Inflorescence Development and Floral Organogenesis in Taraxacum kok-saghyz

Rubber dandelion (Taraxacum kok-saghyz Rodin; TK) has received attention for its natural rubber content as a strategic biomaterial, and a promising, sustainable, and renewable alternative to synthetic rubber from fossil carbon sources. Extensive research on the domestication and rubber content of TK has demonstrated TK’s potential in industrial applications as a relevant natural rubber and latex-producing alternative crop. However, many aspects of its biology have been neglected in published studies. For example, floral development is still poorly characterized. TK inflorescences were studied by scanning electron microscopy. Nine stages of early inflorescence development are proposed, and floral micromorphology is detailed. Individual flower primordia development starts at the periphery and proceeds centripetally in the newly-formed inflorescence meristem. Floral organogenesis begins in the outermost flowers of the capitulum, with corolla ring and androecium formation. Following, pappus primordium—forming a ring around the base of the corolla tube—and gynoecium are observed. The transition from vegetative to inflorescence meristem was observed 21 days after germination. This description of inflorescence and flower development in TK sheds light on the complex process of flowering, pollination, and reproduction. This study will be useful for genetics, breeding, systematics, and development of agronomical practices for this new rubber-producing crop.


Introduction
The genus Taraxacum Wigg. (dandelion) belongs to the family Asteraceae, subfamily Cichorioideae, tribe Cichorieae, and sub-tribe Crepidinae [1]. Asteraceae (Compositae) is a large family of angiosperms. Its members have an inflorescence called a capitulum which is characterized by flowers organized on a receptacle and bracts (phyllaries) forming an involucre [2]. The tribe Cichorieae includes annual and perennial herbs that produce latex, i.e., they are characterized by the presence of lactiferous canals in subterranean and aerial parts [1].
Members of the Taraxacum genus are commonly found throughout the temperate region. These plants form a polyploid complex with both sexual and apomictic modes of reproduction [3][4][5].

Morphological Aspects
Taraxacum kok-saghyz is an herbaceous plant with leaves forming a rosette ( Figure 1A) and produces small flowers that are tightly packed, forming a head-like inflorescence called capitulum ( Figure 1A). The inflorescence-stems (scapes) are leafless, with one solitary capitulum at each apex ( Figure 1A). The inflorescence emergence occurs near the point of the leaf's insertion into the stem Plants 2020, 9,1258 3 of 15 ( Figure 1A). Flowers are all alike (homogamous capitulum; Figure 1B), with the same corolla shape and sexual configuration (bisexual; . The flowers are surrounded by two layers of involucral bracts (phyllaries; Figure 1C,D). The outer series of bracts show a lanceolate and pointed horn at the edge. The inner series of bracts show a lanceolate erect edge ( Figure 1C). These bracts act like sepals and protect the flowers during development. Each capitulum contains dozens of small yellow ligulate flowers that develop from the outside to the inside region of the capitulum ( Figure 1D,E). Flowers have a five-toothed ligule ( Figure 1F) and five epipetalous stamens with rimose dehiscence ( Figures 1G, 3 and 4). The calyx is represented by a circle of unbranched hairs (pappus) forming a ring surrounding the corolla, which is associated with fruit dispersion (Figure 1H,I). The pappus and the corolla are inserted above the inferior ovary ( Figure 1H,I) with two carpels (Figure 3). The style is bifid ( Figure 1J).
Plants 2020, 9, x FOR PEER REVIEW 3 of 15 ( Figure 1A). Flowers are all alike (homogamous capitulum; Figure 1B), with the same corolla shape and sexual configuration (bisexual; . The flowers are surrounded by two layers of involucral bracts (phyllaries; Figure 1C,D). The outer series of bracts show a lanceolate and pointed horn at the edge. The inner series of bracts show a lanceolate erect edge ( Figure 1C). These bracts act like sepals and protect the flowers during development. Each capitulum contains dozens of small yellow ligulate flowers that develop from the outside to the inside region of the capitulum ( Figure  1D,E). Flowers have a five-toothed ligule ( Figure 1F) and five epipetalous stamens with rimose dehiscence ( Figures 1G, 3 and 4). The calyx is represented by a circle of unbranched hairs (pappus) forming a ring surrounding the corolla, which is associated with fruit dispersion ( Figure 1H,I). The pappus and the corolla are inserted above the inferior ovary ( Figure 1H,I) with two carpels ( Figure  3). The style is bifid ( Figure 1J).  In TK plants, successive inflorescences emerge in a spiral distribution surrounding the first inflorescence ( Figure 2) in a basipetal order. Therefore, it is possible to distinguish inflorescences at In TK plants, successive inflorescences emerge in a spiral distribution surrounding the first inflorescence ( Figure 2) in a basipetal order. Therefore, it is possible to distinguish inflorescences at different developmental stages in the same plant (Figures 1 and 2). The first formed capitulum does not show flower primordia arising, even with the second inflorescence already in development ( Figure 2A). In Figure 2B, the first formed inflorescence already shows flower primordia and initial floral organogenesis, whereas the other surrounding younger inflorescences are still beginning to form flower primordia or are even in the stage where no flower primordia are visible. In Figure 2C,D it is possible to observe more developed stages of the inflorescences and the first formed inflorescence formed shows a more advanced stage.
Plants 2020, 9, x FOR PEER REVIEW 4 of 15 different developmental stages in the same plant (Figures 1 and 2). The first formed capitulum does not show flower primordia arising, even with the second inflorescence already in development ( Figure 2A). In Figure 2B, the first formed inflorescence already shows flower primordia and initial floral organogenesis, whereas the other surrounding younger inflorescences are still beginning to form flower primordia or are even in the stage where no flower primordia are visible. In Figure 2C,D it is possible to observe more developed stages of the inflorescences and the first formed inflorescence formed shows a more advanced stage.

Inflorescence Developmental Stages
Nine stages of TK inflorescence (capitulum) development (A-I), from vegetative to reproductive meristem, were identified and detailed in this study ( Figure 3). Stage A ( Figure 3A) refers to the vegetative meristem (shoot apical meristem), which is dome-shaped and surrounded by leaf primordia.
Stage B represents the conversion from vegetative to reproductive meristem ( Figure 3B), where a transition phase is recognizable and first observed in this study at 21 days after germination (DAG) ( Figure S1), representing the beginning of the inflorescence meristem formation. In stage B, the shoot apical meristem dome begins broadening and flattening, and the involucral bract primordia along the periphery (phyllaries) are formed ( Figure 3B). At this stage, there is no sign of flower primordia along the inflorescence meristem.
In stage C ( Figure 3C), the flower primordia begin to emerge in an acropetal (centripetal) order in the inflorescence meristem. For a better view of this floral stage, the involucral bracts have been removed in stages C to I ( Figure 3C-I).

Inflorescence Developmental Stages
Nine stages of TK inflorescence (capitulum) development (A-I), from vegetative to reproductive meristem, were identified and detailed in this study ( Figure 3). Stage A ( Figure 3A) refers to the vegetative meristem (shoot apical meristem), which is dome-shaped and surrounded by leaf primordia.
Stage B represents the conversion from vegetative to reproductive meristem ( Figure 3B), where a transition phase is recognizable and first observed in this study at 21 days after germination (DAG) ( Figure S1), representing the beginning of the inflorescence meristem formation. In stage B, the shoot apical meristem dome begins broadening and flattening, and the involucral bract primordia along the periphery (phyllaries) are formed ( Figure 3B). At this stage, there is no sign of flower primordia along the inflorescence meristem.  In stage C ( Figure 3C), the flower primordia begin to emerge in an acropetal (centripetal) order in the inflorescence meristem. For a better view of this floral stage, the involucral bracts have been removed in stages C to I ( Figure 3C-I).
All flowers are visible in stage D ( Figure 3D), completing the remaining uncommitted meristematic region of the inflorescence. At stage D, the peripheral flowers start the organogenesis of the corolla whorl, showing the beginning of concavity in the center of the flower.
At stage E ( Figure 3E), all flowers show the center concavity. Organogenesis development continues and the peripheral flowers present corolla ring and early androecium.
At stage F ( Figure 3F), organogenesis proceeds acropetally on the capitulum, and the peripheral flowers show more visible development of five petals and five stamens. The central flowers in the capitulum show the early formation of the corolla and androecium whorls.
Stage G ( Figure 3G) shows inflorescence, with the peripheral flowers having corolla lobes enlarging and touching each other.
At stage H ( Figure 3H), all the flowers have elongated petals, covering the stamen. During stage I ( Figure 3I), flowers are closed, longer than in previous stages, and numerous pappus members are visible.

Floral Organogenesis
When the flower arises in the inflorescence meristem, it has a convex-round shape ( Figure 4A). The first sign of whorls is seen in Figure 4B, where the flower center becomes depressed, forming a concavity, and the corolla ring primordium is distinguishable. Next, corolla lobes start emerging at the border of the flower, the center concavity increases, and five stamen primordia appear in the center alternately to the five petal primordia ( Figure 4C). It is notable that all five corolla lobes develop simultaneously in a flower. Similarly, all five stamens are developed simultaneously in a single flower. At this stage, pappus is initiated (shown in Figure 3F).
Subsequently, petals and stamens are clearly distinct, corolla lobes become enlarged, and stamens become round-shaped on the top ( Figure 4D). Subsequently, corolla lobes elongate, with their edges touching each other ( Figure 4E). Afterward, the corolla lobes bend towards the floral apex and approach each other until they meet at their margins, where they interlock by epidermal cells ( Figure 4F). Finally, corolla lobes fuse postgenitally and the congenitally fused portion of the tube elongates ( Figure 4F).

Timing of Inflorescence and Floral Organogenesis Correlated to Plant Development
All nine inflorescence stages of development were observed and correlated to the age of the plants growing in the greenhouse (in DAG; Figure S1). At 5, 10, 15 and 18 DAG, the observed plants showed only vegetative meristems. However, at 21 DAG, the transition to reproductive/inflorescence meristem (stage B) and the initiation of flowers in some inflorescence meristems (stage C) were first observed in some plants. Stages D to H were first observed in plants at 24 DAG. Finally, stage I only appears at 30 DAG. Stage A (vegetative meristem) appears on each sampling date ( Figure S1).
The first trait visually analyzed was the number of leaves ( Figure 5A). At 5 DAG, plants showed a mean of 0.6 leaves, while 7.2 leaves were visible at 21 DAG when some plants began the transition from vegetative to reproductive meristem (Stage B). Finally, at 48 DAG, 21.1 leaves were visible.
The results regarding the longest leaf measurements are shown in Figure 5B. The development of leaves was observed at 5 DAG, with the longest leaf measuring 0.2 cm. The longest leaf was 4.0 cm when the transition to reproductive meristem occurred (21 DAG), and finally reached a mean of 8.5 cm at 48 DAG.
The number of visible inflorescence buds in each of the DAG can be observed in Figure 5C. The overall development of plants in the greenhouse, including the number of leaves, length of the longest leaf, and number of visible buds for all DAG when plants were collected, are found in Figure S2.

Timing of Inflorescence and Floral Organogenesis Correlated to Plant Development
All nine inflorescence stages of development were observed and correlated to the age of the plants growing in the greenhouse (in DAG; Figure S1). At 5, 10, 15 and 18 DAG, the observed plants showed only vegetative meristems. However, at 21 DAG, the transition to reproductive/inflorescence meristem (stage B) and the initiation of flowers in some inflorescence meristems (stage C) were first observed in some plants. Stages D to H were first observed in plants at 24 DAG. Finally, stage I only appears at 30 DAG. Stage A (vegetative meristem) appears on each sampling date ( Figure S1).
The first trait visually analyzed was the number of leaves ( Figure 5A). At 5 DAG, plants showed a mean of 0.6 leaves, while 7.2 leaves were visible at 21 DAG when some plants began the transition The results regarding the longest leaf measurements are shown in Figure 5B. The development of leaves was observed at 5 DAG, with the longest leaf measuring 0.2 cm. The longest leaf was 4.0 cm when the transition to reproductive meristem occurred (21 DAG), and finally reached a mean of 8.5 cm at 48 DAG.
The number of visible inflorescence buds in each of the DAG can be observed in Figure 5C. Until 21 DAG, no buds are externally visible. At 24 DAG, a mean of 0.1 buds are visible, while 4.8 buds are visible at 48 DAG.
The overall development of plants in the greenhouse, including the number of leaves, length of the longest leaf, and number of visible buds for all DAG when plants were collected, are found in Figure S2.

Inflorescence Bud Appearance and Anthesis
The first inflorescence bud externally visible in the center of the plant appeared on average at 32.2 DAG. However, it was noted that the first inflorescence bud appeared in some plants as early as at 24 DAG ( Figure S3). The appearance of further inflorescence buds occurred in a spiral, basipetally. The second inflorescence bud appeared at 37.6 DAG, the third bud at 38.4 DAG, and the fourth bud

Inflorescence Bud Appearance and Anthesis
The first inflorescence bud externally visible in the center of the plant appeared on average at 32.2 DAG. However, it was noted that the first inflorescence bud appeared in some plants as early as at 24 DAG ( Figure S3). The appearance of further inflorescence buds occurred in a spiral, basipetally. The second inflorescence bud appeared at 37.6 DAG, the third bud at 38.4 DAG, and the fourth bud at 41.1 DAG. Finally, the timing of anthesis was observed to be 49.2 DAG. However, anthesis was first observed in some plants at 42 DAG, while other plants showed no inflorescence bud until 60 DAG. There was a mean interval of 17 days from the first externally visible bud to anthesis.
The external morphology of inflorescence development can be observed in Figure 6, where inflorescences are numbered in the order of their initiation (from 1 to 11) and collected from the same plant at 53 DAG. The first inflorescence bud was visible at 30 DAG in this particular plant. The inflorescence buds numbered 2, 3, and 4 appeared together at 35 DAG. The inflorescence bud number 5 appeared at 42 DAG, while the bud number 6 appeared at 46 DAG. DAG. There was a mean interval of 17 days from the first externally visible bud to anthesis.
The external morphology of inflorescence development can be observed in Figure 6, where inflorescences are numbered in the order of their initiation (from 1 to 11) and collected from the same plant at 53 DAG. The first inflorescence bud was visible at 30 DAG in this particular plant. The inflorescence buds numbered 2, 3, and 4 appeared together at 35 DAG. The inflorescence bud number 5 appeared at 42 DAG, while the bud number 6 appeared at 46 DAG. Observation of one TK plant revealed that when the first inflorescence reaches anthesis, several (as many as 6) inflorescence buds were already externally visible. Besides the visible buds, there were additional inflorescence buds in the center of the basal rosette that were smaller in size and did not appear externally ( Figure S2).

Discussion
This study describes the transition from vegetative shoot apical meristem to reproductive meristem in TK plants growing under controlled conditions. The study of floral development explores a key step in inflorescence and floral initiation, the transition from vegetative meristems to inflorescence and floral meristems [34]. Our results provide a description of inflorescence development based on SEM images in TK. Nine stages of inflorescence development are proposed in Observation of one TK plant revealed that when the first inflorescence reaches anthesis, several (as many as 6) inflorescence buds were already externally visible. Besides the visible buds, there were additional inflorescence buds in the center of the basal rosette that were smaller in size and did not appear externally ( Figure S2).

Discussion
This study describes the transition from vegetative shoot apical meristem to reproductive meristem in TK plants growing under controlled conditions. The study of floral development explores a key step in inflorescence and floral initiation, the transition from vegetative meristems to inflorescence and floral meristems [34]. Our results provide a description of inflorescence development based on SEM images in TK. Nine stages of inflorescence development are proposed in this research, starting with the vegetative meristem, the conversion process to the reproductive stage, and further development of the inflorescence meristem, showing flowers initiation and organ formation in the capitulum.
Our data show that TK presents a homogamous capitulum composed of ligulate flowers in accordance with the Cichorieae tribe characteristics [2]. This structure contrasts with the frequent arrangement of the Asteraceae capitulum represented by a peripheral ring of attractive ray flowers encircling a compact cluster of disc flowers [35], as seen in sunflower [36,37] and Gerbera [37,38].
The transition of vegetative to inflorescence meristem (transition process) was observed here in plants at the age of 21 DAG, showing 7.2 leaves, the longest leaf measuring 4.0 cm, and 0.1 buds appearing externally in the center. Asteraceae has marked characteristics regarding the transition of vegetative meristem to inflorescence meristem. The vegetative meristematic apex is small and domed. During transition, the meristem enlarges, becoming broader and flatter [39]. Unlike observations in TK, the transition process in Xeranthemum squarrosum occurs with no sign of flower primordia [40].
After the transition process, the inflorescence developmental stages occurred in an acropetal order in TK. These findings are consistent with most of the Asteraceae plants [39], especially those with homogamous capitulum such as Stilpnolepis centiflora [41]. This acropetal sequence can also occur within some heterogamous capitula, as described in Gerbera [42,43]. Conversely, most species with heterogamous capitulum [e.g., Osteospermum ecklonis [44], X. squarrosum [40], Senecio vernalis [45], Chrysanthemum lavandulifolium, and Ajania achilleoides [41] have an acropetal order within the disk flowers, while the ray flowers (in the periphery of the capitulum) lag behind in development.
The single-born capitulum on scapes described here for TK, although present in some species of the Cichorieae tribe, is not a common feature in the tribe [1]. However, it is also described in other Asteraceae such as Gerbera hybrida and Helianthus annuus [37]. The capitulum usually presents involucral bracts (phyllaries) with the function of sepals, protecting the developing capitulum [40,46]. In TK, these involucral bracts were described and began to appear at stage B in the transition phase, forming a closed protective barrier.
The formation of pappus in Asteraceae could follow one of three developmental paths: sequential (many bristle-like pappus initiate in five points alternate to the petals), random process (pappus member primordia appear wherever space is available), and the appearance of a pappus ring [39]. In the present study, the initiation of numerous pappus primordia, forming a ring around the flower was observed for TK. The same observations were made in S. vernalis, where a great number of pappus bristles are formed arranged in a ring meristem [45]. The pappus formation is an important feature related to dispersal or defense against herbivory [44,47]. In Gerbera, the pappus bristles are modified sepals that aid dispersal [42]. In Asteraceae, sepals lost their protective function (now exerted by bracts) and became narrower and more numerous, being modified into pappus [48].
The floral organogenesis in TK is described in the present work, showing the formation of the corolla ring whorl followed by formation of the androecium. This is similar to what was reported in the common dandelion flowers [49]. This study also observed pappus primordia initiation just prior to gynoecium development. Additionally, during whorl initiation, organs in the same whorl developed simultaneously. These descriptions are in accordance with most Asteraceae species [39]. However, organ initiation in some Asteraceae is not simultaneous and instead follows a successive formation of the organs in the same whorl, in bidirectional order, as described in S. vernalis [45] and X. squarrosum [40].
The morphological evaluations of plants in the greenhouse showed that the first bud appeared externally in the center of the plants, on average, at 32.2 DAG, and anthesis was observed at 49.2 DAG. Thus, anthesis occurred on average 17 days following the first external appearance of the bud. This is consistent with observations in the common dandelion (Taraxacum officinale), where this period ranged from 12.5 to 18.9 days [50]. However, T. officinale took longer to show the first inflorescence bud externally, varying from 88.9 to 96 days after being transplanted to the greenhouse, i.e., 102.9 to 110 days after planting [50]. These differences could be explained in part by differences in the reproductive biology of the analyzed species (i.e., T. officinale being an apomictic polyploid and TK being a sexual self-incompatible diploid), despite them being in the same genus. Some degree of individual variation in flowering time was observed. This may be due to the genetic control of flowering in TK [31], enhanced by the extent of heterozygosity currently exhibited in TK germplasm, regardless of the use of a maternal half-sib family. Based on the evidence gathered in this study, flowering time segregation exists in the current TK breeding germplasm and should be further analyzed. More robust conclusions may be obtained by starting from a series of homogeneous genotypes showing distinct flowering times, including extremely early and biennial phenotypes. In addition, the understanding of the relationship of flower-development traits with rubber production and concentration in the roots is fundamental for the development of TK as a crop. TK plants flowering early in the first year showed lower rubber content, and their seeds generated a lower rubber-content population (Whaley et al., 1947). Moreover, it has been seen that some plants developed flowers in the first year while others did not, with the latter showing larger roots, higher productivity, and higher rubber content (Whaley et al., 1947).
The floral development of some genera in the Asteraceae family have been characterized. However, as far as we know, this study represents the first description of the Taraxacum genus using scanning electron microscopy. The results presented here will enable the design of studies focusing on the determination of the best floral developmental stages for the collection of ovules and pollen grains explants for somatic embryogenesis induction in vitro, and thus the production of haploid plants. Molecular studies can also be developed to understand the regulatory mechanisms in gene expression during flowering and inflorescence development in TK and related diploid sexual species. The understanding of floral development also has relevant implications for the determination of appropriate cultural practices of agronomic management (e.g., fertilization and pesticide application), for the production of the rubber cropping system, and for commercial seed production. Therefore, the characterization of floral development in TK is of crucial importance for establishing this new crop in large scale production and industrial supply.

Plant Material
Seeds from the maternal half-sibling family 305-05 of TK were used for this study.

Morphological Evaluation
Three hundred and sixty plants from three planting dates (15 September, 29 September, and 15 October 2017) were collected for morphological and microscopy analysis, at every 3-or 5-day intervals after germination. Stages of development were recorded based on days after germination (DAG): observed at 5, 10,15,18,21,24,27,30,33,36,39,42,45,and 48 DAG. For sampling,26,30,30,21,31,31,28,28,28,29,23,27,13 and 16 plants were sampled, respectively. These plants were evaluated based on the total number of true leaves in the plant, length of the longest leaf (cm), and number of externally visible bud inflorescences (in the center of the plant rosette) at the time of collection. Pictures of the whole plants were taken in the greenhouse using a Canon EOS Rebel T6i 750D camera with a Canon 50 mm EF lens (Canon Inc., Melville, NY, USA).
To evaluate the timing of inflorescence development and floral organogenesis, a total of 98 plants were evaluated considering the developmental stage of the oldest inflorescence in the plant. The developmental stage was assigned according to the nine stages proposed herein for inflorescence development in TK.

Timing of Inflorescence Bud Appearance and Anthesis
The visual analysis of 58 plants was conducted in the greenhouse considering "time to bud" (in DAG), which represents the day when the first inflorescence bud was visible in the center of the rosette of leaves. Forty-four plants were evaluated for "time to anthesis" (when the inflorescence was completely opened). The second (n = 35 plants sampled), third (n = 23), and fourth (n = 19) inflorescences were recorded when they appeared in the center of the plant. Plants were evaluated every 1, 2, or 3 days from the date of planting until 60 DAG.

Conclusions
The present study shows a description of inflorescence and floral development in TK, proposing nine stages of inflorescence development using SEM techniques. The study showed that flowers were formed in an acropetal order in the capitulum. In relation to floral development, the first formed organ whorl was the corolla ring, followed by the androecium, pappus primordia, and finally, the gynoecium. During the initiation of whorls, organs in the same whorl developed simultaneously. This study also showed that the pappus primordium in TK was initiated forming a ring around the flower. The inflorescence stages were correlated to the morphological description of the flowers, and the first conversion of the vegetative meristem to a reproductive meristem was noticed at 21 DAG in plants showing, on average, 7.2, the longest leaf measuring 4.0 cm, and 0.1 buds appearing externally in the center of the plant. On average, the first floral bud appeared externally in the center of the plants at 32.2 DAG, while anthesis was noticed at 49.2 DAG. The work presented here represents basic knowledge of fundamental interest for the neo-domestication of TK, concerning flowering, pollination, seed production, and rubber accumulation. Furthermore, it is expected that the results presented here can yield information for further experiments and deeper knowledge regarding TK development, physiology, nutrition, morphology, biotechnology, plant breeding, crop management, and the development of agricultural practices. This research can contribute to enhance the knowledge related to this important natural rubber-and latex-producing alternative crop.