Christine Strullu-Derrien
email: c.strullu-derrien@nhm.ac.uk

Michel Ballèvre

Alan R T Spencer

Erwan Guillou

Jean-Paul Gislard

Hubert Lardeux

Christopher J Cleal

The Carboniferous (Serpukhovian) macroflora from the "Coteaux du Pont Barré", Beaulieu-sur-Layon (Maine-et-Loire), South of the Armorican Massif, France

Keywords: Carboniferous, Serpukhovian, coal swamps, macrofloras, Maine-et-Loire

The "Coteaux du Pont Barré" in Beaulieu-sur-Layon is a Regional Natural Reserve which is home to exceptional flora and fauna. Recently fossil plants have been discovered at the site adding to its natural heritage significance. The exposure which is part of the "Sillon Houiller de la Basse-Loire" contains Serpukhovian-age (330-320 Ma) remains of sphenophytes, ferns, and lyginopteridopsid and cycadopsid pteridosperms, which are described for the first time as well as the geology of the site. Most Carboniferous macroflora previously described from the Maine-et-Loire has been based on ex situ specimens from now abandoned and inaccessible coal mines. The newly described macroflora shows evidence of in situ remains and differs somewhat in composition from those reported from the coal workings, especially in the total absence of lycopsids and Calymmotheca pteridosperms, and instead having abundant medullosalean foliage (Neuralethopteris). The co-occurrence of Sphenopteris elegans and Neuralethopteris densifolia suggests a slightly younger age compared to the macrofloras documented from the coal-bearing deposits. However, this could be an ecological consequence of the flora growing in better-drained substrate habitats.

Introduction

The coal-swamps that extended over large areas of palaeotropical Euramerica during the Pennsylvanian and Cisuralian (late Carboniferous and early Permian) was one of the most important Palaeozoic terrestrial biomes (e.g. [START_REF] Dimichele | Response of late Carboniferous and Early Permian plant communities to climate change[END_REF][START_REF] Greb | Evolution and importance of wetlands in earth history[END_REF][START_REF] Cleal | Plant biodiversity changes in Carboniferous tropical wetlands[END_REF]Cleal 2021a, b), providing the habitat in which many biotic groups diversified including insects, arachnids, reptiles and gymnosperms, and having a major impact on atmospheric composition and climate through enhanced transpiration and carbon sequestration. As a result, this biome has attracted considerable interest from geologists and palaeontologists, to try to determine its long-term impact on Earth history.

However, the earliest evolution of the coal swamp biome in the latest Mississippian (Serpukhovian) is less well understood. Palaeobotanical remains (hereafter referred to as macrofloras -see [START_REF] Pardoe | Palaeobotanical experiences of plant diversity in deep time. 2: how to measure and analyse past plant biodiversity[END_REF] of the Serpukhovian proto-coal swamps are well preserved in the Pays-de-Loire region of northwestern France and were documented in detail notably by [START_REF] Bureau | Bassin Houiller de la Basse Loire[END_REF][START_REF] Bureau | Bassin Houiller de la Basse Loire[END_REF] and more recently by [START_REF] Strullu-Derrien | A rare late Mississippian flora from Northwestern Europe (Maine-et-Loire Coalfield, Pays de la Loire, France)[END_REF], 2022). Most of this evidence was derived from coal workings that are no longer accessible and, although many of the fossils are still available in museum geoheritage collections, it is difficult to interpret the depositional context of the macrofloras.

In this paper, we document a macroflora that was recently collected from an exposure, the "Coteaux du Pont Barré", along the Layon River in Beaulieu-sur-Layon, 20 km south of Angers (Maine-et-Loire). The site is well known, especially for its extant flora long studied by the botanist Robert Corillion (1908Corillion ( -1997)), who first established the protection of the area [START_REF] Corillion | Sur le caractère méridional et la richesse de la flore de Pont-Barré[END_REF]. The macroflora was discovered some years ago by the winemaker who owned the site and his collaborators. Nowadays, most of the site belongs to the "Domaine Belargus", a leading biodynamic winery in the Loire Valley, and is managed for its protection by the "Ligue de Protection des Oiseaux (LPO) Anjou".

This macroflora differs in composition from those reported earlier from the coal workings in this area, which we have interpreted as representing vegetation that grew in comparatively less welldrained habitats.

Geological setting

The studied outcrop is part of the "Sillon houiller de la Basse-Loire" [START_REF] Bureau | Bassin Houiller de la Basse Loire[END_REF][START_REF] Bureau | Bassin Houiller de la Basse Loire[END_REF], a narrow (0.5 to 2 km) zone of Carboniferous coal-bearing outcrops extending over a length of about 100 km. This zone is bounded between two major domains, the Saint-Georges-sur-Loire Unit to the north, and the Mauges Unit to the south (Figure 1a, b; e.g. [START_REF] Cartier | The Hercynian orogeny in the South Armorican Massif (Saint-Geroges-sur-Loire Unit, Ligerian Domain, France): rifting and welding of continental stripes[END_REF][START_REF] Cartier | The Saint-Georges-sur-Loire olistostrome, a key zone to understand the Gondwana-Armorica boundary in the Variscan belt (Southern Brittany, France)[END_REF][START_REF] Ballèvre | Palaeozoic history of the Armorican Massif: Models for the tectonic evolution of the suture zones[END_REF][START_REF] Lin | Pre-Variscan tectonic setting of the south margin of Armorica: insights from detrital zircon ages distribution and Hf isotopic composition of the St-Georges-sur-Loire Unit (S. Armorican Massif, France)[END_REF]. The deposition setting for these coal-bearing strata is interpreted as narrow pull-apart basins, deve- loped during dextral displacement along the crustalscale Nort-sur-Erdre Fault [START_REF] Ballèvre | Palaeozoic history of the Armorican Massif: Models for the tectonic evolution of the suture zones[END_REF].

The outcrop is located on the right bank of the Layon River, about 500 m north-west of 2a). It is situated in a Regional Natural Reserve, with the majority of the outcrop located along the middle part of the slope dominating the Layon River, and above the renowned vineyard, the "Coteau des Treilles" (Figure 1d). Two main formations are seen [START_REF] Blaise | Notice explicative de la carte géologique de France, feuille Thouarcé[END_REF]):

(1) To the north-east, mafic schists of the Saint-Georges-sur-Loire Unit display a strongly dipping foliation to the NNE. They consist of mafic volcanics, in most cases fine-grained tuffs and breccias, sometimes containing flattened limestone clasts, up to 10-12 cm long. No fossils have been identified in these fine-grained, fully recrystallized limestones. (2) To the south-west of the schists are found steeply-dipping, alternating layers of Carboniferous conglomerates, sandstones sometimes grading to siltstones, and less frequently shaly layers. Several layers of conglomerates may be seen. Most conglomerates grade from clast-to matrix-supported, the coarser varieties are very poorly sorted while finer layers are much better sorted. They display well-rounded, abundant pebbles of various lithologies, namely milky quartz (derived from quartz veins), rhyolites and tuffs, schists and quartzites. The size of the pebbles is smaller in the southerly layers compared to those close to the contact with the St Georges schists, where an outcrop contains pebbles up to 30 cm. The size of the pebbles, and their nature (with large amounts of phtanites, i.e. siliceous shales) suggest a rather proximal deposit, mainly derived from the northerly situated St-Georges-sur-Loire Unit, consistent with previous observations [START_REF] Blaise | Notice explicative de la carte géologique de France, feuille Thouarcé[END_REF]). Fossil plants have been mainly found within reddish coloured sandy-to -silty layers interbedded within conglomerates (Figures. 1e-g; red star in Figure 2). These are observed in situ in the upper part of the vineyard slope "Coteau des Treilles". A Mesocalamites (cast of stem) occurs in situ (Figure 1g) along the bedding surface of a polygenic conglomerate. This indicates transport and deposition of the plant remains by fast-flowing water (e.g. rivers).

The contact between the St-Georges-sur-Loire and the Carboniferous sediments is not well exposed in the Pont-Barré area. At a regional scale, this contact is generally considered as a major, crustal-scale, fault, as shown on the geological map of Thouarcé [START_REF] Blaise | Notice explicative de la carte géologique de France, feuille Thouarcé[END_REF]. This fault has been called the Nort-sur-Erdre (or Layon) Fault, and displays a long, polyphase Variscan history [START_REF] Cartier | The Saint-Georges-sur-Loire olistostrome, a key zone to understand the Gondwana-Armorica boundary in the Variscan belt (Southern Brittany, France)[END_REF][START_REF] Ballèvre | Palaeozoic history of the Armorican Massif: Models for the tectonic evolution of the suture zones[END_REF][START_REF] Lin | Pre-Variscan tectonic setting of the south margin of Armorica: insights from detrital zircon ages distribution and Hf isotopic composition of the St-Georges-sur-Loire Unit (S. Armorican Massif, France)[END_REF]. In addition, it has been reactivated during several periods during the Mesozoic and the Cenozoic, as indicated in the studied area by the difference in altitude of the base of the transgression by Cenomanian sediments (Figure 2; [START_REF] Poirier | Essai sur la morphologie de l'Anjou méridional (Mauges et Saumurois)[END_REF][START_REF] Louail | La transgression crétacée au Sud du Massif Armoricain. Cénomanien de l'Anjou et du Poitou[END_REF][START_REF] Blaise | Notice explicative de la carte géologique de France, feuille Thouarcé[END_REF][START_REF] Sellier | Essai sur la notion de coteau en Pays de la Loire[END_REF]

Material and methods

Specimens collected on the outcrops were borrowed from Erwan Guillou of the "Ligue de Protection des Oiseaux (LPO) Anjou" where they are currently stored. Later, they will be on exhibit at the "Domaine Belargus" winery located at Saint-Lambert-du-Lattay, Maine-et-Loire, France. Specimens under the names PalB 001 to PalB017 were photographed under natural light, using a Nikon D500 camera with a 180 mm macro lens.

Descriptions

In all cases, genera and species referred to here are fossil-taxa (sensu [START_REF] Cleal | Botanical nomenclature and plant fossils[END_REF][START_REF] Cleal | Naming of parts: the use of fossil-taxa in palaeobotany[END_REF], whereas suprageneric taxa are whole-plant taxa. Following [START_REF] Agosti | Recommendations for use of annotations and persistent identifiers in taxonomy and biodiversity publishing[END_REF], full details of the authorship of all taxa are given.

Class: EQUISETOPSIDA Agardh, 1825 -Genus: Mesocalamites Hirmer (1927) emend. [START_REF] Remy | Calamitopsis n. gen. und die Nomenklatur und Taxonomie von Calamites Brongniart 1828[END_REF] Mesocalamites cistiiformis (Stur) Hirmer (1927) (Figures 1g,e) (≡ Calamites cistiiformis Stur 1877) Description: Linear, unbranched axes up to 20 mm wide, preserved for up to 150 mm long. Fine longitudinal ribs with bluntly acuminate ends, ca. 15 ribs per cm across axis. Clearly marked nodes across axis, with longitudinal ribs sometimes passing directly across or sometimes off-set. Internodes at least 100 mm long, as no preserved stem shows more than one node. No branch scars preserved.

Remarks: These axes represent calamite stems, some definitely as pit-casts, but others as possible stem compressions. They are characterised by long internodes, very fine longitudinal ribs that only sometimes alternate at the nodes, and an absence of branch scars, and are very similar to the specimens figured by Crookall (1969, pl. 111, Figures 1,2), Purkynová (1970, pl. 8, Figure 4) and Josten (1983, pl. 2, Figures 1, 2) as M. cistiiformis. [START_REF] Jongmans | Anleitung zur Bestimmung der Karbonpflanzen West-Europas mit besonderer Berücksichtigung der in den Niederlanden und den benachbarten Länderen gefundenen oder noch zu erwartenden Arten. I Band Thallophytae[END_REF] combined this species with Calamites cistii Brongniart (1828), which also has axes with fine longitudinal ribs, long internodes and few branch scars, but the ribs in the latter species consistently alternate at the nodes [START_REF] Crookall | Fossil plants of the Carboniferous rocks of Great Britain [Second Section]. Part 5[END_REF]. There is also a comparison with Mesocalamites taitianus (Jongmans and Kidston) [START_REF] Hirmer | Handbuch der Paläobotanik[END_REF], but the latter has rather wider ribs, shorter internodes and large, oval branch scars. Remarks: Since this single specimen represents the foliage on a relatively wide stem, it is difficult to definitely ascertain the shape of the leaf whorl. However, the general shape of the leaves and the presence of a basal collar makes it most comparable to A. radiata and Annularia ramosa Stur, (1887). These species of sphenophyte foliage are poorly constrained, and we have used A. radiata as the comparison simply because it is the more widely used name. Description. Strobilus slightly tapered, maximum (proximal) width 9 mm, preserved for a length of 41 mm. Strobilus axis ca. 1 mm wide with whorls of at least 4 bracts at intervals of 4-5 mm. Bracts initially directed proximally, straight for the first half of length and then curve so their distal part is ca 45° to strobilus axis. Large carbonaceous mass (?sporangiophore) attached to axil of bract and strobilus axis.

Remarks. This strobilus clearly shows what are presumably the sporangiophores in an axillary position, indicating that it belongs to Palaeostachya. Most sphenophyte strobili from Namurian macrofloras belong to Calamostachys (e.g. [START_REF] Stockmans | Végétaux namuriens de la Belgique (Texte)[END_REF][START_REF] Josten | Die fossilen Floren im Namur des Ruhrkarbons[END_REF]), but [START_REF] Purkynová | Die Unternamurflora des Beckens von Horní Slezsko (CSSR)[END_REF] described Palaeostachya strobili from the upper Arnsbergian (uppermost Serpukhovian) of Upper Silesia. Although we have only one specimen of this strobilus, it is of generally similar size and shape and so we tentatively compare it with Purkynová's specimens. Description: Bipinnate pinna fragments up to 40 mm long. Penultimate rachis straight and slender (1 mm wide). Ultimate rachises slender (ca. 0.5 mm wide), alternately arranged and oblique to penultimate rachis. Ultimate pinnae up to 15 mm long, spaced 5 mm apart, gradually tapered along most of length. Pinnules alternate, oblique, subtriangular with rounded to acute apex, ca 2 mm long, 1 mm wide. The venation is unclear, but each pinnule appears to have a prominent midvein along most of its length.

Specimen

Remarks: This specimen is very similar to the P. plumosa figured by Dalinval (1960, pl. 11, Figure 1; pl. 12, Figure 2) and Josten (1983, pl. 46, Figure 3), and corresponds to the forma delicatula of that species.

Specimens PalB 007-008

Class: LYGINOPTERIDOPSIDA [START_REF] Novák | rostliny. Tracheophyta[END_REF] -Genus: Sphenopteris (Brongniart) Sternberg KM von (1825) (≡ Filicites (Sphenopteris) Brongniart (1822) Sphenopteris elegans (Brongniart) Sternberg KM von (1825) (Figure 4d) (≡ Filicites (Sphenopteris) elegans Brongniart ( 1822)

Description: Once-pinnate fragments of fronds up to 55 mm long, 12 mm wide, gradually tapered to a deeply incised terminal segment. Rachis slender (<1 mm wide), straight or slightly deflected. Lateral pinnules alternate, obliquely inserted at 45° or less in the distal part of pinna, gradually changing to 60-70° in more proximal positions. Pinnules rhomboidal, up to 6 mm long, 5 mm wide, deeply incised into up to eight segments, each segment especially in more proximal parts of pinnule further divided into up to three subsidiary segments; segments/subsegments narrowly lanceolate with bluntly acuminate apex. Thin, basally decurrent midvein extends for most of the pinnule length and producing thin lateral veins, one for each ultimate segment. Pinnule lamina well developed so that there are only narrow gaps between each segment.

Remarks: These pinna fragments with deeply incised, somewhat obliquely attached pinnules, with bluntly acuminate ultimate segments, compare well with S. elegans as illustrated by [START_REF] Kidston | Fossil plants of the Carboniferous rocks of Great Britain. Parts 1-4[END_REF] and [START_REF] Purkynová | Die Unternamurflora des Beckens von Horní Slezsko (CSSR)[END_REF]. Although the pinnule shape is typical for this species, their size is rather small, but this is probably just a function of them coming from a relatively distal position in the pinna. There are some similarities with the Westphalian Palmatopteris furcata (Brongniart) Potonié ( 1893), but the pinnules of the latter are not so oblique to the rachis and have more bluntly terminated pinnule lobes. S. elegans typically ranges between the upper Cardioneuropteris antecedens and Calymmotheca larischii Zones [START_REF] Wagner | Megafloral zones of the Carboniferous[END_REF][START_REF] Opluštil | Carboniferous macrofloral biostratigraphy: an overview[END_REF] Specimen PalB 009 -Genus: Mariopteris Zeiller (1878) -?Mariopteris sp. (Figure 4c,e)

Description. Linguaeform, pinnatifid pinnules with rounded to bluntly acuminate apex and partly constricted base, 10-15 mm long, 3-5 mm wide. Thin, strongly decurrent midvein extends for much of the pinnule length. Lateral veins alternate and produce a bunch of final veins in each of the pinnule lobes (details of the veining pattern not preserved).

Remarks. These fragments compare with remains of Mariopteris fronds from Pennsylvanian macrofloras such as Mariopteris muricata (Sternberg) [START_REF] Zeiller | Végétaux fossiles du terrain houiller[END_REF], but are too small to make a definite species assignment.

Specimens PalB 010, 011

Class: CYCADOPSIDA Brongniart, 1843 -Genus: Neuralethopteris Cremer (1893) emend. [START_REF] Laveine | Contribution a l'étude de la flore du terrain houiller. Les Neuroptéridées du Nord de la France[END_REF] Neuralethopteris densifolia [START_REF] Josten | Die fossilen Floren im Namur des Ruhrkarbons[END_REF] (Figure 5) Description. Linguaeform, more-or-less bilaterally symmetrical pinnules with parallel margins, broadly rounded apex and constricted base, up to 9 mm wide; largest preserved pinnule 40 mm long. Most pinnules are isolated, but the few that are attached are at 80-90° to the rachis. Prominent midvein extends for most of the pinnule length. Lateral veins alternately arise from midvein and narrow angle, rapidly arched in a somewhat angular pattern and then extend to the pinnule margin, which it meets at 70-90°. Lateral veins fork up to four times depending on pinnule width, producing a vein density of 45-50 per cm on pinnule margin.

Remarks. Although these pinnate fragments are small, the basally constricted, bilaterally symmetrical pinnules with a strong midvein clearly places them within the fossil-genus Neuralethopteris, and the often elongate pinnules with a dense, slightly angular venation indicates N. densifolia. This is a rare species that has only previously been recorded from the Ruhr in north Germany [START_REF] Josten | Die fossilen Floren im Namur des Ruhrkarbons[END_REF][START_REF] Jenchen | Die ehemalige Ziegeleitongrube Voßacker und die Abgrabung am Küchenberg, zwei fossilführende Aufschlüsse im tiefen Oberkarbon[END_REF], where it occurs in the Pecopteris aspera Zone of Kinderscoutian to Yeadonian age [START_REF] Wagner | Megafloral zones of the Carboniferous[END_REF][START_REF] Opluštil | Carboniferous macrofloral biostratigraphy: an overview[END_REF]. Specimens PalB 012-017.

Interpretation of the macroflora

Compared with many of the Maine-et-Loire macrofloras described by [START_REF] Bureau | Bassin Houiller de la Basse Loire[END_REF][START_REF] Bureau | Bassin Houiller de la Basse Loire[END_REF] and [START_REF] Strullu-Derrien | A rare late Mississippian flora from Northwestern Europe (Maine-et-Loire Coalfield, Pays de la Loire, France)[END_REF], which originated from grey, coalbearing sequences, the Pont Barré macroflora is relatively low in diversity. It also differs in the total absence of lycopsids and of Calymmotheca pteridosperms and instead has abundant medullosalean foliage (Neuralethopteris). Similar fern -pteridosperm macrofloras of about the same age from Upper Silesia are interpreted as the remains of vegetation from better-drained substrates and are termed "flöz-fern" macrofloras [START_REF] Havlena | Die flöznahe und flözferne Flora des oberschlesischen Namurs A und B[END_REF]Gastaldo et al. 2009a, b). This ecological interpretation appears to be supported by the Pont Barré macroflora being preserved in coloured, reddish clastic sediment, which could indicate well-drained conditions and a low water table (see, for instance, comments by [START_REF] Besly | Origin of red beds in a moist tropical climate (Etruria Formation[END_REF][START_REF] Besly | Palaeosols in Westphalian coal-bearing and red-bed sequences, central and northern England[END_REF]. As the Pont Barré fossils are relatively fragmentary and preserved in a high-energy water deposited stratigraphical sequence, they are clearly allochthonous. However, since there is no evidence of taxa such as Adiantites, Pseudadiantites and Rhacopteridium that have been interpreted as allochthonous remains of hinterland vegetation (e.g. [START_REF] Wagner | The extrabasinal elements in lower Pennsylvanian floras of the Maritime Provinces, Canada: description of Adiantites, Pseudadiantites and Rhacopteridium[END_REF], it is likely that this macroflora was derived from vegetation growing in well-drained alluvial habitats within the basin (see discussion by [START_REF] Thomas | Distinguishing Pennsylvanian-age lowland, extra-basinal and upland vegetation[END_REF].

It is difficult to place biostratigraphically such a low diversity macroflora and the co-occurrence of Sphenopteris elegans and Neuralethopteris densifolia appears to be in conflict as their previously known ranges do not overlap. However, the lower limit of the range of N. densifolia is not well constrained, being based on the less well-documented floras of the lower Namurian of the Ruhr [START_REF] Josten | Die fossilen Floren im Namur des Ruhrkarbons[END_REF], whereas the upper limit of the range of S. elegans is better constrained to the lower Chokierian such as in Upper Silesia [START_REF] Purkynová | Die Unternamurflora des Beckens von Horní Slezsko (CSSR)[END_REF]. In our view, the Pont Barré macroflora can probably be assigned to the Calymmotheca larischii Zone indicating an Arnsbergian or early Chokierian age in the Heerlen Regional Chronostratigraphy, making it a little younger than the macrofloras documented from the coal-bearing deposits of this area [START_REF] Strullu-Derrien | A rare late Mississippian flora from Northwestern Europe (Maine-et-Loire Coalfield, Pays de la Loire, France)[END_REF]; this would make a closer comparison with the macrofloras from the Poruba Formation of Upper Silesia (e.g. [START_REF] Purkynová | Die Unternamurflora des Beckens von Horní Slezsko (CSSR)[END_REF]), which also includes Neuralethopteris.

Conclusions

The "Coteaux du Pont Barré" at Beaulieu-sur-Layon (Maine-et-Loire, France) has yielded a newly discovered macroflora of late Mississippian age. The allochthonous fossils occur in coloured, reddish clastic deposits suggesting relatively well-drained conditions, and reflect a vegetation dominated by medullosalean pteridosperms, tedeleacean ferns and sphenophytes. It is markedly different from most of the other macrofloras from the "Sillon houiller de la Basse-Loire" (e.g. [START_REF] Strullu-Derrien | A rare late Mississippian flora from Northwestern Europe (Maine-et-Loire Coalfield, Pays de la Loire, France)[END_REF], which are more diverse and tend to be dominated by lycopsids (e.g. Lepidodendron) and Calymmotheca (pteridosperm) fronds, and are probably the remains of vegetation from swampier, poorer-drained habitats. The Pont Barré macrofloras therefore provides a valuable, contrasting insight into the vegetation that formed the origins of the coal swamp biome, which in early Westphalian (early Pennsylvanian) times expanded to cover swathes of tropical Euramerica.

Figure 1 .

 1 Figure 1. (a) Geological map showing the location of the studied area (Beaulieu-sur-Layon). (b) Location of the area (rectangle) within the Armorican Massif (NASZ: North Armorican Shear Zone; SASZ(N) and SASZ(S): respectively, the northern and southern branches of the South Armorican Shear Zone; NSEF: Nort-sur-Erdre Fault). (c) Overview of the "Coteaux du Pont Barré" on the right bank of the Layon River (river visible on the right side). (d) the "Coteau des Treilles" vineyard. (e-g) Different views of the outcrop. Mesocalamites (cast of stem) (g), along the bedding surface of a conglomerate.

Figure 2 .

 2 Figure 2. Geological setting of the studied site, indicated by a red star. (a) Simplified geological map of the studied area, modified from Blaise et al. (1986). The Nort-sur-Erdre Fault (NSEF) separates the Saint-Georges-sur-Loire and the Ardenay Units, to the North and South, respectively. A narrow band of Carboniferous sediments ("Sillon houiller de la Basse-Loire") is located along the southern contact with the Nort-sur-Erdre Fault. Palaeozoic rocks are unconformably covered with a thin sheet of Cenomanian sands and clays. (b, c) Schematic cross-sections of the studied area. Note that the plant-bearing beds are observed in sandy to silty layers interbedded within conglomerates.

Figure 3 .

 3 Figure 3. (a-c, e) Mesocalamites cistiiformis (Stur) Hirmer (PalB 001-003, PalB 005); (a, b) pith casts; (c) ?cast of stem; (e) cast of stem showing leaf-attachment scars. (d) Annularia cf. radiata (Brongniart) Sternberg (PalB 004), leaf whorl. (f) Palaeostachya cf. mostensis Purkynová (PalB 006), part of strobilus showing bracts and axillary ?sporangiophores. Scale bars: 0.42 cm (a); 0.55 cm (b); 0.47 cm (c); 0.84 cm (d); 0.50 cm (e); 0.30 cm (f).

Figure 4 .

 4 Figure 4. (a, b) Pecopteris plumosa (Artis) Brongniart (PalB 007, 008), slender, obliquely attached pinnules. (c, e) ?Mariopteris sp. (PalB 010, 011), linguaeform, pinnatifid pinnules typical of Mariopteris. (d) Sphenopteris elegans (Brongniart) Sternberg (PalB 009), ultimate pinna with typical incised pinnules. Scale bars: 0.37 cm (a), 0.40 cm (b, c); 0.55 cm (d); 0.15 cm (e).

  Pecopteris (Brongniart) Sternberg KM von (1825) emend. Cleal (2015) (≡ Filicites (Pecopteris) Brongniart (1822) Pecopteris plumosa (Artis) Brongniart (1836) (Figure 4a, b) (≡ Filicites plumosus Artis 1825)

Figure 5 .

 5 Figure 5. Neuralethopteris densifolia Josten (PalB 012-017), with pinnules of very variable length-to-breadth ratio, but consistent venation, as is typical for this fossil-genus. Scale bars: 0.47 cm (a); 0,50 cm (b, c); 0.40 cm (d); 0.70 cm (e); 0.30 cm (f).

C. STRULLU-DERRIEN ET AL.

Acknowledgments

We thank Ivan Massonnat (owner) and Jo Pithon (previous owner) of the "Domaine Belargus" vineyard and winery, Valérie Chalumeau from the LPO Anjou and Julien Le Berre (for image 3e). CS-D is supported for her work by the Fondation ARS Cuttoli-Paul Appell-Fondation de France.

Funding

The work has been partly supported by a grant from the Fondation ARS Cuttoli-Paul Appell to C S-D.

Author contributions

CS-D designed the research program; CJC and CS-D contributed to the palaeobotany; MB, HL, ARTS contributed to the geology; J-PG photographed the fossil plants (except 3e) and CS-D photographed the site and the outcrop; CJC, MB, CS-D wrote the first version of the manuscript; all authors contributed to the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.


Ankush S Ranaware

Nandkumar S Kunchge

Smita S Lele

Sergio J Ochatt
email: sergio.ochatt@inrae.fr

Iyyakkannu Sivanesan

Alice Hayward

Jayeni Hiti Bandaralage

Chris M O'brien

Protoplast Technology and Somatic Hybridisation in the Family Apiaceae

Keywords: biotechnological breeding, carrot, celery, cybrids, male sterility, protoplast isolation and fusion

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The rapidly growing population has made crop improvement for enhancing yield and quality to center stage, in order to serve the ever-increasing food needs. Since the onset of crop improvement, almost ten thousand years ago, selection and breeding have been used routinely and continuously for crop improvement, as stressed by the late eminent plant molecular biologist, Lawrence Bogorad from Harvard University [START_REF] Chadipiralla | Plant tissue culture and crop improvement[END_REF]. The limited range of genes accessible through conventional sexual breeding within crossable plants species has hindered the introduction of genes or traits of interest. Thus, for many crops a long period is required for the purification and selection process for a desired trait or gene in a new genotype. Technology advancement in the manipulation of cells in vitro and genetic engineering offer an alternative to conventional plant breeding and provide novel approaches for gene pooling that hitherto were not available naturally in the environment [START_REF] Suenaga | Basic studies on transfer of cytoplasmic male sterility by means of cytoplasmic hybridization in carrot: (Daucus carota L.)[END_REF].

At present, the role of agriculture in ensuring food security is strategic to meet the increasingly growing population demand and simultaneously address the very many up-surging environmental challenges resulting from global warming [START_REF] Kopittke | Soil and the intensification of agriculture for global food security[END_REF]. According to the most recent United Nations estimates (https://www.worldometers.info/; accessed on 3 February 2023), the world population reached 7.93 billion in March 2022. However, it would more likely exceed 9 billion according to an AsiaNews (PIME) report that already in most recent United Nations estimates (https://www.worldometers.info/; accessed on 3 February 2023), the world population reached 7.93 billion in March 2022. However, it would more likely exceed 9 billion according to an AsiaNews (PIME) report that already in 2017 about a billion people around the world had no identities and were hence invisible. Still, their need for food adds to the huge demand that already exceeds the current production globally (https://www.asianews.it/news-en/About-a-billion-people-are-invisible,-one-third-of-them-children-42131.html) (accessed on 9 December 2022). It is therefore necessary to rapidly boost food output without expanding agricultural lands but also using less water, fertilizers, pesticides and herbicides per cultivated hectare in order to reduce emissions from production processes and environmental pollution.

Crops of importance for our diet under cultivation on the planet cover different surfaces and produce variable yields (Figure 1). Amongst them, open-pollinated cultivars are majoritarian in the market for members of the family Apiaceae, such as carrot (Daucus carota), celery and celeriac (Apium graveolens var. dulce and var. rapaceum, respectively). Such cultivars, although productive, often lack uniformity and yield regularity and these problems may be resolved by developing and using hybrid seeds. The availability of highquality male sterile lines is of paramount importance in this respect and remains currently a bottleneck for hybrid breeding of crops in the family Apiaceae. In this respect, male sterility results from the failure to produce either dehiscent anthers, functional pollen, and/or viable gametes. Its evolutionary importance has long been recognized but its breeding utility was initially ignored, until the breeding potential of hybrid vigor as a means of studying the influence of cytoplasm on plant development was acknowledged. However, if the offspring of any cross between two genetically different In this respect, male sterility results from the failure to produce either dehiscent anthers, functional pollen, and/or viable gametes. Its evolutionary importance has long been recognized but its breeding utility was initially ignored, until the breeding potential of hybrid vigor as a means of studying the influence of cytoplasm on plant development was acknowledged. However, if the offspring of any cross between two genetically different plants can be considered to be a hybrid, the process is not that simple. Indeed, in self-fertile crops it requires emasculation of flowers, which is laborious and costly or, when performed Plants 2023, 12, 1060 3 of 21 chemically, is environmentally unfriendly. Hence the great interest of male-sterile genotypes for such crops [START_REF] Chen | Male sterility and fertility restoration in crops[END_REF].

In this review, we focus particularly on both genetic (GMS) and cytoplasmic (CMS) male sterility [START_REF] Chen | Male sterility and fertility restoration in crops[END_REF][START_REF] Yu | Analysis of genetic diversity in cytoplasmic male sterility, and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee)[END_REF], on the candidate genes involved in the process, and on the production of such male sterile genotypes through somatic hybridization. Thus, we review the pretreatments of donor plants, the different tissues used as sources of the parental protoplasts, the methods for obtaining somatic hybrids and cybrids as well as their subsequent culture. Crucial steps for understanding cell wall regeneration in species of the family Apiaceae are also discussed.

The Role of Male Sterility in Plant Breeding

There are two sources of male sterility, genetically encoded (GMS) and cytoplasmic (CMS), which bypass the time-consuming work of flower emasculation in umbels of crops in the family Apiaceae. GMS is determined only by genes encoded in the nuclear genome [START_REF] Chen | Male sterility and fertility restoration in crops[END_REF], while CMS is due to mitochondrial genes that affect nuclear gene functions directly or indirectly.

Genetically Encoded Male Sterility (GMS)

GMS is under the control of various nuclear male sterility genes that are not influenced by cytoplasmic sequences. As a result of this, in a simple genetic situation with a Mendelian inheritance pattern there will be three possible genotypes for the nuclear locus MS (Male Sterility), where the male sterile phenotype is determined by the allele at its recessive (ms) status (Figure 2). Therefore, for such a male sterile genotype (which in breeding would represent the female line), the offspring may be 100% male fertile (for a parental line homozygous for the nuclear restorer-of-fertility locus) or segregate 1:1 yielding 50% male sterile: 50% male fertile plants (for a heterozygous parent).

Using GMS for breeding requires availability of a male sterile line (the female parent), a maintainer line (isogenic but for the presence of a dominant MS allele), and a restorer line (the male parent, which has dominant restorer-of-fertility alleles Rf and thus produces fertile F1 hybrids). In this context, the segregation in the cross with the maintainer line introduces an additional selection step among male sterile phenotypes to remove all heterozygotes for the production of hybrid seeds, which has limited the use of GMS in crops where CMS is not available. Cytoplasmic male sterility (CMS) has been observed in more than 300 species to date [START_REF] Yu | Analysis of genetic diversity in cytoplasmic male sterility, and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee)[END_REF]. This laborious practice of handling a male sterile line, a maintainer line and a restorer line simultaneously in the GMS system can be overcome by environment-sensitive genic male sterility (EGMS). Thus, in rice 5 to 10% higher yield is obtained with respect to land area based on EGMS system. EGMS plays a crucial role in selection of male sterility dependent on sensitivity and adaption to the environmental factors such as high or low temperature, humidity and photoperiod [START_REF] Virmani | Environment-sensitive genic male sterility (EGMS) in crops[END_REF]. In a rapeseed TGMS (thermosensitive genic male sterile) line, a temperature above 20 • C resulted in an alteration of gene expression due to a defect of homologous pairing in meiosis I [START_REF] Liu | TGMS in rapeseed (Brassica napus) resulted in aberrant transcriptional regulation, asynchronous microsporocyte meiosis, defective tapetum, and fused exine[END_REF].

In wheat, a TGMS line was fertile at 20 • C and sterile at 10 • C as a result of miRNA differential expression [START_REF] Kim | Molecular control of male fertility for crop hybrid breeding[END_REF]. Recently, Zhu and colleagues hypothesized that in an Arabidopsis TGMS line low temperature slowed down pollen development which in turn helped in restoring fertility of the thermo-sensitive male sterile plant [START_REF] Zhu | Slowing development restores the fertility of thermo-sensitive male-sterile plant lines[END_REF].

Cytoplasmic Male Sterility (CMS)

CMS, being maternally inherited, is the preferred material since it permits a better control of hybrid seed production [START_REF] Bohra | Cytoplasmic male sterility (CMS) in hybrid breeding in field crops[END_REF]. It has been increasingly applied to a number of major cereal, vegetable, legume, oilseed, industrial and ornamental crops, including various members of the family Apiaceae on which this review is focused. However, it must be stated that in general the hybrids developed have a narrow genetic base due to the use of a very limited number of sources of CMS in plant breeding. Therefore, future research in this domain should concentrate on widening the range of cytoplasmic genes that produce male sterile phenotypes as also their respective nuclear-encoded restorer-of-fertility genes. The recent advent of new genetic tools to study the dynamics of the mitochondrial

Cytoplasmic Male Sterility (CMS)

CMS, being maternally inherited, is the preferred material since it permits a better control of hybrid seed production [START_REF] Bohra | Cytoplasmic male sterility (CMS) in hybrid breeding in field crops[END_REF]. It has been increasingly applied to a number of major cereal, vegetable, legume, oilseed, industrial and ornamental crops, including various members of the family Apiaceae on which this review is focused. However, it must be stated that in general the hybrids developed have a narrow genetic base due to the use of a very limited number of sources of CMS in plant breeding. Therefore, future research in this domain should concentrate on widening the range of cytoplasmic genes that produce male sterile phenotypes as also their respective nuclear-encoded restorer-of-fertility genes.

The recent advent of new genetic tools to study the dynamics of the mitochondrial genome as well as its interaction with the nuclear genes offers novel experimental frameworks to efficiently address these challenges [START_REF] Gandini | The complete organelle genomes of Physochlaina orientalis: Insights into short sequence repeats across seed plant mitochondrial genomes[END_REF][START_REF] Sanchez-Puerta | Homologous recombination and retention of a single form of most genes shape the highly chimeric mitochondrial genome of a cybrid plant[END_REF].

CMS phenotypes have often arisen in wild populations through spontaneous mutations involving rearrangements of the mitochondrial genome (mtDNA), which resulted from intragenomic homologous or non-homologous recombination events and created new open reading frames (ORFs) [START_REF] Dufaÿ | Modelling the maintenance of male-fertile cytoplasm in a gynodioecious population[END_REF]. Disruption of the nuclear gene Msh1 by a transgenic RNAi approach in both tomato and tobacco led to stoichiometry in plant mitochondria which allowed expression of the CMS phenotype ORFs which were at low copy number [START_REF] Sandhu | Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants[END_REF]. Yet, in onion the existence of the CMS ORF in fertile lines at extremely low concentrations plays an important role for the expression of ORF [START_REF] Kim | Identification of a gene responsible for cytoplasmic male-sterility in onions (Allium cepa L.) using comparative analysis of mitochondrial genome sequences of two recently diverged cytoplasms[END_REF]. A full understanding of the molecular mechanisms underlying the CMS condition remains difficult because of its uniqueness in terms of the mitochondrial genes associated with the male sterility condition [START_REF] Heng | Cytological and iTRAQ-based quantitative proteomic analyses of hau CMS in Brassica napus L[END_REF]. Furthermore, there are, for instance, CMS lines frequently used in rice breeding where a specific restorer line was developed but the identity of the gene responsible for the male sterility condition is still not known [START_REF] Huang | Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility[END_REF][START_REF] Kazama | Whole mitochondrial genome sequencing and re-examination of a cytoplasmic male sterility-associated gene in Boro-Taichung-Type cytoplasmic male sterile rice[END_REF].

Figure 2 also depicts a scheme of the genetic model of CMS. Mitochondrial dysfunction is caused by nuclear MADS-box genes that control whorls 2, 3 and even whorl 1. Out of 16 MADS-box genes, DcPI and DcAG-like genes showed a significantly lowered expression in petaloid CMS lines. Among 12 differentially expressed genes of oxidative phosphorylation, the two genes DcNad2 and DcNad5, which encode NADH dehydrogenase had a significantly lower expression in a petaloid CMS line of carrot as compared to the maintainer line. This led to the formation of a petaloid or carpeloid CMS system, due to energy deficiency in mitochondria which constitute the cell powerhouse [START_REF] Liu | Differentially expressed genes between carrot petaloid cytoplasmic male sterile and maintainer during floral development[END_REF]. This shows a larger energy requirement of reproductive organs compared to other organs. Likewise, in species of other families e.g., wild beet, constitutive mRNA expression of orf129 of CMS protein was observed in flowers, leaves and roots, but it only affected the anther development and led to male sterility. In maize, too, CMS-T protein also governs cytotoxic activity resulting in male sterility, while it is also toxic to Escherichia coli and eukaryotic cells. Thus, to date, CMS proteins were reported in sunflower, radish, rice, Brassica sp., among others [START_REF] Chen | Male sterility and fertility restoration in crops[END_REF].

Programmed cell death (PCD) is a necessary developmental event that regularly happens in animals and plants. For plants, it involves developmental processes such as seed germination, root tip elongation, xylem and aerenchyma formation, organ development, senescence and disease resistance, among others [START_REF] Reape | Apoptotic-like regulation of programmed cell death in plants[END_REF]. PCD also plays an important role in CMS development through a co-operative interaction between the anther wall and the microspores. Such interaction requires the cellular degradation of the innermost cell layer of the anther (tapetum) which surrounds the microspores [START_REF] Ma | Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants[END_REF]. In a carpeloid CMS line of carrot, the expression of two nuclear located MADS-box genes DcMADS2 and DcMADS3 was suppressed in developing flowers of whorls 2 and 3, which are homologues to GLOBOSA and DEFICIENS of the Antirrhinum genes [START_REF] Linke | Flower development in carrot CMS plants: Mitochondria affect the expression of MADS box genes homologous to GLOBOSA and DEFICIENS[END_REF]. In Citrus, expression analysis found an involvement of miRNAs targeting transcription factors involved in floral development of Citrus grandis (male sterile cybrid pummelo), such as auxin response factors (ARFs), MYB, SQUAMOSA promoter binding protein box (SBP-box), APETALA2 (AP2), basic region-leucine zipper (bZIP) and transport inhibitor response 1 (TIR1). This indicated a strong interaction between mitochondrial CMS genes and nuclear MADS-box genes, while nuclear-cytoplasmic retrograde signaling must be playing an important role in CMS flower development through miRNA expression as well [START_REF] Fang | High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis)[END_REF].

Alternatively, a fertile line and a CMS line can be combined through somatic hybridization to give a male-sterile plant with a chimeric mitochondrial genome, as the limited homologous recombination yields a mtDNA with few regions from the CMS parent, whereby candidate genes for CMS may be proposed [START_REF] Arimura | Frequent fusion and fission of plant mitochondria with unequal nucleoid distribution[END_REF]. Conversely, if mtDNA rearrangements generate numerous new ORFs, a differential expression assay and/or a segregation analysis will be required for the identification of the CMS candidate [START_REF] Makarenko | Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower[END_REF]. On the other Plants 2023, 12, 1060 6 of 21 hand, some CMS systems have recently been linked to intron splicing and RNA editing, where cytidines are changed to uridines in specific editing sites located in diverse positions of mitochondrial mRNAs [START_REF] Dufaÿ | Modelling the maintenance of male-fertile cytoplasm in a gynodioecious population[END_REF]. This alters organellar protein products, and renders RNA editing essential as it allows the synthesis of functional organellar proteins crucial for plant and seed development [START_REF] Yang | The pentatricopeptide repeat protein EMP9 is required for mitochondrial ccmB and rps4 transcript editing, mitochondrial complex biogenesis and seed development in maize[END_REF]. Thus, impairing mitochondrial function through a deficient RNA editing induces the synthesis of abnormal proteins that, in turn, may induce male-sterility. Interestingly, the exact relationship between the candidate CMS gene and the observed phenotype has been assessed rarely, while the mechanisms of action of the restorer-of-fertility loci remain poorly understood. Comparing directly the mtDNAs, although difficult in such highly rearranged genomes, could help in identifying the gene(s) responsible for the CMS condition [START_REF] Kim | Identification of a gene responsible for cytoplasmic male-sterility in onions (Allium cepa L.) using comparative analysis of mitochondrial genome sequences of two recently diverged cytoplasms[END_REF].

Cytoplasmic Male Sterility in the Family Apiaceae

In the family Apiaceae, the Iranian accession 'P1229526 is the first male sterile line in celery and was reported by Quiros et al. in 1986 [27]. In two successive publications, Gao et al. first attributed CMS to a ms-1 single recessive gene in 2006 [START_REF] Gao | Discovery and botanical characters of celery male sterile material[END_REF]. However, in 2009 they concluded that in fact two recessive genes controlled nuclear male sterility [START_REF] Gao | Genetic characters of 01-3A male sterile celery[END_REF]. Recently in the Chinese celery "tanzhixiangqin", comparative analysis of the mitochondrial genome between the CMS line and its maintainer line opened the door to identifying 21 unique regions with 15 ORFs in the CMS line. Only one chimeric gene was found in ORF768a that had a 1497 bp sequence of the cox1 gene and an unknown sequence of 810 bp. Probably, ORF768a coded for the 11 transmembrane domain of protein, which led Cheng et al. [START_REF] Cheng | Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in celery (Apium graveolens L.)[END_REF] to indicate in 2021 that ORF768a might be a good candidate gene for cytoplasmic male sterility in celery. In carrots, a petaloid CMS line was associated with a novel ORF of 651 bp (orfB-CMS) and an additional 170 bp unknown sequence. This orfB-CMS chimeric gene has a functional role in atp8-like membrane protein [START_REF] Chen | Male sterility and fertility restoration in crops[END_REF][START_REF] Nakajima | A novel orfB-related gene of carrot mitochondrial genomes that is associated with homeotic cytoplasmic male sterility (CMS)[END_REF]. Unfortunately, the number of stable male sterile lines available in species of the family Apiaceae remains limited [START_REF] Quiros | Cytological and genetical studies of a male sterile celery[END_REF][START_REF] Gao | Discovery and botanical characters of celery male sterile material[END_REF], and none is presently used for commercial seed mass production. However, the need for growth uniformity is testified by the increasing number of hybrid cultivars available in the market despite the lack of an efficient CMS system.

Protoplast Technology in the Family Apiaceae

Compared to sexual reproduction, somatic hybridization has many advantages to transfer or generate the CMS condition de novo, in particular because it avoids unwanted/uncontrolled traits coming from the simultaneous transmission of genes other than those responsible for CMS. However, a number of prerequisites exist for the successful exploitation of somatic hybridization in this context. They include an efficient and reliable isolation of large yields of highly viable protoplasts of both partners, and the establishment of reproducible strategies for the high frequency regeneration of plants from the cultured protoplasts of at least one of the prospective fusion partners. This must also be coupled with efficient procedures for obtaining viable heterokaryons after protoplast fusion that, in turn, will be competent for division and ultimate plant regeneration. The information available on these various steps for members of the family Apiaceae will be discussed in the sections below.

Protoplast Isolation from Different Tissue Sources in Species of the Family Apiaceae

In 1880, Hanstein first coined the term protoplast representing the entire cell without its wall. Thereafter, Klercker (1892) for the first time used mechanical means to obtain protoplasts from water warrior (Stratiotes aloides) [START_REF] Bajaj | Potentials of protoplast culture work in agriculture[END_REF]. The pioneering discovery by Prof. Edward Cocking in 1960 [START_REF] Cocking | A method for the isolation of plant protoplasts and vacuoles[END_REF] that protoplast isolation could be achieved by enzymatic methods led to gigantic progress in the engineering of somatic cell genetics by making easier the entry of foreign genes into cells lacking the cell wall.

To date, isolation of protoplasts was reported in the family Apiaceae in Daucus carota (Carrot), Apium graveolens (Celery), Coriandrum sativum (Coriander), Foeniculum vulgare (Fennel), and Petroselinum Hortense (Parsley) (see Reference Table 1) [START_REF] Bruznican | An asymmetric protoplast fusion and screening method for generating celeriac cybrids[END_REF][START_REF] Mujib | Plant regeneration from protoplasts in Indian local Coriandrum sativum L.: Scanning electron microscopy and histological evidence for somatic embryogenesis[END_REF][START_REF] Ali | Protoplast isolation and plant regeneration in two cultivated coriander varieties, Co-1 and RS[END_REF][START_REF] Miura | Direct somatic embryogenesis from protoplasts of Foeniculum vulgare[END_REF][START_REF] Dudits | Intergeneric gene transfer mediated by plant protoplast fusion[END_REF]. A wide variety of tissues and organs have been used for isolation of protoplasts, viz., leaves, petals, petioles, cotyledons, shoot apices, epicotyl, hypocotyls, coleoptiles, stems, embryos, microspore mother cells, microspore tetrads, pollen grains, tubers, roots, root nodules, fruits, endosperm, aleurone layer, crown-gall tissue, callus and cell suspension cultures [START_REF] Bajaj | Potentials of protoplast culture work in agriculture[END_REF][START_REF] Mujib | Plant regeneration from protoplasts in Indian local Coriandrum sativum L.: Scanning electron microscopy and histological evidence for somatic embryogenesis[END_REF][START_REF] Eriksson | Protoplast isolation and culture[END_REF][START_REF] Crepy | Callus production from leaf protoplasts of various cultivars of bean (Phaseolus vulgaris L.)[END_REF]. Plants grown in the field, in a greenhouse and aseptically in vitro are the materials routinely used as source tissue for the isolation of protoplasts. They have their own limitations and benefits, as the use of source tissues grown in a controlled environment and physiological state influences both the yield and the viability of isolated protoplasts. Greenhouse-grown crops are often available but they are dependent on temperature, light and supply of nutrients which are not always controlled enough for optimum growth. Environmentally controlled chambers help in reducing the associated variation of greenhouse and field-grown plants [START_REF] Eriksson | Protoplast isolation and culture[END_REF]. Different enzyme mixtures have been used to date along with combinations of various concentrations for obtaining better yield (see References Tables 1 and2). In the case of both carrot [START_REF] Grzebelus | An improved protocol for plant regeneration from leaf-and hypocotyl-derived protoplasts of carrot[END_REF] and cabbage [START_REF] Kielkowska | An alginate-layer technique for culture of Brassica oleracea L. protoplasts[END_REF], leaf-derived protoplasts gave yields three times those of hypocotyl protoplasts. In this respect, most authors preferred to use cell suspensions as the protoplast source rather than differentiated tissues such as with leaves or hypocotyls of celery [START_REF] Bruznican | Regeneration of cell suspension derived Apium graveolens L. protoplasts[END_REF]. In non-Apiaceae species like Sowbread cyclamen, embryogenic cell suspensions gave higher yields compared to somatic embryos [START_REF] Reed | Protoplast regeneration and its use in new plant breeding technologies[END_REF]. Alstroemeria friable embryogenic callus was found to be a better source of protoplasts than leaves or compact embryogenic callus, maybe due to the exposure of a larger surface area to the enzyme solution, resulting in better digestion [START_REF] Kim | Isolation of protoplasts, and culture and regeneration into plants in Alstroemeria[END_REF]. Similarly, for the silk tree, leaves digested with 1.5% cellulase gave higher protoplast yields compared to hypocotyl-derived callus although these were digested with a higher (2%) cellulase concentration, and this was coupled with higher viability (of 87% and 85%, respectively) even if enzyme digestion time was 6 h and 16 h, respectively [START_REF] Rahmani | Protoplast isolation and genetically true-to-type plant regeneration from leaf-and callus-derived protoplasts of Albizia julibrissin[END_REF]. In Brassica oleracea, selection of the tissue source and age of explant was one of the important steps in protoplast isolation, and 4 to 6 week old leaf explants and 1 week old hypocotyls gave the highest protoplast viability compared to 2 week old tissues of either source [START_REF] Kielkowska | An alginate-layer technique for culture of Brassica oleracea L. protoplasts[END_REF].

For a successful isolation of viable protoplasts, pre-treatments applied to the source tissues before enzyme digestion play a crucial role. These may include both a physical disruption (i.e., chopping of tissues) and/or coupling with a pre-plasmolysis in a solution consisting of either sucrose or metabolically inert sugars, among which mannitol and sorbitol are often preferred [START_REF] Reed | Protoplast regeneration and its use in new plant breeding technologies[END_REF][START_REF] Cocking | Plant cell protoplasts-isolation and development[END_REF][START_REF] Yamada | Examination of the conditions for protoplast isolation from tobacco cells cultured in vitro[END_REF]. Interestingly, in carrot, plating efficiency of hypocotyl protoplasts was twice that of leaf protoplasts, while the routinely used suspension culture-derived protoplasts exhibited a plating efficiency even lower than that of those two differentiated tissue sources [START_REF] Grzebelus | An improved protocol for plant regeneration from leaf-and hypocotyl-derived protoplasts of carrot[END_REF].

Understanding Cell Wall Regeneration for an Improved Efficiency of Protoplast Technology in Carrot

The key to an efficient exploitation of protoplast technology for breeding includes a necessary step of de novo regeneration of the cell wall after protoplast isolation. Knowledge on this fundamental stage has been the object of several studies for many years, including in carrot which has, in this respect, sometimes even been considered as a model system.

Thus, in an early work, Shea et al. [START_REF] Shea | Structural analysis of the cell walls regenerated by carrot protoplasts[END_REF] examined the structure of the cell wall regenerated by carrot protoplasts for a better understanding of the mechanisms contributing to their subsequent viability and division competence in culture. They found that callose, one of the (1 → 3)β-d-glucan fractions that constitute hemicelluloses whose synthesis is generally associated with tissue and cell wounding, was not a component of the incipient wall of carrot protoplasts. Conversely, callose synthesis could be induced by intentional wounding, while the acid-resistant cellulose was formed more slowly. As a result, the complete regeneration of the wall required 3 days and the resulting protoplast-derived cell was unable to cope with turgor before at least 5 days. On the other hand, the pectic substances synthesized by protoplasts were less anionic than those of parent cells, and they became more highly charged during wall regeneration. The authors proposed that de-esterification of the carboxyl groups of pectin uronic-acid units permits the formation of a gel that envelops the protoplast, and then the rigid cellulose-hemicellulose framework is formed along with this gel matrix.

Soon after this, Emmerling and Seitz [START_REF] Emmerling | Influence of a specific xyloglucan-nonasaccharide derived from cell walls of suspension-cultured cells of Daucus carota L. on regenerating carrot protoplasts[END_REF] isolated a xyloglucan oligosaccharide isolated from the walls of suspension-cultured carrot cells and examined its impact on regenerating carrot protoplasts. This nonasaccharide named XG9 (Glc4Xyl3GalFuc) exhibited anti-auxin properties, whereby XG9 addition in nanomolar concentration to media containing 2,4-D influenced both the viability of the isolated protoplasts and the activity of glycan synthases, with effects similar to those of omitting 2,4-D from the regeneration medium.

Phytosulfokine-α (PSK) is a plant specific disulfated pentapeptide known to be involved in the initial steps of cellular dedifferentiation, proliferation, and re-differentiation, that exhibits a biological function similar to that of plant hormones when added at nanomolar concentrations [START_REF] Ryan | Polypeptide hormones[END_REF][START_REF] Ochatt | Phytosulfokine-alpha, an enhancer of in vitro regeneration competence in recalcitrant legumes[END_REF]. Since PSK increased the plant regeneration efficiency through somatic embryogenesis and this trait is linked to a thinning of the cell walls [START_REF] Ochatt | Cell morphometry and osmolarity as early indicators of the onset of embryogenesis from cell suspension cultures of grain legumes and model systems[END_REF], it was legitimate to assess whether there were any modifications of the cell wall fractions associated with the onset of the regeneration process. Thus, Godel-Jędrychowska et al. [START_REF] Godel-Jedrychowska | Composition of the reconstituted cell wall in protoplastderived cells of Daucus is affected by phytosulfokine (PSK)[END_REF] examined the correlation between the fractions (pectin, arabinogalactan protein and extension epitopes) involved in the regeneration of the walls of protoplast-derived cells of carrot and the presence of PSK in the culture medium. They directed various antibodies against the wall components and observed a variable response to PSK in terms of protoplast-derived cell development of the three Daucus taxa studied, as well as a diversity in chemical composition of the cell walls between the control and the PSK-treated cultures.

Protoplasts and Asymmetric Somatic Hybridization in the Family Apiaceae: State-of-the-Art

Generally, desired protoplast fusion could be performed either by chemical or electrical fusion. Due to the charged surface, spontaneous fusion happens very rarely; chemical fusing agents mainly used in the family Apiaceae are 15% Dextran, 10% DMSO, Glycine, 5% to 56% Polyethylene glycol (PEG M.W.1540, 4000, 6000) that have different formula weights, along with osmotic stabilizers such as mannitol or sorbitol. Usually, a mixture of a different combination of compounds such as mannitol, sorbitol, sodium chloride, calcium chloride, calcium nitrate, glycine, sodium hydroxide and buffers is used (see References in Table 2).

In 1972, Carlson et al. reported the first successful production of somatic hybrids, of Nicotiana glauca and N. langsdorffii, showing that organelles transfer is possible and therefore transfer is not limited to genetic material only [START_REF] Carlson | Parasexual interspecific plant hybridization[END_REF]. Among other strategies available, CMS can be experimentally induced through protoplast fusion and somatic hybridization, which is a technique additively combining the somatic cells from two different genotypes, cultivars, species, or genera aimed at regenerating an entirely novel genotype [START_REF] Guo | Somatic cybrid production via protoplast fusion for citrus improvement[END_REF]. In general, a standard somatic hybridization strategy comprises four steps (Figure 3): (a) isolation of parental protoplasts, (b) their chemical or electrical fusion, (c) culture of the heterokaryons obtained to regenerate first hybrid callus then plants, and finally (d) identification and selection of the somatic hybrid lines of interest. Such protoplast fusions may be symmetric, when the contribution of each parent is equivalent (2n = 2x + 2x), or asymmetric when one parent contributed the nuclear and the other the cytoplasmic genetic information (2n = 2x). The latter requires limiting the genetic contribution of one of the parents, for instance by inactivating the nucleus of the donor parent using radioactivity or chemicals (Figure 3) giving rise to a cytoplasmic hybrid or cybrid [START_REF] Liu | Intergeneric somatic hybridization and its application to crop genetic improvement[END_REF].

tion and selection of the somatic hybrid lines of interest. Such protoplast fusions may be symmetric, when the contribution of each parent is equivalent (2n = 2x + 2x), or asymmetric when one parent contributed the nuclear and the other the cytoplasmic genetic information (2n = 2x). The latter requires limiting the genetic contribution of one of the parents, for instance by inactivating the nucleus of the donor parent using radioactivity or chemicals (Figure 3) giving rise to a cytoplasmic hybrid or cybrid [START_REF] Liu | Intergeneric somatic hybridization and its application to crop genetic improvement[END_REF] Therefore, a cybrid is the type of asymmetric somatic hybrid in which the nuclear genome comes from a single parent whereas the cytoplasmic genomes are inherited from both parents. However, with few exceptions, following cell divisions the chloroplast genome tends to become uniparental while the mitochondrial genome remains recombinant, with segments of both parental mtDNAs [START_REF] Morgan | Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids[END_REF][START_REF] Mohapatra | Random chloroplast segregation and mitochondrial genome recombination in somatic hybrid plants of Diplotaxis catholica + Brassica juncea[END_REF][START_REF] Arimura | The mitochondrial genome of an asymmetrically cell-fused rapeseed, Brassica napus, containing a radish-derived cytoplasmic male sterility-associated gene[END_REF][START_REF] Przetakiewicz | The use of RAPD and semi-random markers to verify somatic hybrids between diploid lines of Solanum tuberosum L[END_REF]. One feature that justifies the appeal of cybrids for breeding programs is the maintenance of cultivar integrity given that the entire nuclear genome comes from one parent [START_REF] Guo | Targeted cybridization in citrus: Transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness[END_REF]. Noteworthy, cybrids are often male-sterile, and allow for the transfer of genomic fragments from wild plants with interesting agronomic traits to commercial crops. Additionally, protoplast fusion permits to sidestep existing barriers to sexual hybridization and hence combine sexually incompatible germplasms between phylogenetically close or distant plants, but also to transfer to a commercial crop those desirable traits that are encoded by the plastid or mitochondrial genomes of an uncultivated genotype. Therefore, a cybrid is the type of asymmetric somatic hybrid in which the nuclear genome comes from a single parent whereas the cytoplasmic genomes are inherited from both parents. However, with few exceptions, following cell divisions the chloroplast genome tends to become uniparental while the mitochondrial genome remains recombinant, with segments of both parental mtDNAs [START_REF] Morgan | Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids[END_REF][START_REF] Mohapatra | Random chloroplast segregation and mitochondrial genome recombination in somatic hybrid plants of Diplotaxis catholica + Brassica juncea[END_REF][START_REF] Arimura | The mitochondrial genome of an asymmetrically cell-fused rapeseed, Brassica napus, containing a radish-derived cytoplasmic male sterility-associated gene[END_REF][START_REF] Przetakiewicz | The use of RAPD and semi-random markers to verify somatic hybrids between diploid lines of Solanum tuberosum L[END_REF]. One feature that justifies the appeal of cybrids for breeding programs is the maintenance of cultivar integrity given that the entire nuclear genome comes from one parent [START_REF] Guo | Targeted cybridization in citrus: Transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness[END_REF]. Noteworthy, cybrids are often male-sterile, and allow for the transfer of genomic fragments from wild plants with interesting agronomic traits to commercial crops. Additionally, protoplast fusion permits to sidestep existing barriers to sexual hybridization and hence combine sexually incompatible germplasms between phylogenetically close or distant plants, but also to transfer to a commercial crop those desirable traits that are encoded by the plastid or mitochondrial genomes of an uncultivated genotype.

Recently, Gieniec et al. developed a protocol for the real-time detection of somatic hybrids with stable non-toxic fluorescent protein (FP) tagging of mitochondria during electrofusion using carrot protoplasts [START_REF] Gieniec | Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria[END_REF]. Thus, either cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, were introduced to carrot cell lines by Agrobacterium-mediated transformation and were subsequently used as a source of protoplasts for electrofusion, after selection to confirm stable labelling. First, the authors assessed the effect of various direct current (DC) parameters on protoplast integrity and on their ability to form heterokaryons. They found that the protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterokaryons (GFP + RFP or YFP + RFP) were identified through their dual-color fluorescence [START_REF] Gieniec | Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria[END_REF]. A similar approach based on dual-color fluorescence was developed many years ago for somatic hybridization in protein legumes [START_REF] Durieu | Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts[END_REF] using two dyes and non GMO material, but this study using FP stable tagging of mitochondria was the first of its kind in carrot. Three hybrids were produced after the symmetric fusion between carrot root and celery mesophyll protoplasts [START_REF] Wang | Somatic hybrids obtained from fusion of carrot and celery protoplast[END_REF].

The Production of Cybrid CMS Lines via Protoplast Fusion

As indicated above, cybrids are obtained through the fusion between donor protoplasts, whose nuclei are irradiated by ionizing or non-ionizing radiation treatment such as Gamma-rays, X-rays or Ultraviolet (UV) rays, and recipient protoplasts whose cytoplasm organelles are metabolically inactivated by treatment with chemicals such as iodoacetamide (IOA) or iodoacetate. Ultimately, this leads to the production of a fusion product with an intact cell nucleus and a cytoplasm from recipient and donor, respectively.

The fusion events are obtained through chemical (PEG-mediated) and/or electrical methods to give somatic hybrids or cybrids. A 15 mM IOA treatment was necessary to achieve 90% true hybrids in the Brassicaceae as although a 7.5 mM IOA treatment given to inactivate B. oleracea protoplasts was sufficient, when fused with non-treated B. campestris these yielded only 43% true hybrids. This shows the nurse effect that helps treated protoplasts to tolerate even the double dose of IOA needed to achieve 90% true hybrids over a time of 15 min [START_REF] Terada | Somatic hybrids between Brassica oleracea and B. campestris: Selection by the use of iodoacetamide inactivation and regeneration ability[END_REF]. Bruznican and co-workers increased the exposure time from 20 min to 25 min to overcome such nursing effects at 10 mM IOA [START_REF] Bruznican | An asymmetric protoplast fusion and screening method for generating celeriac cybrids[END_REF]. In 2000, Yamamoto et al. reported the conversion of a MS-1 CMS line into a fertile line with iodoacetamide treatment and X-ray irradiation of fertile cultivars, through electrofusion [START_REF] Yamamoto | Morphological changes in homeotic cytoplasmic male-sterile carrots combined with fertile cytoplasm by asymmetrical cell fusion[END_REF].

In 1982, Maliga et al. [START_REF] Maliga | Cytoplast-protoplast fusion for interspecific chloroplast transfer in Nicotiana[END_REF] first reported cytoplast-protoplast fusion, where cytoplasts were the cells whose nucleus was removed. Before that, two methods had been successfully devised to obtain such cytoplasts, i.e., either by application of cytochalasin B [START_REF] Wallin | Enucleation of plant protoplasts by cytochalasin B[END_REF], or by ultracentrifugation using a discontinuous percoll/mannitol gradient [START_REF] Lörz | Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells[END_REF]. These methods have potential for the transfer of organelle-mediated traits to obtain the desired cybrids [START_REF] Guo | Somatic cybrid production via protoplast fusion for citrus improvement[END_REF] but are yet to be explored in carrot. Another, still unexplored approach in the family Apiaceae is cytoplast-protoplast fusion and asymmetric fusion, as symmetric hybridization of such partners also produces cybrids, this being a common phenomenon in a few species like citrus and tobacco. Thus, in tobacco half of the regenerated plants produced by interspecific symmetric somatic hybridization were cybrids [START_REF] Guo | Somatic cybrid production via protoplast fusion for citrus improvement[END_REF]. This lack is rather surprising considering that in carrot intergeneric, intraspecific and interspecific somatic hybridization have been well known for many years [START_REF] Dudits | Somatic hybridisation of Daucus carota and D. capillifolius by protoplast fusion[END_REF][START_REF] Cella | Characterization of intraspecific somatic hybrids of carrot obtained by fusion of iodoacetateinactivated A2CA-resistant and sensitive protoplasts[END_REF]100,101].

In this context, Wang et al. [START_REF] Wang | Somatic hybrids obtained from fusion of carrot and celery protoplast[END_REF] regenerated three hybrids after a symmetric fusion between celery mesophyll and carrot root protoplasts, while Tan et al. [START_REF] Tan | Preliminary study of asymmetric protoplast fusion between celery (Apium graveolens L.) and CMS carrot (Daucus carota L.)[END_REF] regenerated 11 petaloid celery CMS plants following the asymmetric fusion between IOA-treated celery and UV-treated carrot protoplasts. Most recently, CMS hybrids were also recovered after the fusion between carrot UV-inactivated donor protoplasts and inactivated acceptor protoplasts of celeriac, while the use of coriander or celery donor protoplasts did not permit the recovery of CMS cybrids in this study [START_REF] Bruznican | An asymmetric protoplast fusion and screening method for generating celeriac cybrids[END_REF].

The cybridization approach combines the cytoplasm of a donor protoplast of one species with the nucleus of an acceptor protoplast belonging to another species, thus permitting the generation of novel interspecific nucleo-cytoplasmic genomic configurations. The obtained cybrid tissues result, therefore, from an asymmetric protoplast fusion and, on occasions, plants regenerated from them may display CMS due to the incongruent expression of nuclear and cytoplasmic DNA. This was shown in several species in the Brassicaceae with the nuclear genome of the donor partner inactivated by UV [START_REF] Terada | Somatic hybrids between Brassica oleracea and B. campestris: Selection by the use of iodoacetamide inactivation and regeneration ability[END_REF]102,103] or iodoacetamide (IOA), and rearrangements of the mitochondrial genes [104]. In the Solanaceae, too, alloplasmic CMS has been obtained in Nicotiana using X-rays [105,106], while in tomato this was achieved with IOA-treated protoplasts fused with γ or X-ray irradiated protoplasts of potato [107]. Here, the authors generated novel CMS types in tomato after fusing Lycopersicon esculentum IOA-treated protoplasts with Solanum acaule or S. tuberosum. Fertile plants were only recovered when the donor was S. lycopersicoides, despite the presence of donor mitochondria fragments in the acceptor protoplasts. This underlined the importance of the donor genotype for the generation of a novel CMS type.

One example where asymmetric protoplast fusion is used routinely concerns the genus Citrus, a model for somatic hybridization. Thus, Grosser et al. [108] transferred CMS from Satsuma mandarin to other seeded cultivars, and Aleza et al. [109] recovered cybrids with different mitochondrial and chloroplast combinations following symmetric fusions between two mandarine genotypes (Chios and Clementine) and between Chios mandarine and Sanguinelli orange.

The Requisites for a Successful Somatic Hybridization

For any asymmetric protoplast fusion to succeed, the availability of efficient plant regeneration methods is a pre-requisite, particularly for the acceptor species. A second pre-requirement is the reproducibility of a reliable method to inactivate the nuclear DNA from the donor and the cytoplasmic DNA from the acceptor. The existence of a routinely working method for protoplast fusion is needed to create interspecies hybrids. Finally, the cytoplasmic genome characterization of plants regenerated from the fusion events is essential when asymmetric fusions are employed to integrate chloroplast or mitochondria into the acceptor, and the hybridity of shoots regenerated from such fusions is assessed using molecular markers from both the nuclear and cytoplasmic genomes, to identify those regions in the genome of the hybrids that are polymorphic between the donor and the acceptor species. Alternatively, plasmotype discrimination can also be undertaken by high resolution melting analysis based on the presence of SNPs, insertions and deletions (INDELS) or SSRs [110].

To date, a number of methodologies for the regeneration of plants from protoplasts have been developed in different species, and there are various techniques with promising applications in breeding, such as increasing the genetic diversity through somaclonal variation, the transfer of cytoplasmic or nuclear genes through somatic hybridization, or the direct introduction of foreign genes through the transformation of isolated protoplasts [109,[111][112][113][114]. In this context, the use of protoplast fusion and somatic hybridization for the production of novel hybrids should focus on desirable agricultural traits, aiming at combinations that can only be achieved through protoplast fusion, subsequently using the somatic hybrids for conventional breeding and broadening the range of crops where protoplast technology is exploitable [115]. In turn, this context restricts the potential for the utilization in fusion experiments of protoplasts carrying specific traits that were introduced following genetic transformation. Indeed, they would be considered GMOs and hence would be subject to culture regulations for the somatic hybrids ultimately recovered, and their progenies, in several countries, particularly in the European Union [116,117].

In the 1980s, somatic hybridization was considered as "the" biotechnique with the potential to remodel crop improvement and agricultural research for years to come. However, this assumption is yet to become true, which resulted in a shift of research focus to molecular-based techniques. This failure of a major impact of somatic hybridization on crop development can be ascribed to many reasons. Among them are the difficulty in the isolation and culture of protoplasts added to that encountered for plant regeneration from protoplasts in many crops where it has proved highly genotype-dependent, and also the fact that somatic hybrid plants exhibit a higher ploidy level or chromosome complement since they did not result from a crossing and gamete recombination but from a genomeaddition process. Nevertheless, this drawback of using somatic hybridization has not been paramount with citrus where it is presently applied for breeding of novel genotypes [118].

Miscellaneous Studies with Carrot Protoplasts

In an early study, Chen and Wang [119] succeeded in cryopreserving both cell suspensions and protoplasts of carrot by vitrification. Both were precultured in liquid MS medium with 0.175 M sucrose for 3 d followed by 0.4 M sorbitol for 1 d, and they were quenched in liquid nitrogen following loading in 25% PVS2 at room temperature of precultured cells (for 5 min) and protoplasts (for 3 min) and treatment with 100% PVS2 at 0ºC (for 7.5 min and 5 min, respectively). As compared to this, only 47% of the untreated control survived after cryopreservation.

Other studies concerned somaclonal (protoclonal) variation, which is a means to recover novel genotypes with interesting traits, and has been used in many species for this purpose. In carrot, Grzebelus et al. [120] subjected freshly isolated protoplasts and 5-day-old protoplast-derived microcolonies to in vitro selection using culture filtrates from the fungus responsible for black rot disease (Alternaria radicina). They revealed that the fungal culture filtrate decreased the viability and plating efficiency of isolated protoplasts and inhibited cell divisions in the microcolonies at all concentrations tested above 5% (v/v). However, the authors also observed that these responses were genotype-dependent and a few plants which were regenerated with 1%, 2% and 3.5% fungal culture filtrate were hence deemed to be stress tolerant. Noteworthy among such regenerants up to 19% were tetraploids, while only 5% of tetraploid plants were regenerated from the control protoplasts. However, RAPD markers did not reveal any large chromosomal rearrangements between the controls and the regenerants obtained after selection. Some of the plants regenerated from both control protoplasts and cultures selected with the fungal culture filtrate showed a lower susceptibility to A. radicina compared to seed-derived plants, but those derived from fungal culture treatments were less capable of flowering and showed a higher rate of male sterility.

The study above confirmed the potential interest of protoclonal variation, both spontaneous and induced, for the generation of genetic novelties of interest in carrot, and prompted another study by the same team where Kiełkowska et al. [121] undertook experiments on in vitro selection for salt tolerance with carrot protoplast cultures. Thus, they exposed protoplasts of three carrot accessions to increasing concentrations of NaCl up to 400 mM and found that NaCl at 50 mM or more reduced plating efficiency while 200 mM NaCl or higher completely arrested cell division. Thereafter, they subjected the protoplast-derived plants from the control and 50-100 mM NaCl treatments to an 8-week salt stress in greenhouse conditions induced by salinized soil (electrical conductivity, EC, of 3 and 6 mS cm -1 ). They found that the 50 mM NaCl stress in vitro induced polyploidy among regenerants, but also that the plants from the 50 and 100 mM NaCl-treated protoplast cultures had a higher survival rate under salt stress compared to the controls. These salt-stressed plants also accumulated anthocyanins in the petioles, produced denser hairs on the leaves and petioles, and exhibited altered pollen viability and seed setting compared to control plants.

The Impact of Artificial Intelligence on Protoplast Technology-Based Approaches in Future

Some of the drawbacks above can be removed by employing state-of-the-art technologies such as artificial intelligence (AI), neuronal networks and robotic platforms. In this respect, the use of computational approaches may have a valuable impact in improving trial and error in regeneration systems [122]. To optimize and develop regeneration protocols, AI models can be taken into consideration. Several studies reported the accuracy and reliability of artificial intelligence methodology to optimize various processes such as callogenesis, protoplast and cell growth, somatic embryogenesis, androgenesis, shoot regeneration, rhizogenesis, hairy root cultures, sterilization, temperature inside the culture containers, plant virus detection, secondary metabolite production, microshoot length, in vitro physiological disorders, shoot organogenesis, in-vitro rooting and acclimatization in several crops [123,124]. Shiotani et al. used Multilayer perceptron (MLP) of artificial neural networks (ANNs) to classify the alive or dead cell status of Arabidopsis thaliana cultured protoplasts using digitalized imaged shape and color of cells [125]. In the family Apiaceae, MLP was used for the classification of somatic embryos and non-embryogenic structures of Apium graveolens that can be selected for transfer to the next culture phase [126,127]. Likewise, MLP was also applied for cell growth prediction and optimization in order to determine the final biomass level of Daucus carota by using initial inoculum and sugar concentration data [128]. AI models could be applied for the emerging field of genetic engineering and genome editing, i.e., via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). Genetic transformation mainly relies on multiple factors such as nutrients, light, temperature and bacterial optical density but not the least on the plant genotype [124]. Recently, robotic platforms were used for isolation of protoplasts, minimizing the time duration compared to agrobacterium mediated or biolistic methods for early detection of transformed plants [129].

Most recently, protoplasts were used for production of transgene-free carrot following PEG-mediated transformation using CRISPR technology, for the centromere-specific Histone H3 (CENH3) gene [START_REF] Meyer | Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation[END_REF]. Protoplasts were also used earlier to verify the blockage of the anthocyanin synthesis in a model purple carrot callus following the knock-out of F3H gene to demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems [130]. In this respect, the new plant breeding technologies (NPBT) approach must be utilized in the species lacking host susceptibility to transformation mediated by Agrobacterium [START_REF] Reed | Protoplast regeneration and its use in new plant breeding technologies[END_REF]. Use of protoplasts for CRISPR/Cas system, artificial intelligence (AI), neuronal networks, and robotic platforms for isolation, transformation and screening of protoplast isolation are emerging fields in agriculture [131].

Conclusions

The rising population remains challenging due to the increased demand for food, leading to a simultaneous need for high production and yield, coupled with a good quality of crops. The family Apiaceae contributes a major share to the human diet and hence shows a need for higher production. The use of protoplasts for somatic hybridization increased due to commercially important traits that are governed by mitochondria and plastids. To maintain the uniformity in quality and yield, CMS lines are one of the good sources for hybrid seed production. Other traits of agronomic and economic relevance that could benefit from the availability of somatic hybrids in members of the family Apiaceae are those associated with nutrition, disease tolerance and even natural color production. For protoplast fusion, well established isolation, pre-treatment and regeneration from different tissue sources are the primary needs of today. Protoplast fusion has often remained a bottleneck for obtaining hybrids due to the unavailability of nuclear irradiation facilities including Gamma rays and X-rays. As an alternative, the more easily available ultra violet radiation has been used. In addition, fusion efficiency with chemical and electrical fusion needs to be improved. To date, a number of studies have reported somatic hybridization in the family Apiaceae, but the transfer of a trait and its commercialization have remained challenging. In future, use of evolving technologies such as CRISPR and AI will be helpful when associated with protoplast technology.

Figure 1 .

 1 Figure 1. Yield data of primary crops of the year 2020 from Food and Agriculture Organization of the United Nations (https://www.fao.org/faostat/en/#data/QCL) (accessed on 10 February 2022).

Figure 1 .

 1 Figure 1. Yield data of primary crops of the year 2020 from Food and Agriculture Organization of the United Nations (https://www.fao.org/faostat/en/#data/QCL) (accessed on 10 February 2022).

Figure 2 .

 2 Figure 2. (A) Simple genetic model of Genic male sterility (GMS), (B) Maintenance of parent line and hybrid seed production for GMS, (C) Simple genetic model of Cytoplasmic Male Sterility (CMS), (D) Maintenance of parent line and hybrid seed production for CMS.

Figure 2 .

 2 Figure 2. (A) Simple genetic model of Genic male sterility (GMS), (B) Maintenance of parent line and hybrid seed production for GMS, (C) Simple genetic model of Cytoplasmic Male Sterility (CMS), (D) Maintenance of parent line and hybrid seed production for CMS.

Figure 3 .

 3 Figure 3. Symmetric and asymmetric protoplast fusion. Donor mitochondria (Green), acceptor mitochondria (Red). Recently, Gieniec et al. developed a protocol for the real-time detection of somatic hybrids with stable non-toxic fluorescent protein (FP) tagging of mitochondria during

Figure 3 .

 3 Figure 3. Symmetric and asymmetric protoplast fusion. Donor mitochondria (Green), acceptor mitochondria (Red).

Table 1 .

 1 Protoplast isolation in the family Apiaceae[START_REF] Ali | Protoplast isolation and plant regeneration in two cultivated coriander varieties, Co-1 and RS[END_REF][START_REF] Grzebelus | An improved protocol for plant regeneration from leaf-and hypocotyl-derived protoplasts of carrot[END_REF][START_REF] Grzebelus | Effect of beta-lactam antibiotics on plant regeneration in carrot protoplast cultures[END_REF][START_REF] Maćkowska | Plant regeneration from leaf-derived protoplasts within the Daucus genus: Effect of different conditions in alginate embedding and phytosulfokine application[END_REF][START_REF] Meyer | Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation[END_REF].

	Plant Genotypes (Daucus carota = Dc)	Tissue Source	Enzyme Mixture and Condition	Yield and Viability	References
			1% Cellulase Onozuka R10, 0.1% Pectolyase	Leaves: 3.21 × 10 6 gfw -1 ,
	Carrot	Leaves and Hypocotyls	Y-23, 0.6 M Mannitol, 5 mM CaCl 2 , 20 mM MES, 14-18 h, 30 rpm, 26 • C	0.96 × 10 6 gfw -1 74% Viability; Hypocotyls:	[41]
			1% Cellulase Onozuka R10, 0.1% Pectolyase
		Leaves	Y-23, 0.6 M Mannitol, 5 mM CaCl 2 , 10 mM MES, 14-16 h, Dark, 30 rpm, 26 • C	Not reported	[49]
		Leaves	1% Cellulase Onozuka R10, 0.1% Pectolyase Y-23, 0.6 M Mannitol, 5 mM CaCl 2 , 20 mM MES, 12-16 h, Dark, 30 rpm, 26 • C	2.8 × 10 6 gfw -1 , 72-93% Viability	[50]
			2% Cellulase Onozuka R10, 0.1% Pectolyase Y-23
		Leaves	and 1% Macerozyme R-10, 0.6 M Mannitol, albumin, 15 h, Dark, 30 rpm, 26 • C 10 mM CaCl 2 , 10 mM MES, 0.8% Bovine serum	Not reported	[51]
	Coriander (Coriandrum sativum vars.)	Embryogenic cell suspension	2% Cellulase Onozuka R10, 1% Pectinase and 0.2% Macerozyme R-10, 0.6 M Mannitol, 5 mM CaCl 2 , 14-18 h, Dark, 50 rpm	4.81 × 10 6 gfw -1 , 90-93.8% Viability	[36]

Table 2 .

 2 Examples of protoplast fusion and somatic hybridization with the family Apiaceae[START_REF] Suenaga | Basic studies on transfer of cytoplasmic male sterility by means of cytoplasmic hybridization in carrot: (Daucus carota L.)[END_REF][START_REF] Bruznican | An asymmetric protoplast fusion and screening method for generating celeriac cybrids[END_REF][START_REF] Dudits | Intergeneric gene transfer mediated by plant protoplast fusion[END_REF].

	Plant Genotypes (Daucus carota = Dc)	Tissue Source a	Enzyme Mixture and Condition	Pre-Treatment b	Chemical/Electrofusion	Fusion Type	References
	Dc	CS	2% Driselase, 0.4 M Sorbitol (2-3 h)	-		S	[52]
			4% Onozuka P 1500, 1% Driselase, 1% Pectinase, 1% Rhozyme,
	Dc × Hordeum vulgare L	CS × L	0.35 M Sorbitol and 0.35 Mannitol, 6 mM CaCl 2 .2H 2 O, (4 h, 50 rpm, 26 • C) NaH 2 PO 4 .H 2 O, 3 mM MES (Mixed with Equal Volume of CS)	-	Chemical	HK/S	[53]
			4% Onozuka P 1500, 1% Driselase, 1% Pectinase, 1% Rhozyme,
	Hordeum vulgare L., cv. Taplavni tavaszi × (Dc L., cv. Nantaise Slender)	L × CS	0.35 M Sorbitol and 0.35 Mannitol, 6 mM CaCl 2 .2H 2 O, (5 h, 50 rpm, 25 • C) NaH 2 PO 4 .H 2 O, 3 mM MES (Mixed with Equal Volume of CS)	-	Chemical	HK/S	[54]
			4% Onozuka P 1500, 1% Driselase, 1% Pectinase, 1% Rhozyme,
	(Dc) × D. capillifolus	CS × CS	0.35 M Sorbitol and 0.35 Mannitol, 6 mM CaCl 2 .2H 2 O, (4 h, 50 rpm, 26 • C) NaH 2 PO 4 .H 2 O, 3 mM MES (Mixed with Equal Volume of CS)	-	Chemical	HK/S	[55]
			4% Onozuka P 1500, 1% Driselase, 1% Pectinase, 1% Rhozyme,
	Dc L. (albino) × Aegopodium podagraria	CS × L	0.35 M Sorbitol and 0.35 Mannitol, 6 mM CaCl 2 .2H 2 O, (4 h, 50 rpm, 26 • C) NaH 2 PO 4 .H 2 O, 3 mM MES (Mixed with Equal Volume of CS)	-	Chemical	HK/S	[56]
			4% Onozuka P 1500, 1% Driselase, 1% Pectinase, 1% Rhozyme,
	Dc × Petroselinum Hortense	CS × L	0.35 M Sorbitol and 0.35 Mannitol, 6 mM CaCl 2 .2H 2 O, (16 h, 4 h for Parsley, 50 rpm, 26 • C) NaH 2 PO 4 .H 2 O, 3 mM MES (Mixed with Equal Volume of CS)	X-irradiation of 9kR (Parsley)	Chemical	HK/A	[38]
	Dc (C123) × D. capillifolius	CS R line × CS Seedling	0.1% Macerozyme R-10, 0.1% Cellulase R-10, 10% Mannitol, 0.1% CaCl 2 .2H 2 O (12 to 24 h, 25 • C)	-	Chemical	HK/S	[57]
	Dc L. var. Danvers (Line C81) × (line C123)	CS both	2% Cellulase Onozuka-R10, 1% Macerozyme R10, 1% Driselase purum, 0.4 M Mannitol, 10 mM CaCl 2 , 0.5% MES (4.5 h, 25 • C)	-	Chemical	HK/S	[58]
	(Dc L. cv. Lunga di Amsterdam) (C1) × (Dc A2CA-N)	CS both	2% Cellulase Onozuka R-10, 1% Macerozyme, 0.4 M Mannitol (4 h, 25 • C, gentle shaking)	IOA 0.2 mM and incubated at 25 • C for 20 min	Chemical	HK/S	[59]
	Spinacia oleracea L. (cv. Hybrid 102) × Dc L. (cv. Western Red)	L × R	4% Onozuka Cellulase R-10, 0.1% Pectinase, 0.8 M Mannitol, 7 mM CaCl 2 (16-20 h, Dark, 25 • C, 25 rpm)	-	Chemical	HK/S	[60]
	(Dc L. cv. Lunga di Amsterdam) × rice (Oryza sativa L. cv. Roncarolo)	CS (Carrot R) × Rice (seedling callus)	1% Cellulase Onozuka RS, 2% Pectinase, 0.4 M Mannitol (5 h, 26 • C, gentle shaking)	-	Chemical	HK/S	[61]
				Carboxyfluorescein, Scopoletin,
	Dc	ECS	2% Driselase, 0.4 M Sorbitol, 2.5 mM EGTA, 1 mM MES (2 h)	FITC, RITC, RHO 123, RHO B	Chemical	HK/S	[62]
				ethyl ester
	Dc	ECS	2% Driselase, 0.4 M Sorbitol, 1 mM MES (2 h, 125 rpm, 25 • C)	-	Chemical	HK/S	[63]

Table 2 .

 2 Cont.

	Plant Genotypes (Daucus carota = Dc)	Tissue Source a	Enzyme Mixture and Condition	Pre-Treatment b	Chemical/Electrofusion	Fusion Type	References
	Dc	ECS	2% Driselase, 0.4 M Sorbitol, 2.5 mM EGTA, 1 mM MES (2 h, 125 rpm, 25 • C)	fluorescent dyes and RHO B ethyl ester) (carboxyfluorescein, RHO 123,	Chemical	HK/S	[64]
	Dc	CS	2% Driselase, 0.4 M Sorbitol, 1 mM MES	1 mM DFMA final concentration (Inhibitor)	Chemical	HK/S	[65]
	Dc L. cv. Lunga di Amsterdam (A2CA r -N × E2A1)	CS	2% Cellulase Onozuka R-10, 1% Macerozyme, 0.4 M Mannitol (4 h, 25 • C, gentle shaking)	-	Chemical	HK/S	[66]
	Dc L. X D. capillifolius Gilli	CS (H derived calli) × CS	1% Driselase, 0.5% Cellulase Onozuka RS, 0.01% PectolyaseY-23, 0.5 M Mannitol, 0.1% MES (4 h, 25 • C, Occasional Shaking)	(15 mM IOA, 10 min at RT) × (X-irradiated of 60 Krad)	Chemical	HK/Cybrids	[67]
	N. plumbaginifolia Viviani (NX1) × Dc cv. Danvers (strain, PR)	L × R	2% Cellulase Onozuka R10, 0.1% Macerozyme, 10% Mannitol, 0.1% CaCl 2 .2H 2 O (14-16 h, 30 rpm)	-	Chemical	HK/S	[68]
	Dc	CS	2% Driselase, 0.4 M Mannitol (5 h)	-	Electrofusion	HK/S	[69]
			2% Cellulase R-10 or 2% Cellulysin, 1% Pectinase, 0.5%
	Dc × (Nicotiana tabacum)	CS × L	Pectinase, 0.5% Driselase, 0.5% Rhozyme, 0.35 M Mannitol, 0.35 M Sorbitol, 3 mM MES, 6 mM CaCl 2 .2H 2 O, 0.7 mM NaH 2 PO 4 (Overnight, 25 • C, 50 rpm)	53 Gy, 18%; 107 Gy, 2%; 166 Gy, 0.5%. X Non-irradiated	Chemical	HK/A	[70]
	Dc 28K CMS line × Dc cv. Kikuyo gosun (K5)		Occasional Shaking) 1% Driselase, 0.5% Cellulase Onozuka RS, 0.01% PectolyaseY-23, 0.5 M Mannitol, 0.1% MES (4 h, 25 • C,	15 mM IOA for 10 min. X-irradiated with a total dosage of 60 Krad (1 Krad/min) ×	Chemical	Cybrids	[71]
	Dc CMS line, 28A1 (brown anther type) × fertile Dc cultivar 'K5'	CS × H	1% Driselase, 0.5% Cellulase Onozuka RS, 0.01% PectolyaseY-23, 0.5 M Mannitol, 0.1% MES (4 h, 25 • C, Occasional Shaking)	X-irradiated at 60 Krad (1 Krad/min) × 15 mM IOA	Chemical	HK	[72]
	D. capillijolius and Dc ssp. Gummijer × Dc cv. NS and 35B	CS	1% Driselase, 0.5% Cellulase RS, 0.01% Pectolyase Y-23, 0.5 M Mannitol, 0.1% MES (1-6 h, 25 • C, Shaking)	X-irradiated with a total dosage 15 mM lOA of 60 Krad (1 Krad/min) ×	Chemical	Cybrids	[2]
	Nicotiana tabacum (KR-SR) × Dc L. and Nicotiana tabacum (KR-SA) × Dc L.	L × CS and CS × CS	1.6% Cellulase Onozuka R-10, 0.3% Macerozyme R10, 8% Mannitol, 0.1% CaCl 2 .2H 2 O (3 h, 25 • C)	Irradiated with 7 Krad or 10 Krad of X-rays	Electrofusion	Somatic hybrid	[73]
	Dc L. × Oryza sativa L.	CS	1.6% Cellulase Onozuka R-10, 0.3% Macerozyme R10, 8% Mannitol, 0.1% CaCl 2 .2H 2 O (3 h, 25 • C)	50 Krad X-rays × 10 mM IOA for 20 min	Electrofusion	HK/A	[74]
	Barley (Hordeum vulgare L.) × Dc L.	L × CS	1.6% Cellulase Onozuka R-10, 0.3% Macerozyme R10, 8% Mannitol, 0.1% CaCl 2 .2H 2 O (3 h, 25 • C)	_	Electrofusion	HK/A	[75]
	Daucus carota ssp. sativus (Hoffm.) Arcang. (Eight fertile cultivars × MS-1 CMS line)	CS young L or H	1% Driselase, 0.5% Cellulase RS, 0.01% Pectolyase Y-23, 0.5 M Mannitol, 0.1% MES (3 h, 30 • C, 50 rpm)	Irradiated with 85 Krad X-ray × 15 mM IOA for 20 min at 4 • C	Electrofusion	Cybrids	[76]

Table 2 .

 2 Cont.

	Plant Genotypes (Daucus carota = Dc)	Tissue Source a	Enzyme Mixture and Condition	Pre-Treatment b	Chemical/Electrofusion	Fusion Type	References
				6 mmol.L -1 IOA for 7 min ×
	Celery (Apium graveolens L.) × CMS Dc			(20 µmol.m -2 .s -1 ) for 9 min irradiated with UV rays	Chemical	HK/A	[77]
	Dc var. sativus Hoffm. × American ginseng (P. quinquefolius L.)	CS × calli	1.5% Cellulase Onozyka RS, 0.3% Pectolyase Y-23, 0.6 M Mannitol, 5 mM CaCl 2 (4 h, RT, 70 rpm)	-	Chemical	HK/A	[78]
	Dc ssp. sativus Hoffm. × Amsterdamska (A) and Koral (K)	CS	1% Cellulase Onozuka R-10, 0.1% Pectolyase Y-23, 0.6 M Mannitol, 5 mM CaCl 2 , 20 mM MES (14-18 h, Dark, 30 rpm, 26 • C)		Electrofusion	Homo-fusant fusant/ Homo-	[79]
	'Diamant' celeriac (acceptor) and 'Parmex' carrot, coriander or 'WL253' white celery (donors)	CS × L and P	1.5% Cellulase R10, 0.1% Macerozyme (Overnight, Dark, 30 rpm, 22 • C)	10 mM IOA for 20 min × UV (257 µWcm -2 ) for 6 min	Electrofusion	Hetero-fusant	[34]

a Tissue sources, CS: Cell Suspension, ECS: Embryogenic Cell Suspension, H: hypocotyl, L: leaf, P: Petiole, R: root; b IOA: iodoacetamide, RHO: Rhodamine, S: Symmetric, A: Asymmetric, HK: Heterokaryon.

Acknowledgments:

The authors are grateful to MDPI for having waved the APC. A.S.R. also would like to thank the Institute of Chemical Technology, Marathwada Campus, Jalna for Doctoral fellowship.

Funding: This research was funded by Kalash Seeds Pvt. Ltd. The APC were waived by Plants.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement:

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Author Contributions: All authors participated in the writing, original draft preparation, review and editing. All authors have read and agreed to the published version of the manuscript.