Vol.:(0123456789) 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-018-1399-4 ORIGINAL ARTICLE Efect of thermal shock in the development of androgenic plants of Anemone coronaria L.: infuence of genotype and fower parameters A. Copetta 1  · F. Dei 1  · I. Marchioni 1  · A. Cassetti 1  · B. Rufoni 1 Received: 4 October 2017 / Accepted: 21 February 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018 Abstract Anemone is an allogamous species marketed as a cut fower. Pure lines formation in this species is expensive and inefcient because progeny sufers from inbreeding depression. Pure lines obtained from plant anthers are a widely used material to produce new hybrids in short time. The aim of this study is the optimization of production of A. coronaria androgenic plants. Flower buds of eight cultivars were measured, disinfected for 20′ in 1% sodium hypochlorite and rinsed with distilled sterile water. Development stage of microspores and their size were evaluated. Anthers were distributed in Petri plates containing a double layer constituted of Nitsch and Nitsch agarized substrate enriched with activated charcoal (1%) and with or without sucrose (3%) and the same substrate in liquid phase without activated charcoal. Anthers were shocked with a preculture at 5 or 33 °C for 5 days and then incubated in the dark at 23 °C. After 21 weeks, the number of regenerants was counted. Plants were transferred into the fask with modifed MS substrate and placed at 18 °C. After 3 months of in vitro culture, the survival rate of plants was evaluated. Among the varieties, signifcant diferences in anther number, microspore size, and presence of abnormal microspores were observed. Heat shock pretreatment at 33 °C increases the number and survival of embryos and plants, but the varieties respond to the thermal shock in diferent ways. The infuence of genotype and the maturation phase of fower buds, anthers and microspores are discussed related to the androgenic plant development. Keywords Anemone · Anther culture · Heat shock · Breeding · Anomalous microspores Introduction Anemone coronaria L. is an herbaceous perennial species cultivated for cut fowers or garden plants. The interest in this species has grown with new improved varieties with fowers with larger petals and sepals, robust stems and early fowering for cut fower types, while garden types should produce numerous fowers and erect leaves with short peti- ole (Laura and Allavena 2007). Anemones are allogamous, predominantly cross-pollinated because stigmas are recep- tive 10 days before pollen releasing and dispersion by insect or wind. Pollen diameter is lesser than 40 µm, it is dry and able to stores carbohydrates. Self-pollination is possible among fowers of the same plant but the progeny often shows inbreeding depression symptoms (Horovitz et al. 1975). For this reason, existing cultivars and sub-cultivars are highly heterozygous, genetically non-uniform and often diferent in ploidy level (Laura et al. 2006; Laura and Allavena 2007). An in vitro protocol for rapid micropropagation of anem- one hybrids was established (Rufoni et al. 2005), although pure lines are needed for new hybrid production with stable genotype. The use of androgenic plant from anther culture is a method widely consolidated for the production of new hybrids in several cereal, horticultural, fruit and ornamental species included in diferent families as Asteraceae, Brassi- caceae, Cucurbitaceae, Poaceae, Ranunculaceae, Solanaceae and some species belonging to Fabaceae family (Germanà 2006, 2011; Ferrie and Caswell 2011). In vitro anther culture permit to develop androgenic embryos deriving from respon- sive microspores contained in anthers in specifc stage; in this case androgenic plants regenerate. Androgenic plants are useful for studies about gene expression and genetic Communicated by Sergio J. Ochatt. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11240-018-1399-4) contains supplementary material, which is available to authorized users. * A. Copetta andrea.copetta@crea.gov.it 1 CREA – Research Centre for Vegetable and Ornamental Crops, Corso Inglesi 508, 18038 Sanremo, IM, Italy