PRODUCTION OF HAPLOID PLANTS FROM ANTHER CULTURES AND SECONDARY EMBRYOIDS OF WINTER OILSEED RAPE, BRASSICA NAP US SSP. OLEIFERA

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1 Phytol. (9) 9, PRODUCTION OF HAPLOID PLANTS FROM ANTHER CULTURES AND SECONDARY EMBRYOIDS OF WINTER OILSEED RAPE, BRASSICA NAP US SSP. OLEIFERA BY LOH, CHIANG SHIONG AND D. S. INGRAM The Botany School, University of Cambridge, Downing Street, Cambridge CB EA, U.K. {Accepted 9 January 9) SUMMARY Following anther culture of a range of cultivars of winter oilseed rape, Brassica napus ssp. oleifera, approximately 5 to 7 % of the resulting embryoids developed directly into plants. The rest grew abnormally in culture, giving rise to abnormal plantlets which formed large numbers of secondary embryoids on their surfaces (up to per abnormal plantlet). The secondary embryogenic plantlets were haploid, and the secondary embryogenic potential was maintained for over a year in culture without diminution. More than 7% of the secondary embryoids, and the plants regenerated from them, were haploid. These results are discussed in connection witb tbe possibility of producing large numbers of baploid plants tbrougbout tbe year for breeding purposes and for tbe production of novel disease resistant plants tbrougb genetic manipulation in culture. INTRODUCTION Brettell and Ingram (979) have discussed the possibility of using cultured haploid plant cells or tissues as substrates for genetic manipulation, leading to the production of novel, disease-resistant lines of crop species. As a preliminary to exploring the feasibility of such an approach to the improverhent of winter oilseed rape, Brassica napus ssp. oleifera, the generation of haploid plants through anther culture was investigated. Following anther culture of spring oilseed rape (Thomas et al., 976; Keller and Armstrong, 977; Renard and Dosba, 9) haploid plants were produced successfully. However, only a certain proportion of the embryoids which emerged from the anthers developed directly into plants, while a larger proportion developed swollen hypocotyls and grew abnormally, producing secondary embryoids on their surfaces. Similar secondary embryoids were also observed by Sacristan and Hoffmann (979) on regenerating plantlets from protoplast cultures of haploid androgenic spring oilseed rape. The secondary embryoids apparently originated from single epidermal cells ( homas et al., 976). They could be subcultured on to a medium without addition oi any plant growth substances, and then gave rise to another generation of secondary embryoids in a similar way (Thomas et al., 976; Sacristan and ^offmann, 979). After four to six such passages, the secondary embryoids differentiated into seemingly normal plantlets without further secondary embryoid ormation (Thomas et al., 976). These plantlets were predominantly haploid \n = 9)^ i^^ diploids were found only rarely. In this paper we report further on ^ther culture, secondary embryogenesis and the nuclear cytology of secondary i regenerants of cultivars of winter oilseed rape. ^-646X//757 + $./ 9 The New Phytologist

2 5o LoH, CHIANG SHIONG AND D. S. INGRAM Anther culture MATERIALS AND METHODS Six cultivars of B. napus ssp. oleifera (winter oilseed rape) were used, namely Brinke,, Primor, Quinta, and Rapora. The seed was sown in the field in polyethylene tunnels at the Botany School Field Station, Cambridge during September 979 and 9, and fioral buds were harvested for anther culture during March and April 9 and 9. Unopened floral buds were surface sterilized in % (v/v) sodium hypochlorite solution, with a drop of Tween as wetting agent, for min. They were then washed with sterile distilled water and the anthers isolated aseptically and transferred to solidified culture medium contained in 5 cm plastic Petri dishes ( ml, six anthers per dish). The basal medium used was that of Keller, Rajhathy and Lacapra (975), with the addition of sucrose (%, w/v), Difco agar (-%, w/v), -naphthylacetic acid (NAA; - mg "^),,4-dichlorophenoxyacetic acid (, 4-D; - mg ~^), glutamic acid ( mg ~^) and serine ( mg "^). The ph was adjusted to 5* before autoclaving at 5 p.s.i. (-5 kpa) for min. Cultured anthers were incubated at C in darkness for 4 days and were then transferred to 5 C, also in darkness, until the emergence of embryoids. Maintenance of embryoids and secondary embryogenic cultures Embryoids which emerged from anthers were transferred to the same medium as described above, with the sucrose level reduced to % (w/v), and contained in 9 cm Petri dishes sealed with Nescofilm.They were then incubated in a growth chamber at 5 C under continuous light from fluorescent tubes (-695 mw cm"^ at the surface of the cultures). Subsequently, green plantlets and secondary embryogenic tissues were maintained on Murashige and Skoog's basal medium (96) supplemented with sucrose ( % w/v) and Difco agar (- % w/v), but without plant growth substances. Such cultures were incubated in the growth chamber at C with a h photoperiod. When the plantlets became too large for the Petri dishes, they were transferred to fresh medium contained in ml Elenmeyer flasks closed with aluminium foil. When it was necessary to regenerate whole plants, plantlets of about 5 cm height were taken out of the flasks and planted in compost in 4- cm plastic pots. They were then kept in the growth chamber at C and to 9 % relative humidity with a 6 h photoperiod for to 4 weeks, before being transferred to a glasshouse. They were finally planted out in soil in open-ended polyethylene tunnels in the field. Cytology Secondary embryoids or root tips were detached from parental tissues with forceps and fixed in ethanol: glacial acetic acid (:) for to h. They were then washed with N HCl before hydrolysis in IN HCl for 9 min at 6 C. After hydrolysis the tissues were rapidly washed with 45 % acetic acid and stained overnight in % natural orcein in 45 % acetic acid. Squashes were prepared in % natural orcein in 45 % acetic acid and counts of chromosomes made using a light microscope. In the case of secondary embryoids, counts were usually obtained from at least five dividing nuclei per embryoid. In the case of plantlets and plants, normally three to five root tips were examined per individual, and counts obtained from at least five dividing nuclei per root tip.

3 Haploid plants from secondary embryoids 59 RESULTS Anther culture and the establishment of secondary embryogenic tissues A preliminary experiment was made to determine the relationship between bud length and the production of embryoids from anthers. The best results were obtained from anthers taken from buds of - to 5 mm length (Table ). Phase contrast microscopy showed that the pollen grains from such buds were either uninucleate or binucleate. Thus in subsequent experiments, anthers from buds - to -5 mm long were always used. Table. The production of embryoids by cultured anthers taken from different sizes of unopened floral buds of winter oilseed rape cv. Primor Bud length (mm) and above No. of anthers cultured No. of anthers producing embryoids (% total) (-7%) 7(-7%) 4 (- o) (7-%) (%) After to 6 weeks in culture, embryoids were successfully obtained from a relatively small proportion of anthers of all six cultivars of oilseed rape tested (Table ). A proportion of the embryoids of each cultivar, except Brinke, differentiated into plants directly, but to 6% grew on to produce secondary embryoids on their hypocotyl surfaces (Table ). Development of these secondary embryogenic plantlets was abnormal to varying degrees: the hypocotyls were usually swollen and white rather than green in colour, and normal leaves were seldom produced. Secondary embryoids became visible to the naked eye on the surfaces of the swollen hypocotyls to 4 weeks after the embryoids first emerged from anthers. They were green in colour and easily observed against the whitish background of the swollen hypocotyls (Fig. ). In some cases embryoids emerged from the surfaces of apparently normal hypocotyls (i.e. not swollen) and also from the surfaces of green cotyledons, but this was unusual. All secondary embryoids developed through globular, heart and cotyledonary stages [Fig. l(d), (e) and (f)]. After being detached from the parental tissue and transferred to fresh medium, roots were initiated from the radicle end and cotyledons from the proximal end of each secondary embryoid, thus demonstrating their bipolar nature [Fig. l(a)]. Usually, between 4 and % of the plantlets of secondary embryoid origin developed abnormally and were capable of producing urther secondary embryoids on their hypocotyls. The rest developed into plants r»d did not produce further secondary embryoids [Fig. l(c)]. The number of secondary embryoids produced on each secondary embryogenic culture ranged ro one to over a hundred, but was usually forty to fifty at any one time. Monthly lre of secondary embryogenic cultures or detached secondary embryoids in maintenance of the secondary embryogenic potential for over a year Without diminution.

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5 Haploid plants from secondary embryoids 5 '.. b».^ -V.. ' " -*w Fig.. Secondary embryoids arising from the swollen hypocotyls of abnormal, pollen-derived haploid plantlets of winter oilseed rape, (a) many small secondary embryoids are visible on the surface of the abnormal plantlet while others have been removed to the surrounding agar. A further embryoids have been removed from this plantlet and transferred to fresh medium to be grown on into plants (not illustrated), (b), (c) secondary embryoids growing-on into plantlets while still attached to the abnormal parental plantlet. Small secondary embryoids are also visible on the surface of the abnormal plantlet in (b). (d), (e), (f) close-up views of secondary embryoids on the surfaces of the swollen hypocotyls of abnormal parental plantlets.

6 5 LoH, CHIANG SHIONG AND D. S. INGRAM Table. The ploidy of winter oilseed rape tissues with secondary embryoids Number of cultures Cultivar examined* Haploid Other ploidy levels Primor Quinta Rapora Total * Three to five root tips were fixed for each culture and counts made on at least five dividing nuclei per tip. Cytology The ploidy of secondary embryogenic tissues. Secondary embryogenic cultures with roots and with visible secondary embryoids on their surfaces were selected at random and root tips fixed for cytological examination. All were haploid {n = 9) [Table ; Fig. (c)]. The ploidy of secondary embryoids. Newly emerged secondary embryoids were obtained from secondary embryogenic cultures andfixedfor cytological examination I year after anther culture. A large proportion (*4%) were haploid {n = 9) [Table 4; Fig. (a)]. Eight (-6%) were found to be diploid {n = ) [Fig. (b)]. However, these were all from a single secondary embryogenic culture; haploid embryoids were also formed on the same culture. The ploidy of regenerants. A total of nearly plants were regenerated from secondary embryoids year after anther culture. Forty-three of these plants were selected at random and sample root tips excised and fixed for cytological examination. Thirty-two plants (74-4%) were found to be haploid [Fig. (d)] and II (5-6%) were diploid [Table 5; Fig. (e)]. No polyploids were detected. A total of 55 regenerants were planted out in polyethylene tunnels in the field during the summer of 9. Of the which lowered (Table 6), (9-9%) had characteristic haploid flowers and only (*%) had characteristic diploid flowers [Table 6; Fig. (e) and (f)]. It is notable that some of the regenerants flowered without artificial vernalization (Table 6). DISCUSSION In oilseed rape, the frequencies of naturally occurring haploids are low, ranging from - to -65 % (Thompson, 975; Renard and Dosba, 9). During recent years several attempts have therefore been made to obtain haploid plants through anther culture (Thomas and Wenzel, 975; Wenzel et al., 977; Keller and Armstrong, 977, 97; Keller and Stringam, 97; Hansson, 97; Renard and Dosba, 9). A breeding scheme to utilize anther-derived haploids has been devised and a timetable for cultivar development formulated (Keller and Stringam, 97). Regrettably, except for a small number of spring cultivars, the frequencies of embryoid production through anther culture have also been low (Tables and 7). In order to obtain sufficient numbers of haploids for breeding programmes and for genetic manipulation, large numbers of anthers have to be plated in a relatively

7 Haploid plants from secondary embryoids :. -.-?r-^; "...'" ' '^V':' '.' -.,.' '.- :. "I. - - " :» '. \ I}.. Fig.. (a) to (e) Chromosomes in cells of winter oilseed rape at metaphase of mitosis. All were stained with % natural orcien in 45% acetic acid, (a) Cell from a haploid (K = 9) secondary embryoid. (b) Cell from a diploid {In = ) secondary embryoid. (c) Cell from a root-tip excised from a haploid {n 9) secondary embryogenic plantlet. (d) Cell in a root tip excised from a haploid {n = 9) plant regenerated from a secondary embryoid. (e) Cell in a root tip from a diploid (K = ) plant regenerated from a secondary embyroid.(f) Plants regenerated from secondary embryoids of of winter oilseed rape, year after anther culture. The plant labelled A is diploid and that labelled B is haploid. (g) Flowers from plants regenerated from secondary embryoids. The flower labelled A is haploid and that labelled B is diploid. Scale bar = cm.

8 54 LoH, CHIANG SHIONG AND D. S. INGRAM Table 4. The ploidy of winter oilseed rape secondary embryoids obtained year after anther culture No. of parental No. of secondary No. No. Cultivar cultures embryoids examined haploid diploid Rapora Total 4 4 5(-4%)t * (-6%)t * All from a single secondary embryogenic culture, which also produced haploid secondary embryoids. t Percentage of total number of embryoids examined. Table 5. The ploidy of winter oilseed rape regenerants obtained from secondary embryoids year after anther culture No. of regenerants No. No. Cultivar examined haploid diploid Rapora Total / \# (74-4 o) 4 4 (5-6 /o) * Percentage of total number of regenerants examined. short flowering season. Therefore, the success in the long term maintenance of secondary embryogenic tissues following anther culture described here has important implications in winter oilseed rape breeding. Our results show that such a potential can be maintained for at least a year after anther culture, without diminution. Large quantities of secondary embryoids and regenerants can be obtained in a relatively short period, and only a small number of anthers need be plated to initiate embryogenic cultures. Further, previous workers (Wenzel et al., 977; Keller and Armstrong, 97) have found that a maximum of only % of plants regenerated from anther cultures were haploid, the rest being diploid. Renard and Dosba (9) reported that 4- % of regenerants from anthers of the cross R XL 7 were haploid, but the numbers involved were very small since only 9 regenerants of this cross were examined. In our experiments, by contrast, -4% of secondary embryoids of winter oilseed rape and 74-4 % of regenerants were haploid. A high yield of haploids such as we obtained is essential for the purposes of breeding and genetic manipulation. The haploids regenerated from secondary embryoids were obtained year after anther culture, which clearly indicates the stability of haploidy in the system. The presence of secondary embryoids on cultured plantlets derived from embryoids and other secondary embryoids could serve as a useful marker for the presence of haploidy, although we have now frequently observed secondary embryoid formation on anther-derived tissues diploidized by treatment with colchicine (unpublished data). By subculturing secondary embryogenic tissues at regular intervals it should be possible to maintain a continuous supply of large numbers of haploids over a very

9 Haploid plants from secondary embryoids 55 Table 6. Elowering of winter oilseed rape plants regenerated from secondary embryoids Cultivar Original embryoid from anther (embryoid no.) Total no. of regenerants No. of regenerants with characteristic haploid flowers* No. of regenerants with characteristic diploid flowersf No. of regenerants without flowers Batch A Rapora Batch B Rapora Totals Batch C Rapaora Totals (9-7%)t 4 4 ( %)t Vernalization: Batch A, regenerants vernalized at 5 C for 7 weeks with a 7 h photoperiod before planting out; Batch B, regenerants planted out on 5 May 9, without artificial vernalization, and scored on July 9 (temperatures below 5 C recorded on several nights during late May and early June 9); Batch C, regenerants planted out on July 9 without artificial vernalization and scored on September 9 (temperatures below 5 C not recorded during the growing period). * Approximately half the diameter of diploid flowers, not extruding pollen and seeds not set. t Anthers extruding pollen and seed set. X Percent of total number of regenerants. lable 7. Frequencies of embryoid production through anther culture of oilseed rape Cultivar No. of embryoids per anthers cultured References spring type varieties and winter lines Tower and Midas (spring) Tower (spring) Tower (spring) Summer rape Tower (spring) Ospal X Bronowski (spring) XL7 (winter) R X L7 (winter) -65 O-5--O Wenzel ef a/., 977 Keller and Armstrong, 977 Keller and Armstrong, 97 Keller and Stringam, 97 Hansson, 97 Renard and Dosba, 9 Renard and Dosba, 9 Renard and Dosba, 9 Renard and Dosba, 9

10 5i6 LoH, CHIANG SHIONG AND D. S. INGRAM long period of time. These would have the advantage over naturally occurring haploids and haploids produced direct from anthers in being clonal, since each secondary embryogenic culture probably originates from a single pollen grain. It is not yet known whether genetic variation develops spontaneously or can be induced artificially in secondary embryoid producing cultures, but plants regenerated from such cultures do show phenotypic variation with regard to various characters (unpublished results). Experience with other crop species (Brettell and Ingram, 979; Bretell, Ingram and Thomas, 9) suggests that this form of variation may be common. Further research with oilseed rape will now be directed towards identifying novel forms of disease resistance in plants regenerated from secondary embryoids, with and without prior treatment with mutagens and other agents, and incorporating any novel resistant lines so produced in breeding programmes. ACKNOWLEDGEMENTS We thank the British Council and the Lucy Ernst Scholarship Trust for the support of one of us (C.-S.L.), B. Goddard and M. Seekings for technical assistance, Dr R. Michelmore for advice on cytology and Dr K. Thompson of the Plant Breeding Institute, Cambridge, for the provision of growth room facilities. REFERENCES BRETTELL, R. I. S. & INGRAM, D. S. (979). Tissue culture in the production of novel, disease-resistant crop plants. Biological Reviews, 54, BRETTELL, R. I. S., INGRAM, D. S. & THOMAS, E. (9). Reversion of Texas male-sterile cytoplasm maize in culture to give fertile, toxin-resistant plants. Theoretical and Applied Genetics, 5, HANSSON, B. (97). Temperaturchocker - ett satt att kraftigt hoja frekvensen embryoidbildningar vid antherkultur av raps {Brassica napus). Sveriges Utsddesforenings Tidskrift,, 4-4. KELLER, W. A., RAJHATHY, T. & LACAPRA, J. (975). In vitro production of plants from pollen in Brassica campestris. Canadian Journal of Genetics and Cytology, 7, KELLER, W. A. & ARMSTRONG, K. C. (977). Embryogenesis and plant regeneration in Brassica napus anther cultures. Canadian Journal of Botany, 55, -. KELLER, W. A. & ARMSTONG, K. C. (97). High frequency production of microspore-derived plants from Brassica napus anther culture. Zeitschrift Pflanzenziichtg,, -. KELLER, W. A. & STRINGAM, G. R. (97). Production and utilization of microspore-derived haploid plants. In: Frontiers of Plant Tissue Culture (Ed. by T. A. Thorpe) pp. -. The International Association for Plant Tissue Culture, Calgary. MuRASHiGE, T. & SKOOG, F. (96). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiological Plantarum, 5, RENARD, M. & DOSBA, F. (9). Etude de l'haploide chez le Colza {Brassica napus L. var. oleifera Metzger). Annales des Ameliorationes des Plantes,, 9-9. SACRISTAN, M. D. & HOFFMANN, F. (979). Direct infection of embryogenic tissue cultures of haploid Braw^'' napus with resting spores of Plasmodiophora brassicae. Theoretical and Applied Genetics, 54, 9-. THOMAS, E., HOFFMANN, F., POTRYKUS, I. & WENZEL, G. (976). Protoplast regeneration and stem embryogenesis of haploid androgenetic rape. Molecular and General Genetics, 45, THOMAS, E. & WENZEL, G. (975). Embryogenesis from microspores of Brassica napus. Zeitschrift fh^ Pflanzenzuchtg, 74, 77-. THOMPSON, K. F. (975). Homozygous diploid lines from naturally occurring haploids. In: Proceedings Fourth International Rapeseed Conference, pp Giessen, West Germany. WENZEL, G. F., HOFFMANN, F. & THOMAS, E. (977). Anther culture as a breeding tool in rape. I. Ploidy level and phenotype of androgenetic plants. Zeitschrift fiir Pflanzenziichtg, 7,

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