Impaginato 155 Adv. Hort. Sci., 2022 36(2): 155­159 DOI: 10.36253/ahsc­12818 Observation of unexpected neo like­fruit development from Cakile maritima calli D. Arbelet­Bonnin (*), S. Cangémi, P. Laurenti, F. Bouteau Université Paris Cité, LIED, F‐75013 Paris, France. Key words: Cell differentiation, in vitro culture, totipotency. Abstract: Parthenocarpy, the ability of some plants to undergo fruit growth in absence of fertilization, is an important question of basic science and the sub­ ject of much interest due to its possible agricultural benefits. In the context of our cellular biology studies on a halophyte of interest, Cakile maritima, we gen­ erated calli, pluripotent cell masses, that unexpectedly allowed the appearance of parthenocarpic fruits without any floral tissues. These observations raise the hope to develop an in vitro model to study parthenocarpic fruit development. 1. Introduction For several years, we are studying Cakile maritima, a promising model of halophyte in a worldwide context of increasing salinization of lands due to climate change (Arbelet­Bonnin et al., 2019). In addition to whole plant studies (Debez et al., 2006; Ellouzi et al., 2014; Ben Hamed­Louati et al., 2016 b; Arbelet­Bonnin et al., 2020) we underwent cellular biology stud­ ies (Ben Hamed­Laouti et al., 2016 a; Arbelet­Bonnin et al., 2018) for which we have developed a C. maritima cultured cell suspension that starts by calli generation as a first step (Ben Hamed et al., 2014). It is now admitted that in vitro cultured cells do not undergo a full dedifferentia­ tion but rather a transdifferentiation, leading to increased developmental potency and/or cell proliferation (Sugimoto et al., 2011; Fehér, 2019). The supposed totipotency capacity of plant cells (Haberlandt, 1902) is largely operated for the in vitro culture of plants. Calli are thus frequently consid­ ered as transient tissue dedicated to somatic embryogenesis allowing the plant regeneration (Sugimoto et al., 2011; Fehér, 2019). Interestingly, hormonal balances play an important role in many developmental proces­ ses in plant (Molesini et al., 2020), and in particular the balance between auxin and cytokine seems to be crucial during in vivo parthenocarpic fruit development (Pandolfini, 2009; Joldersma and Liu, 2018; An et al., 2020; Sharif et al., 2022). Parthenocarpy is the ability of some plants to undergo fruit growth in absence of fertilization. Parthenocarpy has contributed to some of humanity’s domestication of plants such as breadfruit or banana (Zerega et al., 2004; Kislev et al., 2006; Sardos et al., 2016). Furthermore, it repre­ sents a highly desirable trait in agronomy since seedless fruits are highly (*) Corresponding author: delphine.bonnin@u­paris.fr Citation: ARBELET­BONNIN D., CANGÉMI S., LAURENTI P., BOUTEAU F., 2022 ­ Observation of unexpected neo like‐fruit development from Cakile maritima calli. ­ Adv. Hort. Sci., 36(2): 155­159. Copyright: © 2022 Arbelet­Bonnin D., Cangémi S., Laurenti P., Bouteau F. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 17 February 2022 Accepted for publication 26 April 2022 AHS Advances in Horticultural Science Short note https://doi.org/10.36253/ahsc-12818 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2022 36(2): 155­159 156 appreciated by consumers and fruit set is less affect­ ed by environmental factors in absence of fertiliza­ tion (Ruan et al., 2012). Therefore, parthenocarpy is the subject of much interest and leads to many stud­ ies (Sharif et al., 2022). However, to our knowledge, all studies published so far have been conducted in whole plants but not at the level of cell cultures so far. We report here the first appearance of neo­like fruits directly on calli obtained and long maintained on callus­inducing medium (CIM). These observations raise the hope to develop a cellular model that will allow in vitro studies of parthenocarpic fruit develop­ ment. 2. Materials and Methods Plant material and establishment of calli Cakile maritima seeds used in this study come from Raoued (North of Tunisia). The seeds cultivation in vitro conditions were established as described by Ben Hamed et al. (2014). Briefly, bleached seeds were placed in petri­dishes containing Murashige and Skoog medium (1962) hormone­free (MS, Sigma), supplemented with 30 g.L­1 sucrose, 8 g.L­1 agar and the pH was adjusted to 5.8. This medium induced seed germination under a light cycle of 12 h light and 12 h dark with 40 µE m2 s­1 at 22°C. The stems of 14­ days­old seedlings were then chopped finely and placed on an agar­callus­induced medium (CIM) (Valvekens et al., 1988) and then put in a growth chamber, in the same conditions that for seed germi­ nation. CIM medium contain 6.2 g.L­1 Gamborg B5 (Gamborg et al., 1968) from Sigma supplemented with 20 g.L­1 glucose, 8 g.L­1 agar and phytohormones : cytokinin and auxin (Valvekens et al., 1988; Akama et al., 1992). The precise hormone balance depends of plant species (Pacheco et al., 2012; Thomas and Hoshino, 2015). For C. maritima we used 9.06 µM of 2,4­dichlorophenoxyacetic acid (2.4 D) and 0.46 µM of kinetin (Kn) that were shown to be efficient (Ben Hamed et al., 2014). The pH was adjusted to 5.7. After two weeks, calli were formed from the frag­ ments of seedlings tissues. When the size of the calli reached 1 cm in length, they were subcultured on a fresh CIM medium. For some experiments, the hormonal balance was modified with 9 µM of Kn and 2­4D, or 0.46 µM of Kn and 2­4D and calli were grown during two months. To assess the putative role of volatile organic com­ pounds (VOCs) emitted from mature fruit, a surface sterilized fruit of C. maritima was added in petri­dish­ es with calli during 2 months. 3. Results and Discussion Cakile maritima calli were subcultured every four weeks on the CIM medium, otherwise they began to brownish and lose their ability to be sub­cultured. During a callus subculture, a spontaneous green excrescence from calli easily visible (Fig. 1a) was observed. As the subcultures went along on fresh medium CIM, green excrescences grew on calli and more excrescences appeared on different calli (Fig. 1b and 1c). Each appeared excrescence grew all­long the time (Fig. 1d). After 58 days, numerous green structures resem­ bling C. maritima differentiated tissues arise (Fig. 2). 40% of them are clearly green differentiated tissue although we cannot identify specific organ structures (Fig. 2A, B). On the contrary, the other 60 % of green differentiated structures resemble neo like­fruits (Fig. 2C­E). Indeed, they present two asymmetric seg­ ments reminiscent of the typical dimorphic fruit that Fig. 1 ­ Emergence and development of parthenocarpic fruits on Cakile maritima callus. First observation (a), observations after 30 days (b); 49 days (c) and 58 days (d). Arbelet‐Bonnin et al. ‐ Neo like‐fruit from C. maritima calli 157 develops on whole plant of C. maritima (Fig. 2F). These neo like­fruits did not contain seeds and are thus parthenocarpic. Such parthenocarpic fruits have never been reported in C. maritima in the literature. More interestingly, this represents not only the first report of parthenocarpic fruits forming from calli, but also forming from non­floral tissues. These partheno­ carpic fruit appeared without any change in the hor­ mone balance, a mean generally used to triggered shoot or root regeneration in vitro in the context of somatic embryogenesis and plant multiplication (Thomas and Hoshino, 2015; Das et al., 2018; Shin et al., 2020). Parthenocarpy can be artificially obtained by applying synthetic growth factors to unpollinated ovaries (Pandolfini, 2009; Molesini et al., 2020). Auxins seem to play a prominent role in triggering and coordinating the transition from flower to fruit, and exogenous supplies of auxins to unpollinated flowers could induce fruit growth in various plants, suggesting that these hormones can replace the sig­ nals provided by pollination and fertilization (Pandolfini, 2009; Molesini et al., 2020). Cytokinin could also induced parthenocarpy but probably through modulation of auxin metabolism (Molesini et al., 2020; Sharif et al., 2022). We tried to modify the hormones concentrations and the balance between Kn and 2­4D by using 0.46 µM or 9 µM of both of these hormones but unfortunately, no new sponta­ neous green excrescences development were observed on the 20 calli present on each petri dish (n=2 per conditions). Since we observed a multiplica­ tion of these parthenocarpic fruits in the same petri­ dishes, we asked ourselves if this phenomenon could be reminiscent of fruit ripening triggered by volatiles organic compounds (VOCs) such as ethylene released from already developed fruits (Tohge et al., 2014). Even if the role of ethylene still appeared unclear (Sharif et al., 2022), ethylene responses could also lead to parthenocarpic fruit development (Pandolfini, 2009). We thus put a mature C. maritima fruit har­ vested from a fully developed plant in a petri­dish with calli, as a putative VOCs furnisher to calli. One more time no excrescences appeared on the 20 dif­ ferent calli present in the petri dish. Although we have no clear explanation at the moment, the ques­ tion of genetic homogeneity of callus cells is not solved and only certain cells of a callus could be regarded as totipotent and thus involved in organ regeneration (Fehér, 2019). Moreover, this ability to develop parthenocarpic fruits disappeared after three months and more subcultures. This suggests that only freshly prepared calli are able to develop parthenogenetic fruits. Accordingly, if callus tissue can express a wide variety of genes especially at the early phase of their development, their transcrip­ tome seems to be homogenize along time (Fehér, 2019). Fig. 2 ­ Various green structures observed on Cakile maritima callus. A­B. Undefined differentiated green structures. C­E. Typical dimorphic fruit like structures. F. Typical dimorphic fruit developed on a whole plant of C. maritima (arrows indicated the two asymmetric segments). Adv. Hort. Sci., 2022 36(2): 155­159 158 Although we cannot control the appearance and development of parthenocarpic fruits on calli at this time, these data certainly, deserve more investiga­ tions. Recent genomic studies have greatly con­ tributed to elucidate the role of phytohormones in regulating fruit initiation, providing at the same time genetic methods for introducing seedlessness in hor­ ticultural plants. Moreover, that some plants may produce fruit without the need for fertilization by the male gamete remains an important question of basic science. Therefore, the development of an in vitro model of parthenocarpic fruits will certainly be an essential tool to understand how a fruit could form without the need of fertilization nor floral tissues. 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