 O, 83; KH 2 PO 4, 200; MgSO 4 7H 2 O, 93; Na 2 EDTA, 37.3; FeSO 4 7H 2 O, 27.8; H 3 BO 3, 5; CoCl 6H 2 O, ; CuSO 4 5H 2 O, ; KI, 0.4; MnSO 4 4H 2 O, 5; Na 2 MoO 4 2H 2 O, ; ZnSO 4 7H 2 O, 5; myoinositol, 50; nicotinic acid, 0.5; pyridoxine-hcl, 0.5; thiamine- HCl, 1.0; maltose, 90,000; glutamine, 500; kinetin, 0.2; 2,4-dichlorophenoxyacetic acid, 0.2; phenylacetic acid (PAA), 1.0; filter-sterilized with the ph adjusted to 6.5]. The mesh filter was

3 687 Fig. 1a d Embryogenesis by microspores first isolated and then treated with 0.18 mm 2- HNA. All photos are of microspores isolated from Pavon 76. a Microspores immediately after isolation, b embryogenic microspores induced by 2- HNA for 48 h, c multi-cellular structures 7 days in induction culture, d proembryoids 14 days in induction culture then inverted and microspores gently rinsed off of the mesh into a sterile mm petri dish. The microspores were equally distributed to various petri dishes of the same size with a minimal density of /ml, as determined with a hemocytometer, and with a total volume of 4 5 ml of NPB-99. Induction culture After the density was adjusted with NPB-99, 8 10 fresh wheat ovaries were transferred aseptically to each petri dish. Although ovaries from many genotypes were found to be effective in our microspore culture system, an awnless cultivar, Chris, was selected as ovary donor for the ease of ovary excision. Boots were sampled in mid-uninucleate to early binucleate stage when the spikes were still fully encased by boots. Sampled boots were then sprayed with 80% alcohol on the surface until saturation. Sprayed boots were wrapped with one to two sheets of Kimwipes and allowed to dry off for min. The spikes were then taken out of the boots, and the ovary was picked out of each floret with a pair of fine forceps. Once the ovaries were added, all dishes were then sealed with Parafilm and incubated in the dark at C for embryoid development. After approximately 30 days in culture, embryoids reaching the size of 2 mm in diameter or larger were transferred to petri dishes containing medium (Zhuang and Xu 1983) solidified with Phytagel for plant regeneration. Approximately embryoids/calli were transferred to each mm petri dish. Sealed petri dishes were incubated at room temperature under a 16/8-h (day/night) photoperiod with light supplied by incandescent and fluorescent lighting at an intensity µmol m 2 s 1 for. Plantlets were ready for potting 2 weeks after the embryoids were transferred for regeneration. The survival rate of the microspores was evaluated 2 days after culture initiation by mixing a sub-sample of 0.05 ml from the culture with an equal volume 0.01 mm fluorescein diacetate in 0.5 M sucrose. After the microspore suspensions were mixed well with the dye, live and dead cells were counted using a hemocytometer under a fluorescence microscope. The survival rate for each treatment was a pooled mean from four replicates. Once a sub-sample was drawn, the petri dishes were re-sealed and placed back into the incubator for embryoid development. Cultures were monitored and pictures were taken 36 h, 7 days, 10 days, 21 days, and 28 days after the initiation of induction culture. Data collection All of the data presented were pooled means from four replicates, and a t-test was performed for data in Table 2. The plant regeneration frequency (PRF) was calculated as the total number of plants regenerated from 100 embryoids/calli actually transferred for regeneration. The percentage of green plantlets represents the percentage of green plants among the total number of regenerated plants. The frequency of DH is defined as the percentage of green plants that exhibit normal fertility in the greenhouse. Results and discussion Basic pattern in microspore embryogenesis Microspores isolated from the spikes of cv. Pavon 76 were mostly at the mid- to late-uninucleate stage (Fig. 1a). Figure 1b shows the appearance of the microspores after a 48-h pretreatment with 0.18 mm 2-HNA in

4 688 Fig. 2a d Plant regeneration by embryoids derived from microspores first isolated and then treated with 0.18 mm 2-HNA. All photos are from microspore cultures initiated from Pavon 76. a Embryoids became visible after 21 days in induction culture with a diameter of 0.2 mm; b massive mature embryoids after 28 days in induction culture, ready to be transferred for plant regeneration; c germinated embryoids (plantlets) are ready for potting at 2 weeks; the petri dish is 10 cm in diameter; d plants derived from embryoids showed over a 95% survival rate following the transfer to pots (25 cm in diameter) in the greenhouse solution A. Following pretreatment, the microspores were switched to the induction medium NPB-99 (Konzak et al. 1999). The first cell divisions occurred within about 36 h, which was somewhat slower than has been reported in other systems (Reynolds 1993; Touraev et al. 1996a). Multi-cellular structures were clearly formed after 7 days in culture (Fig. 1c), with all cells still enclosed within the exine. Even though many cell divisions occurred, the size of the multi-cellular microspores increased only slightly, from about 50 µm to 65 µm in diameter. The size versus cell number relationship during this period was analogous to that occurring during the cleavage of a fertilized egg in animals. The sharp increase in cell numbers with a slight increase in size during the early stage of embryogenesis seems to be a basic underlining mechanism, even for in vitro embryogenesis by cultured microspores. Pro-embryoids began to emerge from the exine 10 days after culture initiation (Fig. 1d). From approximately 21 days onwards, embryoids became visible (Fig. 2a), and these continued to expand in size rapidly. Mature embryoids (approx. 2 mm in diameter) were obtained after 4 weeks in culture (Fig. 2b). Upon transfer to medium, mature embryoids germinated into plantlets within 2 weeks (Fig. 2c). These plantlets had a survival rate of more than 95% following transfer to pots in the greenhouse (Fig. 2d). Due to the large numbers involved, we could only transfer a small portion of the embryoids/calli derived from the microspores of a single spike to regeneration medium. Our estimate, based upon the regeneration rate, suggests that at least 1,500 green plants could be regenerated from microspores isolated from a single spike for some genotypes (e.g., cvs. Chris and Pavon 76) if all the embryoids/calli were to be transferred to regeneration medium. Effects of some inducer chemicals Other inducer chemicals had effects on microspore embryogenesis similar to that of 2-HNA (Table 1). We report here only a set of data on survival rate studies. These early experiments were conducted to determine if the presence of these candidate inducer chemicals was able to increase the number of microspores that survive the pretreatment and show the characteristic embryogenic structures. For experiments with 2-HNA, the final density of microspores during the 2-day treatment was 6,100 microspores per milliliter. After transfer to culture

5 689 Table 1 The effect of chemical inducers on microspore survival during a 38- to 52-h-long pretreatment. Microspores were all isolated from a spring wheat, WED 202:16-12 Chemical Concentrations in Optimum Microspore Percentage Inducer a millimoles (mm) concentration density/ml of viable (mm) microspores No chemical 0 0 4, HNA 0.06; 0.12; 0.18; , b AA 0.09; , b BT 0.09; , b B-5-CA 0.09; , b 2,3-BM 0.09; , b DL-H 0.09; , b 2,4-DPCA 0.09; , ,3-PCA 0.09; , b SA 0.045; 0.09; 0.13; , b VAM 0.09; , b a AA Anthranilic acid, BT benzotriazole, B-5-CA benzotriazole-5-carboxylic acid, 2,3-BM 2,3-butanedione monoxime, 2,4-DPCA 2,4-dihydroxy pyrimidine 5-carboxylic acid, DL-H DL-histidine, 2-HNA 2-hydroxynicotinic acid, 2,3PCA 2,3-pyridine carboxylic acid, SA sulfanilamide, VAM violuric acid monohydrate b Statistically significant at P=0.01 between each treatment and the control based upon a t-test Table 2 Culture response of microspores isolated from one spike and treated with 0.18 mm 2-HNA at 25 C for 48 h prior to the induction culture a The number in parenthesis is the control for each variety b No data on the frequency of DH are available for the controls as too few green plantlets were regenerated Genotype Initial number Total number Number Plant Percentage Frequency of microspores of embryoids transferred regeneration of green of DH (%) or calli to medium frequency plantlets Pavon 76 23,000 4,326 (147) a 520 (40) 76 (71) 43 (35) 78 b Chris 26,000 4,914 (126) 484 (40) 89 (91) 95 (89) 42 Calorwa 21,000 1,894 (31) 210 (25) 67 (70) 15 (13) 39 WED ,000 1,805 (54) 180 (30) 85 (81) 58 (60) 80 Waldron 27, (3) 105 (3) 64 (0) 78 (0) 55 WPB , (0) 180 (0) 72 (0) 86 (0) 47 medium NPB-99, the microspores were incubated for 2 days at 27 C. The data showed that there was a marked effect of 2-HNA on microspore survival, particularly at concentrations of mm. The positive effect peaked around 0.18 mm (Table 1). When two concentrations, 0.09 mm and 0.18 mm, of anthranilic acid, benzotriazole, benzotriazole-5-carboxylic acid, 2,3-butanedione monoxime, DL-histidine, 2,4- dihydroxy pyrimidine 5-carboxylic acid, 2,3-pyridine carboxylic acid, and violuric acid monohydrate and various concentrations ( mm) of sulfanilamide were tested for their effect on microspore survival, all of the compounds, except 2,4- dihydroxy pyrimidine 5-carboxylic acid, at one or more concentrations helped maintain a significantly higher survival rate compared to the control. Among the compounds that were beneficial to microspore survival, 2-hydroxynicotinic acid, benzotriazole-5-carboxylic acid, and violuric acid monohydrate were also found to increase the frequencies of embryogenic microspores substantially. Their inducing effects have since been confirmed in repeated experiments being carried through plant regeneration. Fresh microspores treated with benzotriazole-5-carboxylic acid and violuric acid monohydrate exhibited a developmental pattern that was almost identical to those treated with 2- HNA (documented in Figs. 1and 2). This suggests that once triggered for embryogenesis, microspores usually follow the same developmental pattern toward mature embryoid formation under the same culture conditions. The effectiveness of a fresh microspore system with various genotypes To determine if treating freshly isolated microspores with 2-HNA in solution A was effective over a range of genotypes, cultivars were selected on the basis of previous work at NPB or other reports using anther cultures. Included in the study were highly responsive cultivars such as Pavon 76 and Chris, moderately responsive cultivars Alpowa and WED 202:16-12, and recalcitrant cultivars, Waldron and WPB 926. Although differences in the number of embryoids, percentage green plants, and frequency of doubled haploids did exist among genotypes, the use of 2-HNA in solution A clearly had a positive effect in triggering microspore embryogenesis (Table 2). For all six varieties, the inclusion of 2-HNA during the 48-h pretreatment yielded a significantly better response. These results also showed that the fresh microspore system was applicable to genotypes ranging from the naturally responsive to the non-responsive. In this system, microspores were first isolated by blending at 16,000 rpm for 20 s and then subjected to a 48 h chemical treatment in a 0.18 mm 2-HNA solution at

6 C. The treated microspores were then purified as described earlier, via filtration and gradient centrifugation at 450 g on 21% maltose. Purified microspores were cultured in NPB-99 medium at 27 C in the dark for embryoid development. Fresh ovaries were included in each petri dish at a density of two ovaries for each milliliter of induction medium. Mature embryoids (approx. 2 mm in diameter) were transferred to Phytagel-solidified medium for plant regeneration under incandescent and fluorescent light [light intensity: µmol m 2 s 1 and a 16/8-h (day/night) photoperiod, and room temperature (23 C)]. This system offers great potential for a variety of reasons. First, the technology has been proven to be a direct and easy method for evaluating the potential triggering effects on embryogenesis by some inducer chemicals and/or their formulations. Second, with microspores already released into the culture medium, the technology can be employed widely in basic cell biology research and in studies aimed at unraveling the pathway of embryogenesis. Finally, the fresh microspore culture system may fulfill the requirement of genetic transformation and transgenic experiments, as they are amenable to affecting changes at the single cell level. We anticipate being able to continue improving the percentages of green plants as well as doubled haploids through amending the culture conditions. Nevertheless, the current technology itself may be widely applicable to wheat breeding. References Bruins MBM, Rakoczy-Trojanowska M, CHA Snijders (1996) Isolated microspore culture in wheat (Triticum aestivum L.): the effect of co-culture of wheat or barley ovaries on embryogenesis. Cereal Res Commun 24: Chuang CC, Ouyang JW, Chia H, Chou SM, Ching CK (1978) A set of potato media for wheat anther culture. In: Proc Symp Plant Tissue Cult. 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