Plant Cell Reports (1999) 18: 858Ð862 © Springer-Verlag 1999

B. Barnabás · B. Obert · G. Kovács Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero

Received: 15 June 1998 / Revision received: 17 September 1998 / Accepted: 3 December 1998

Abstract The construction of maize genotypes with high haploid induction capacity made it possible to study the ef- Introduction fect of colchicine on maize androgenesis in vitro. Anther cultures of three hybrids were treated with 0.02% and Anther culture seems to be a useful tool for creating maize 0.03% colchicine for 3 days at the beginning of microspore inbred lines, since homozygous progeny can be produced induction. Colchicine added to the induction medium had from heterozygous parents in one generation. However, the no negative influence on the androgenic responses (anther application of anther culture in plant breeding is strongly induction, induction of structures of microspore origin dependent on the production of large numbers of micro- and their regeneration ability) of the genotypes examined. spore-derived plants and on high-frequency induction of However, significantly higher fertility was observed in chromosome doubling. Since the pioneer work of Chinese plants originating from colchicine-treated microspores, es- scientists (Kuo et al. 1978), numerous research teams in pecially at 0.03%. Cytological examinations showed that China, the USA and Europe (Miao et al 1978; Genovesi colchicine treatment before the first microspore division and Collins 1982; Dieu and Beckert 1986; Petolino and efficiently arrested mitosis and resulted in homozygous Jones 1986) have studied the factors leading to a greater doubled-haploid microspores. Under the experimental androgenic response and an increased number of doubled- conditions, the antimitotic drug had no later effect on the haploid plants. In general, the frequency of spontaneous division symmetry of the microspore nucleus, and unequal chromosome doubling in maize haploids has been low and divisions remained dominant. Callus formation from the extremely unreliable (Wan et al. 1991). induced microspores seemed to be more typical (ranging The occurrence of spontaneous diploids has been re- between 60Ð70%), but embryo frequency was increased by ported from anther cultures of various agronomically im- approximately 10%, especially at the higher colchicine portant species, such as barley (Subrahmanyam and Kasha concentration. These results suggest that the mechanism 1975), tobacco (Burk et al. 1972), wheat (Hu et al. 1978), of colchicine action in premitotic maize microspores may rice (Chen and Li 1978), maize (Ku et al. 1981) and Bras- differ from that previously observed in wheat. sica napus (Charne et al. 1988). Spontaneous doubling might be caused by endomitosis, endoreduplication or Key words Zea mays L. á Anther culture á nuclear fusion between vegetative and generative nuclei Genome doubling á Colchicine within the pollen grain during the early stages of anther culture (Sunderland et al. 1974; Keller and Amstrong 1978). Due to the lack of critical cytological and biochem- ical observations, it is not known at exactly which phase of in vitro culturing the chromosome doubling takes place. Communicated by K. Glimelius As spontaneous diploidisation occurs infrequently and in- consistently, colchicine is the agent most commonly used B. Barnabás (½) · G. Kovács Agricultural Research Institute to induce chromosome doubling. of the Hungarian Academy of Sciences, Colchicine disrupts mitosis by binding to tubulin, thus Brunszvik u. 2, H-2462, Martonvásár, Hungary inhibiting the formation of microtubules and the polar mi- Fax: +36-22-460213 gration of chromosomes, resulting in a cell with a doubled e-mail: [email protected] chromosome number. The methods used for colchicine B. Obert treatment have not changed basically since Levan (1938) Institute of Plant Genetics and Biotechnology, Slovak Academy of Science, soaked plant roots in colchicine solution. Treatment of Akademicka, 2., POB 39A, SK-95007 Nitra, Slovak Republic roots with the drug is still the most frequently used con- 859 ventional technique in practice. In a number of species, ap- Collins 1982) supplemented with 0.1 mg lÐ1 2,3,5-triiodobenzoic acid, 5 g lÐ1 charcoal, 500 mg lÐ1 casein hydrolysate, 120 g lÐ1 su- ical meristems, secondary buds and tillers can also be ex- Ð1 posed to colchicine (Mathias and Röbbelen 1991). These crose and 2.5 g l gelrite at pH 5.4, with or without colchicine. procedures may result in about 50% of responsive plants, but the regeneration of chimeras and aneuploids may also Early genome doubling occur at high frequency (Wenzel et al. 1977; Zhao and Davidson 1984; Barnabás et al. 1991). Colchicine treat- To double the chromosome number of the induced microspores, col- ment of maize seedlings or plantlets may double the chro- chicine (Sigma C3915) at two different concentrations (0.02% and 0.03%) was added directly into the induction medium by ultrafiltra- mosome number in the tassel or the ear, but often not in tion. Anthers were incubated on the colchicine-containing media for both, which will make self-pollination impossible (Wan 3 days at 29¡C in the dark to complete the first microspore mitosis. et al. 1989). High mortality and abnormal plant develop- After this treatment, the anthers were transferred to a colchicine-free ment can also be observed in colchicine-treated plant pop- induction medium and cultured at 29¡C in the dark for 28 days. The number of responding anthers and the frequency of microspore-de- ulations. This could explain the low efficiency of doubled- rived structures (embryo-like structures and calli) was then calculat- haploid production in maize after colchicine treatment of ed as a percentage of the anthers inoculated. Androgenesis under regenerated haploid plantlets. control (colchicine-free) and treatment (0.02%, 0.03% colchicine in Genome doubling in culture before the plantlet stage the medium) conditions was compared. might help to circumvent the above-mentioned problems. Papers by Wan et al. (1989) and Wan and Widholm (1995) Cytological observations reported the recovery of genetically stable doubled-hap- loid maize plants at high frequency through the colchi- Light microscope (Zeiss Axiovert 35) examinations were carried out cine treatment of embryogenic, microspore-derived hap- on squashes of 2-day cultured control anthers and 5-day cultured col- chicine-exposed anthers. The anthers were fixed in Carnoy solution loid callus. In other recent experiments, the application (3:1 mixture of absolute ethanol and cc. acetic acid) and stored in of colchicine treatment together with a 7-day cold shock 70% ethanol until investigation. Acetocarmine-stained mitotic mi- to cultured maize anthers resulted in a considerable in- crospores were examined for their division symmetry. crease in chromosome doubling in microspore-derived plants (Saisingtong et al. 1996; Antoine-Michard and Beckert 1997). Plant regeneration The present paper reports the effects of colchicine in Calli and embryo-like structures were removed from the 1-month the early stages of androgenesis, when anthers of inducible cultured anthers and transferred directly to a differentiation medi- maize genotypes were cultured on a colchicine-containing um. For plant regeneration a revised Murashige and Skoog (1962) medium (MS macronutrients, MS microelements supplemented with medium soon after a 10-day cold pretreatment only for the Ð1 Ð1 myo-inostol 100 mg l L-asparagine 150 mg l , thiamine HCl period of first microspore mitosis. Fertile doubled-haploid 0.5 mg lÐ1, kinetine 1 mg lÐ1, naphthaleneacetic acid 0.05 mg lÐ1, plant production from the cultures is also estimated. sucrose 2 g lÐ1 and agar 7 g lÐ1 at pH 5.8. Plantlet differentiation was carried out under 16-h illumination (50 mmol sÐ1 mÐ2 light intensity) at a constant 26¡C. Plantlets were placed in larger glass containers for further growth. The plant growth medium was the same, but without hormones. Well-differentiated Materials and methods healthy green plantlets were then transplanted into peat pellets (di- ameter: 5 cm) from AS Jiffy Products Ltd. Finally, the plants were Plant material transferred to soil and grown to maturity in a growth chamber. Plants which produced normal male and female inflorescences were self- In the present experiment the following maize genotypes were used pollinated and the seed set was recorded. as anther donor plants: DH 309×DH 247 (A21), A632×DH309, The experimental data obtained for induction and plant regener- A21×F58. A632 and F58 were non-responsive inbred lines. A21 was ation were statistically analysed by ANOVA (SPSS for Windows, a single-cross hybrid of two highly androgenic double-haploid lines version 6.0) and fertility data were compared using the Mann-Whit- produced in the experimental nursery of the Agricultural Research ney U-test (Goldstein 1965). Institute, Martonvásár. These latter two double-haploid lines were closely related genetically, since they originated from anther culture of a Chinese hybrid 592×A2. The plants were raised in growth cham- bers in a climatic programme designed for growing maize (Tischner et al. 1997). Results

Anther culture Induction of androgenesis in the presence of colchicine

Tassels were collected prior to the emergence of the main leaf blade The results presented in Table 1 show that all the geno- and checked for microspore development. Tassel fragments contain- ing anthers in the late uninuclear microspore development stage were types used in this study gave a relatively high androgenic surface sterilized with 20% sodium hypochlorite for 20 min and then response under the experimental conditions. There were washed three times with sterile distilled water. They were then cov- no significant differences between the genotypes studied, ered with sterile aluminium foil and kept in the cold and dark (at 7¡C and the colchicine treatments did not basically modify the for 10 days) as pretreatment under aseptic conditions. During this period, the microspores in the anthers reached the premitotic devel- anther response of the given genotypes. However, the hy- opmental stage. The anthers were then dissected under sterile con- brid A21 performed better under both untreated and treated ditions and inoculated onto a modified YP medium (Genovesi and conditions. 860 Table 1 Influence of colchicine treatments on the androgenic re- Table 2 Effect of different colchicine concentrations on the divi- sponse of maize genotypes in anther culture (ns not significant) sion symmetry of androgenic microspores in maize anther culture (ns not significant) Genotype Treatment Number Anther Induction of response of micro- Genotypes Treat- Number Types of division (%) anthers (%) spore- ments of divi- inocu- derived- sions Asym- Sym- Atyp- lated structures counted metric metric ical (%) A632×DH309 Control 315 54.2 38.7 7.1 A632×DH309 Control 1100 12.25 22.25 0.02% 201 65.7 ns 24.9 ns 9.4 0.02% colchicine 1100 14.20 ns 20.30 ns 0.03% 232 52.1 ns 42.2 ns 5.7 0.03% colchicine 1100 13.65 ns 21.05 ns A21 Control 203 59.1 39.4 1.5 A21 Control 1100 15.75 41.10 0.02% 281 67.9 ns 28.8 ns 3.3 0.02% colchicine 1100 18.60 ns 46.90 ns 0.03% 312 65.4 ns 30.1 ns 4.5 0.03% colchicine 1100 18.10 ns 35.10 ns A21×F58 Control 243 63.8 31.3 4.9 A21×F58 Control 1100 13.15 27.35 0.02% 219 62.1 ns 32.8 ns 5.1 0.02% colchicine 1100 15.55 ns 32.10 ns 0.03% 218 65.6 ns 27.9 ns 6.5 0.03% colchicine 1100 18.45 ns 38.25 ns

Effect of colchicine on division symmetry Table 3 Effect of different colchicine treatments on the frequency in the induced microspores of different structures induced in maize anther culture (ns not signif- icant)

The cytological observations indicated that different con- Genotype Treatment Number Em- Calli centrations of colchicine did not have the same influence of struc- bryoids (%) on mitotic microspores. The higher colchicine concentra- tures (%) tion significantly increased the frequency of mitotic arrest counted in the microspore population. It appears that the applied A632×DH309 Control 136 35.3 67.4 concentrations of colchicine were not toxic to the micro- 0.02% colchicine 188 34.1 ns 65.4 ns spores; relatively high microspore mortality could gener- 0.03% colchicine 167 41.8 ns 58.2 ns ally be observed even under control conditions due to the A21 Control 204 30.9 69.1 low adaptability of the microspores to in vitro culture 0.02% colchicine 197 43.1 ns 56.9 ns conditions. Data concerning the effect of colchicine on 0.03% colchicine 112 43.8 ns 56.2 ns division symmetry are presented in Table 2. Asymmetri- A21×F58 Control 105 32.4 67.6 cal divisions dominated in all cases, independently of the 0.02% colchicine 142 41.5 ns 58.5 ns 0.03% colchicine 206 40.3 ns 59.7 ns treatments. This phenomenon was dominant in all the genotypes studied. The frequency of atypical divisions was low in every treatment: nuclear fragmentation could occasionally be observed in colchicine-treated micro- spores. quency of spontaneous chromosome doubling was very low except for genotype A21, but according to the pheno- logical observations (data not shown), most of the plants Morphotypes of microspore-derived structures were chimeric, having sectorial fertility in the tassels. The non-treated regenerants had a general tendency to tassel In the genotypes examined, there was no significant in- seed formation and a certain degree of feminisation, which crease in the frequency of embryo-like structures due to resulted in a loss of double-haploid plants due to the lack the treatments with colchicine. However, a slight in- of pollen for self-pollination. crease (maximum 10%) in embryo yield could be ob- The early genome doubling caused by colchicine in the served in the colchicine-treated cultures (Table 3). The induced microspores seemed to be efficient in increasing majority of induced structures were type I (compact) or the fertility of the regenerants. Even at the lower (0.02%) type II (friable) calli, independent of genotypes and treat- concentration, colchicine increased the number of fertile ments. double-haploid plants of a non-chimeric nature. All these double-haploid plants produced a high seed set (more than 70% on average) and no tassel seed formation was ob- Plant regeneration and fertility served. The application of 0.03% colchicine to microspores in the induction phase resulted in significantly higher plant The plant regeneration data (Table 4) demonstrate the fertility, with more than 80% seed set. The only exception problematic nature of in vitro plant differentiation and sur- (A21×F58 treated with 0.03% colchicine) was caused by vival in maize. Colchicine did not influence the plant re- plant loss due to an aphid infection under the artificial cli- generation process of the genotypes examined. The fre- mate. 861 Table 4 Plant regeneration frequency of microspore-de- Genotype Origin Number Plant Number Fertile rived structures. * Significantly of em- regeneration of fertile plants different from the control at bryoids plants (%) P = 0.05; ns not significant cultured n % A632×DH309 Control 48 9 18.75 1 11.1 0.02% colchicine 30 7 23.3 ns 4 57.1 ns 0.03% colchicine 28 6 21.4 ns 6 100.0* A21 Control 63 14 22.2 3 21.4 0.02% colchicine 85 13 15.3 ns 7 53.8 ns 0.03% colchicine 49 5 10.2 ns 4 80.0* A21×F58 Control 34 6 17.6 0 0.0 0.02% colchicine 59 12 20.3 ns 7 58.3* 0.03% colchicine 83 19 22.9 ns 3 15.7 ns

sociated proteins in the microspores would explain the Discussion diverse behaviour of microtubules and microfilaments to antimitotic drugs (Schmit and Lambert 1988). Rather than In recent years, a number of scientific reports have under- reducing the androgenic response of maize hybrids exam- lined the fact that genome doubling in haploids before plant ined, low concentrations of colchicine slightly increased regeneration may be more efficient than the conventional it. In contrast with wheat (Szakács and Barnabás 1995) and plant treatment with colchicine to produce genetically B. napus (Zaki and Dickinson 1995), in maize, colchicine stable double-haploid plants in various species, such as had no significant effect on the division symmetry. In this wheat, Brassica, tobacco and maize (Barnabás et al. 1991; species, the first microspore mitosis in culture remained Takashima et al. 1995; Zaki and Dickinson 1995; Saising- dominantly asymmetric. From the results of the present tong et al. 1996; Antoine-Michard and Beckert 1997). In study, the conclusion can be drawn that the first asymmet- recent research carried out on maize (Wan et al. 1991; Wan ric division of the androgenic microspore leads to primary and Widholm 1995), colchicine seemed to be the most ef- callus formation, since the frequency of symmetric divi- fective chromosome-doubling agent for microspore-de- sions shows a strong correlation with microspore-derived rived calli, helping to reduce mixoploidy and somaclonal embryo development, as was indicated in earlier work on variation in the offspring. Generally, colchicine surpassed wheat (Szakács and Barnabás 1988). the other antimitotic chemicals (oryzalin, pronamide and In maize anther culture, the asymmetric type of first mi- amiprophos methyl) and conventional plant treatments. totic division seems to be characteristic (Barnabás et al. In our recent work, relatively low concentrations (200 1987), together with callus formation. However, as a con- and 300 mg/l) of colchicine were found to efficiently dou- sequence of colchicine treatment on the induced anthers, ble the chromosome number of cold-pretreated premitotic a moderate increase in embryo yield could be observed. maize microspores in anthero at the onset of culturing, as It was found that all the genotypes examined were was similarly observed earlier in wheat (Barnabás et al. able to regenerate plantlets from both calli and embryoids. 1991) and in other small-grain cereals such as rice and tri- The plant regeneration capacity of microspore-derived tordeum (Alemanno and Guiderdoni 1994; Barcelo et al. structures was not influenced fundamentally by the colchi- 1994). The frequency of fertile plants directly regenerated cine treatments. It seemed that a short (3-day) exposure of from maize anther cultures was significantly higher for cold-pretreated anthers to a low concentration (0.03%) of early treatments with colchicine in the present experi- colchicine in the induction phase was sufficient for early ments. However, the action of colchicine on androgenic genome doubling if responsive genotypes and optimal maize microspores was different in nature to that observed treatment conditions were chosen for the cultures. This for the above-mentioned crops. The differences between procedure could help overcome the problems of a possible species in their responses to colchicine might be related to ploidy increase in the microspores caused by a longer the kinetics of mitotic division in culture (Zaki and Dick- (7-day) period of colchicine treatment (Saisingtong et al. inson 1991; Möllers et al. 1994). Different species and 1996; Antoine-Michard and Beckert 1997), which can re- even genotypes within a species require various time inter- duce the plant regeneration capacity of the microspore- vals from anther inoculation to the start of the first mitotic originated structures. division in the microspores. Pretreatments and culture As the extent of spontaneous versus colchicine-induced conditions could also greatly influence the cell cycle of chromosome doubling in maize anther culture could be microspores. Genotypes with high haploid induction ca- genotype and environment dependent, the treatments and pacity and synchronised microspore cultures could help to culture conditions should be adjusted for each individual reduce or eliminate these differences. Possible alterations case. Under the experimental conditions used in the present in the construction of the cytoskeleton in various gramina- work, enough seeds were set on plants showing normal ceous species may also contribute to the different responses morphological features to allow the further cultivation of to colchicine. The identification of tubulin- and actin-as- double haploid plants in the field. 862 The procedure of early genome doubling described Mathias R, Röbbelen G (1991) Effective diploidization of micro- above can be efficiently used in maize breeding pro- spore-derived haploids of rape (Brassica napus L.) by in vitro colchicine treatment. Plant Breed 106:82Ð84 grammes to save time and resources. Miao SH, Kuo CS, Kwei YL, Sun AT, Ku SY, Lu WL, Wang YY, Chen ML, Wu MK, Hang L (1978) Induction of pollen plants of Acknowledgements This work was supported by funds from the maize and observations on their progeny. In: Proceedings of sym- Hungarian Scientific Found (OTKA), grant nos. T16071 and T16072, posium plant tissue culture. Science Press, Peking, pp 23Ð33 and was carried out in connection with the European Cooperative Möllers C, Iqbal MCM, Röbbelen G (1994) Efficient production of Actions COST 824, Working Group 1 and COST 822, Working doubled-haploid Brassica napus plants by colchicine treatment Group 2. Bohus Obert would like to thank the UNESCO BETCEN of microspores. Euphytica 75:95Ð104 Agricultural Biotechnology Center, Gödöllo˝, for a 1-year scholar- Murashige T, Skoog F (1962) A revised medium for rapid growth ship, and for the continuation of the work granted by the Slovakian- and bioassays with tobacco tissue culture. Physiol Plant 15:473Ð Hungarian Bilateral Cooperation. 497 Petolino JF, Jones AM (1986) Anther culture of elite genotypes of maize. Crop Sci 26:1072Ð1074 Saisingtong S, Schmid JE, Stamp P, Büter B (1996) Colchicine-me- diated chromosome doubling during anther culture of maize (Zea References mays L.). 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