Development of haploid embryos and plants of Lactuca sativa induced by distant pollination with Helianthus annuus and H. tuberosus

Abstract

Haploidisation is a biotechnological method used to obtain plants with improved traits that are of use to humans. Lettuce (Lactuca sativa L.), a well-known and popular leafy vegetable, is consumed worldwide. Its haploid form would provide a good basis for producing a pure line of plants (doubled haploids) allowing new varieties to be regenerated. The main aim of this work was to develop an effective haploidisation method for this economically important species. In order to stimulate the development of 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 obtained after pollination did not develop further (despite the presence of cellular endosperm), we implemented the technique of in vitro culture of an isolated embryo sacs (surrounded by endothelium) with parthenogenetic embryos on various, modified Murashige and Skoog media. During the in vitro culture, we observed the formation of callus tissue and, after subsequent cultures of calluses, 23 haploid L. sativa plants were regenerated. The haploid status of the regenerated plantlets was confirmed by estimation of the genome size by flow cytometry, chromosome counting in root tips, stomata cell size and by disturbances in pollen formation resulting from abnormal microsporogenesis. This paper contains the complete protocol for obtaining haploid plants of L. sativa.

Fruit set and plant regeneration in cassava following interspecific pollination with castor bean

“…These results confirm that the castor bean pollen used in this study was viable. Relatedly, in lettuce (Lactuca sativa L.) in vivo pollen germination was also observed following distant pollination with Helianthus annuus L. or H. tuberosus L. (£ukasz, 2012;Piosik et al, 2016). In a study on intergeneric cross-ability, pollen of poplar, Populus species, was seen to germinate on stigmas of willow, Salix viminalis and some pollen tubes were observed growing into the ovary (Zenkteler et al, 2005).…”

“…In other crops, crosses of carrot (Daucus carota L.) x parsley (Petroselinum crispum) haploids and embryo-derived homozygotic diploids were obtained (Kielkowska et al, 2014). Similarly, haploids were obtained in Lettuce (Lactuca sativa L.) x Helianthus annuus L. or H. tuberosus L. (Piosik et al, 2016). On the other hand, cross-incompatibility in interspecific and intergeneric hybridisations has hampered the development of hybrids in Brassicaceae crops (Kaneko and Bang, 2014).…”

“…These results suggest that parthenogenesis, was induced, but the resulting diploid embryos could have been due to spontaneous doubling of chromosomes in the induced embryo from the egg cell, or from fusion of two haploid cells in the embryo sac (either the synergids, or the antipodals, or the polar nuclei). Relatedly, in lettuce, Lactuca sativa, distant pollination technique was successfully used to induce haploid embryos, using pollen of Helianthus species and the ploidy level of the regenerated plants was easily detected by flow cytometry (Piosik et al, 2016). Intergeneric hybrids were also obtained in a cross of Salix aegyptica and Populus caspica (Ahmadi et al, 2010).…”

“…DHs can improve the efficiency and speed of the usually cumbersome, time-consuming, laborious and sometimes rather inefficient conventional breeding methods. They expedite the breeding process thus leading to an increase in crop yield (Piosik et al, 2016). Doubled haploids and/or haploids have been known to originate spontaneously in nature or as a result of various induction techniques (Murovec and Bohanec, 2012).…”

“…This therefore, necessitates early embryo rescue and further in vitro culture of embryos for onward growth and development (Kasha and Kao, 1970). Successful induction of haploids through wide crosses (induced parthenogenesis) has been reported in some crops; for example, in barley, Hordeum vulgare (Berzonsky et al, 2003); lettuce, Lactuca sativa L. (Piosik et al, 2016); carrot, Daucus carota L. (Kielkowska et al, 2014); cucumber, Cucumis sativus L. (Galazka et al, 2013); and wheat, Triticum aestivum (Inagaki and Hash, 1998). No DH cassava has been reported.…”