Euphytica (2016) 208:439–451 DOI 10.1007/s10681-015-1578-x Development of haploid embryos and plants of Lactuca sativa induced by distant pollination with Helianthus annuus and H. tuberosus Ł. Piosik . E. Zenkteler . M. Zenkteler Received: 10 June 2015 / Accepted: 9 October 2015 / Published online: 17 October 2015 Ó The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Haploidisation is a biotechnological estimation of the genome size by flow cytometry, method used to obtain plants with improved traits that chromosome counting in root tips, stomata cell size are of use to humans. Lettuce (Lactuca sativa L.), a and by disturbances in pollen formation resulting from well-known and popular leafy vegetable, is consumed abnormal microsporogenesis. This paper contains the worldwide. Its haploid form would provide a good complete protocol for obtaining haploid plants of L. basis for producing a pure line of plants (doubled sativa. haploids) allowing new varieties to be regenerated. The main aim of this work was to develop an effective Keywords Haploid Á Lettuce Á Parthenogenesis Á haploidisation method for this economically important Callus proliferation Á Distant pollination Á In vitro species. In order to stimulate the development of culture haploid embryos of lettuce based on our previous experience, we conducted in vivo distant pollination with fresh pollen grains of Helianthus annuus L. or H. tuberosus L. Because the haploid proembryos Introduction obtained after pollination did not develop further (despite the presence of cellular endosperm), we Haploid plants (characterized by gametic number of implemented the technique of in vitro culture of an chromosomes) are both significant and necessary isolated embryo sacs (surrounded by endothelium) elements in plant improvement programs. They are with parthenogenetic embryos on various, modified important in many basic research disciplines, such as Murashige and Skoog media. During the in vitro biotechnology, genetics, crop evolution and plant culture, we observed the formation of callus tissue breeding. Moreover, haploids are very useful as a base and, after subsequent cultures of calluses, 23 haploid for the production of homozygous plants in that they L. sativa plants were regenerated. The haploid status expedite the breeding process and can lead to an of the regenerated plantlets was confirmed by increase in crop yield. Lactuca sativa L. (lettuce) is a very popular and economically significant leafy vegetable that belongs to the Asteraceae family (De Vries 1997). Lettuce Ł. Piosik (&) Á E. Zenkteler Á M. Zenkteler leaves are highly nutritious and used widely as a Department of General Botany, Faculty of Biology, garnish for a variety of dishes. This species has Institute of Experimental Biology, Umultowska 89, relatively low requirements with regard to cultivation 61-614 Poznan, Poland e-mail: [email protected] conditions (Bradley et al. 2009). An efficient 123 440 Euphytica (2016) 208:439–451 haploidisation method would have great potential for Because a protocol for the haploidisation of L. sativa improving many phenotypic characteristics of this has not yet been developed, the main aim of the work species. reported here was to find an effective method for Modern biotechnology offers many recognised and obtaining haploid plants of this species. The first report effective techniques for inducing the development of on the development of haploid, globular embryos of L. haploid plants. The most common methods are: sativa after distant pollination and after chemical treatment was published by Piosik (2013). Haploid 1. Self-pollination using inactivated pollen grains: embryos of lettuce were induced by distant pollination for example, a method of deactivating pollen with pollen grains of 18 of 25 species (belonging mainly grains by gamma- or X-rays was used to induce to genera of the family Asteraceae) and by the the development of haploid embryos of Cucurbita application of seven tested chemicals to stigmas of L. moschata Duchesne ex. Poir (Kurtar et al. 2009), sativa (Piosik 2013). The frequency of developed Cucumis melo (Nasertorabi et al. 2012) and parthenogenetic embryos was dependent on the polli- Actinidia deliciosa (Musiał and Przywara 1998, nator species, with the most effective being Helianthus 1999; Pandey et al. 1990). annuus (19 %) and H. tuberosus (16 %), or on the type 2. Distant pollination of species which belong to of chemical inductor, with the most efficient being different species or genera: for example, haploid dicamba (15 %) and picloram (14 %) (Piosik 2013). embryos of Cichorium intybus L. were obtained a Unfortunately, none of the parthenogenetic embryos few days after pollination with Cicerbita alpina that were produced developed further and they degen- Walbr. (Dore et al. 1996) and also in carrot after erated in vivo very quickly. To induce the growth of crossing with parsley with the simultaneous haploid embryos and lettuce plants, we produced application of 2,4-dichlorophenoxyacetic acid cultures of ovaries, ovules and embryo sacs (surrounded (2,4-D) (Kiełkowska et al. 2014). by endothelium) in vitro upon their isolation after cross- 3. The bulbosum method—using in order to induce pollination and chemical treatment. We observed that the elimination of paternal chromosomes of callus tissue was produced from the haploid embryo embryo cells has been applied to several species, (developed after the pollination of L. sativa 9 H. such as Hordeum vulgare L., after crossing with annuus) inside one of the embryo sacs cultured H. bulbosum L. A process of elimination of in vitro. This result was very promising because the bulbous barley chromosomes was observed in callus originated from the dividing cells of the haploid hybrid embryos (Kasha and Kao 1970). Haploid proembryo. Consequently, we focused our experiments embryos were also obtained after crossing wheat on enabling the development of embryos after pollina- and maize (Gu et al. 2008). tion with H. annuus or H. tuberosus and on in vitro 4. Androgenesis this is a method for obtaining cultures of these proembryos inside whole embryo sacs. haploids from in vitro cultures of anthers and Finally, we developed an efficient method of producing isolated microspores; it has been used success- haploid L. sativa plants from haploid calluses that fully in many species, such as Brassica napus proliferated from parthenogenetic embryos produced (Cegielska-Taras 2004; Dubas et al. 2014), Ly- after cross-pollination with Helianthus pollen grains. copersicon esculentum Mill. (Shtereva et al. 1998), Aesculus hippocastanum L. (Radojevic et al. 2000), Triticum aestivum L. (Kyung-Moon Materials and methods and Baenziger 2005) and in Solanaceae crops (Segui-Smiarro et al. 2011). Plant material 5. Gynogenesis this method allows haploids to be obtained from an unfertilised egg cell or synergids The experimental material comprised male-sterile (Mo´l 1999; Musiał et al. 2005) through the (Fig. 1a–d) and fertile (Fig. 1e, control) forms of culturing of unfertilised ovules or ovaries, such Lactuca sativa (obtained from Rijk Zwaan R&D, as in Beta vulgaris L. (Gos´ka et al. 2004), Fijnaart, The Netherlands). Fertile plants of H. annuus Gentiana triflora Pall. (Doi et al. 2011)orAllium and H. tuberosus (growing wild) were used as pollen cepa (Musiał et al. 2001). donors. Male-sterile plants of lettuce were grown in a 123 Euphytica (2016) 208:439–451 441 Fig. 1 Flowers and pollen used in cross-pollination: a male-sterile lettuce plant with numerous flowering inflorescences; b flower head of lettuce composed of florets (c); d receptive stigma of L. sativa; e isolated pollen grain of L. sativa (control) culture room, using a 16/8-h photoperiod, photosyn- microscope with aniline blue (1 g/100 ml of H2O, thetic photon flux density of 250–300 lmol m-2 s-1, pH = 7.2) 1 day after pollination (1 DAP). For humidity of 40 % and appropriate irrigation. Fertile analysis of the development of haploid embryos, forms of lettuce were cultured in a separate culture ovaries and ovules were isolated 6 h after pollination room. (HAP) until 6 DAP, fixed in FAA solution (90 ml of 70 % ethanol ? 5 ml of formalin ? 5 ml of glacial Crossing technique acetic acid) embedded in Paraplast, sectioned with a microtome (Reichert; section thickness: 12 lm), For cross-pollination in vivo, pistils of open inflores- stained with iron haematoxylin (3 g/500 ml of 80 % cences of lettuce were used (Fig. 1a–c). Receptive ethanol) and counterstained with fast green FCF stigmas of L. sativa (Fig. 1d) were hand-pollinated (0.3 g/100 ml of clove oil). Stained permanent slides with freshly collected pollen grains of H. annuus and were enclosed in Entellan (Merck). Chromosome H. tuberosus (Table 1) using a tiny brush. All number was established on mitotic metaphase plates pollinated inflorescences were labelled using coloured of embryos on squashed slides of ovules or isolated threads. embryo sacs (surrounded by endothelium) stained with acetocarmine (1 % solution of carmine in 45 % Analysis of cross-pollination glacial acetic acid). The ratio of the number of haploid proembryos produced after crossing to the total The germination of foreign pollen grains on stigmas of number of analysed ovules is expressed as a L. sativa was analysed under a fluorescence percentage. 123 442 Euphytica (2016) 208:439–451 Table 1 Frequency of haploid embryos after distant pollination with Helianthus pollen Pollen donor species Number of pollinated Germination of pollen grains Frequency of haploid inflorescences on L. sativa stigma embryos (%) Helianthus annuus 6600 ? 15 Helianthus tuberosus 7200 ? 16 Embryo-rescue technique and plant regeneration 2 % PVP-10 (polyvinylpyrrolidone) and 15 nM b- mercaptoethanol and stained with propidium iodide. Enlarged ovaries obtained at 5–10 DAP were har- For analysis of the ploidy level, diploid L. sativa was vested and their surfaces were disinfected by 3-min used as an external standard. The suspensions were treatment with 2 % active chlorine water with con- passed through a nylon filter with a 50-lm mesh size.