Root Causes of Flowering: Two Sides of Bolting in Sugar Beet

Abstract

Sugar beet is an important root crop with a biennial life cycle. In the first year of its life cycle, it produces huge amounts of leaf and root mass used for the production of sugar and bioethanol, livestock feed, confectionery and pharmaceuticals, fertilizers, and soil restoration. Normally, after exposure to cold temperatures during winter storage, in the second year of its life cycle, it enters its reproductive phase. However, during the first year of growth, sugar beet plants may be susceptible to producing flowering shoots, or “bolting”, due to vernalization and long-day conditions. Bolting reduces both the yield and the sugar content of roots. Here, we review the published research works that study the environmental factors influencing bolting, the genetic (including epigenetic) and physiological mechanisms that regulate the transition to the reproductive phase, and the agrotechnical and breeding practices used to prevent bolting. Models of gene networks that regulate the transition to flowering are presented. Methods for selecting non-bolting plants using conventional, marker-assisted, and genomic breeding are demonstrated. Attention is also paid to the speed breeding technology that stimulates bolting and flowering sugar beet plants in an artificial climate. Growing sugar beet plants “from seed to seed” can potentially accelerate the breeding and seed production of sugar beet. This review compares different conditions for inducing bolting in sugar beet in climatic chambers and greenhouses. It examines parameters such as temperature, duration of light exposure, and light intensity during the pre-vernalization, post-vernalization, and vernalization periods. The present review may be useful for specialists in sugar beet cultivation, breeders working on developing cultivars and hybrids that are resistant to bolting, and molecular biologists studying the genetic and physiological mechanisms underlying the transition of plants into the flowering stage.

1. Introduction

Sugar beet (Beta vulgaris L.) is a highly important crop. In its first year of cultivation, it produces huge leaves and roots, which can be processed as a raw material with minimal waste. Thus, its roots are used for sugar and bioethanol production; the molasses derived from it is used in confectionery and the production of yeast, alcohol, citric acid, etc.; and both its pulp and molasses are used for feed. Sugar factory lime can be used as fertilizer. The tops remaining during harvesting are placed in the soil as organic fertilizer and are also used as feed for cattle and pigs [1,2]. Sugar beet is of great importance in crop rotation and serves as a beneficial precursor for crops such as corn, legumes, annual grasses, millet, and early-spring grains. These crops yield higher outputs as a result of the positive effects of sugar beet on soil improvement and phytosanitary conditions. The value of sugar beet as a precursor depends on climatic and soil conditions [3,4]. Sugar beet cultivars are classified into three groups: high-yielding (with low sucrose content and large roots), high-yielding-sugar (with a medium level of sucrose and medium-sized roots), and sugary (with a high level of sucrose and relatively small root vegetables). Owing to the work of breeders, the sugar content in beet roots has increased from 1.3% since its discovery in the roots to 17–20% today [5].

In 2021, approximately 270.16 million metric tons of sugar beet was produced in the world [6], which accounted for around 30% of global sugar production [4]. The European Union is the leading producer of sugar beets, accounting for 42% of global production. The highest contributors to its production in the EU are France (13%), Germany (12%), and Poland (6%). Other world leaders include the Russian Federation (15%), the United States (12%), Turkey (7%), Egypt (5%), Ukraine (4%), and the United Kingdom (3%) [7].

Sugar beet is a biennial plant that undergoes a two-year growth cycle. In temperate climates, sugar beet is sown in the spring and harvested in the late summer or autumn, depending on the latitude, the weather conditions of the year, and the demand from sugar factories. Typically, during its first year of development from the spring to the autumn, the plant develops vegetative organs, including leaves, a shortened stem (top), and a fleshy taproot (Figure 1). In warmer regions such as Morocco, Egypt, and certain southern parts of Italy, Spain, and the southeastern USA, sugar beet is cultivated as a winter crop that remains in the field throughout the winter season [8,9,10]. The possibility of introducing winter sugar beet is being considered in Central Europe, Turkey, Iran, and other countries. This is due to the potential for earlier harvesting, a significant increase in taproot yield, and, in arid zones, a higher availability of soil moisture [9,11,12,13].

During winter storage, the roots are subjected to prolonged exposure to low temperatures, which is known as vernalization. In the second year, under long-day conditions, a stem elongates and becomes a peduncle with flowers and seeds on it (Figure 1). This stage of the sugar beet life cycle is crucial for breeding and seed production purposes. Such technology is called the planting method of seed production. In the first year, root crops are grown from seeds sown at a higher density than that for sugar production. These roots are then dug up and stored until the spring. In the second year, they are planted in a field; in the case of F₁ hybrid seed production, a ratio of one row of the paternal form to three rows of the maternal form is recommended.

Besides the planting method, non-planting and transplanting methods are also used. With the non-planting method, also known as direct seeding or overwintering, the seeds are sown at the end of summer. In the case of F₁ hybrid seed production, the recommended density ratio is one row of the paternal form to three rows of the maternal one. The plants are left in the field to overwinter without digging up the root crops and are covered with soil for better winter protection. The seeds are then obtained in the following year. The most favorable areas for non-planting methods of seed production are in Northern Italy and Southern France, where the world’s leading sugar beet seed producers are located. The transplanting method, also known as steckling method, is a relatively new and interesting technique. A smaller mother root crop compared to a traditional one is referred to as a steckling. It offers a higher yield of planting material per unit area and a rapid rate of seed multiplication. This method consists of sowing seeds in August of the first year at a higher density than that for taproot production. This allows for the identification of the most viable biotypes. In the autumn, the seedlings are dug up and stored until spring. In the second year, during the springtime, they are transplanted to another field for seed production [14,15].

The flowering process can also occur in a plant’s first year of life and is referred to as bolting. Bolting and early flowering in a plant’s first year are unnecessary traits that result in lower yields. It has been established that beet cultivars bred for northern latitudes do not produce bolting plants. However, those bred for middle latitudes can produce up to 10% bolters, while those originating from southern countries can produce 10–50% bolters. Cultivars from southern and warm countries can produce up to 100% flowering plants in the first year of cultivation [16]. Southern European cultivars and hybrids of sugar beet consistently exhibit a high level of bolting in Northwest Europe. However, in Southern Europe, the percentage of bolting is typically very low or may not occur at all [17]. In the Russian Federation, as a country with several different soil and climatic conditions, bolting is a relevant problem for sugar beet production [18]. The United Kingdom is among other countries where bolting is a serious issue. It has been observed that for every 1% increase in bolting plants, there is a corresponding decrease in yield ranging from 0.3% to 0.7%. Additionally, a 1% increase in bolters results in a 0.05% decrease in sugar content, which can sometimes lead to a loss of up to 20% in sugar [19,20,21]. In addition, bolting is relevant for the USA as well as tropical and subtropical countries [22,23,24]. In Japan, transplant cultivation is widely practiced, exposing young plants to low temperatures, which can induce flowering [25]. Moreover, the tendency to bolt limits the widespread use of winter beet due to its requirement for winter vernalization [26,27].

Studies on model plants have shown that the regulation of flowering is complex and includes many regulatory pathways that depend on both environmental and endogenous signals [28], and the causes of flowering can be external (the influence of temperature and light conditions, mineral nutrition, herbicides, etc.) and internal (genetically determined). In order to understand why flowering occurs in field conditions and whether it can be helpful for sugar beet breeding and seed production, it is necessary to consider the biology of beet development and the genetic and physiological mechanisms of its transition from the vegetative to the reproductive stage of its life cycle.

2. Developmental Stages of Sugar Beet

The ontogenetic development of sugar beet is a complex process that has been divided into stages in accordance with morphological and physiological characteristics. The classification of phenophases in the study by Zhuzhzhalova et al. (2007) [29] is somewhat similar to the macrostages reported in Meier et al.’s work (1993) [30], which further introduces more differentiated microstages (

Table 1. Relationship between age-related processes in sugar beet and phases of development and stages of organogenesis as classified in [29] in comparison to classification in [30].

Classification Developed by Zhuzhzhalova et al. (2007) [29]					Classification Developed Meier et al. (1993) [30]
Age Period	Age State	Plant Development Phase		Stage of Organogenesis	Macrostage of Growth
		Phase	Duration, Days
latent	dormant seeds	From 14 days to 7 years or more	-
pre-reproductive (vegetative)	Sprout	shoots “fork”	6–10	I	Macrostage 0 Germination/Seedling development
					Macrostage 1 Leaf development (youth stage)
		1–2 pairs of leaves	12–14	II
	Juvenile	3–5 pairs of leaves	7–9
	immature	11–30 leaves	20–22
	Virginal	rosette of leaves	90–100		Macrostage 3 Rosette growth (crop cover)
					Macrostage 4 Development of harvestable vegetative plant parts Beet root
		dormant root vegetables during storage	95–200	III
reproductive	Early		8–17	IV–V
		regrowth of rosette leaves	20–26	VI
		Bolting	18–20		Macrostage 5 Development of inflorescence/flower buds (2nd year of growth)
		budding	19–20	VII, VIII
	Middle	flowering	25–27	IX, X	Macrostage 6 Flowering
	Late	seed ripening and full maturity	30–35	XI, XII	Macrostage 7 Fruit development
					Macrostage 8 Seed ripening
post-generative	Senile	plant senescence and dying	30	-	Macrostage 9 Dying-off

). The latent period, accompanied by dormancy, occurs in mature seeds. It is important to note that even in the mother plant, seeds can perceive environmental signals, which are then reflected in the developing plant. Once the cotyledons unfold (the “fork” phase), the seedling becomes self-sufficient and begins to live autonomously, relying on its own supply of nutrients. During the development of the first and second pair of true leaves in the true leaf stage, the shoot apex expands, internodes and leaves are formed, and secondary cortex and cork tissue develop, leading to the shedding of the primary cortex, known as root molting. During the juvenile stage, the root completes its molt, and the sequential emergence of leaves ceases. Afterward, the leaf primordia at the base of the shoot apex are arranged in a spiral pattern. During the immature stage, the plant starts to take on the shape of a root crop, with the upper part of the root crop serving as an organ for storing reserve substances. During the virginal period, the root crop experiences an increase in mass, and the neck becomes noticeably rounded. The roots also become thinner and longer. This state continues throughout autumn–winter storage [29,30,31].

During the first year of growth, sugar beet transports sucrose through the phloem from photosynthetic source tissues to storage tissues in the roots, which act as a sink. In their second year, the plants transport carbon from the roots to support new growth and reproduction, making the roots a source of organic matter and energy. The transition from sink to source is controlled by the transcriptomic and functional reprogramming of root tissues, leading to a reversal of the flux direction in the phloem [32].

Under the influence of vernalization (cold temperatures above freezing for 60–100 days) and exposure to light (30–90 days), the growth cone undergoes segmentation and elongation. Biennial plants native to temperate zones must undergo vernalization to avoid a premature transition into a cold-sensitive generative phase and to accumulate enough resources for reproduction during the winter. At the end of the storage period for root crops, the generative phase begins. During this phase, the lateral axes of the inflorescence are formed in the growth buds, spikelet primordia develop on the main axis, and the whorls and individual flowers of the inflorescences differentiate. Additionally, the formation of staminate and ovule primordia takes place. Bolting begins on the 18th to 20th day after the rosette of leaves of the second year appears. During this process, the main stem and 2 to 15 side shoots are formed. The sugar beet is a plant with a monopodial growth pattern and shoot system. Flowering shoots are ribbed, heavily developed at the base, and have abundant foliage. Simultaneously with the growth of flowering shoots, inflorescences develop, and micro- and megasporogenesis occur in the buds. Gametogenesis occurs at stage VII, coinciding with the completion of the formative processes of the generative sphere and the end of the budding phase. The flowering phase signifies the start of the middle generative period. Flowering begins at the main shoot and progresses upwards along each axis, with the highest intensity occurring at the end of the second week. Beetroot can undergo cross-pollination (allogamy), self-pollination within the same plant (geitenogamy), or self-pollination within a single flower (autogamy). The fertilization period and zygote formation coincide with stage IX of organogenesis. Stage X is linked to the development and growth of the embryo, seed, and fruit. During the late generative state (stages XI and XII), seed ripening and fruiting take place. During old age, shoots die, fruits fall off, and root crops are destroyed and rot [29,30,31].

It is interesting to note that while cultivated sugar beet has a two-year development cycle, its wild relatives can have varying life cycles. Sea beet (B. vulgaris ssp. maritima), native to the Mediterranean region, produces peduncles and flowers within one season under long-day conditions and does not require vernalization. In contrast, sea beet from northern latitudes is biennial and only flowers after being exposed to low temperatures for an extended period, typically during the winter. Perennial sea beet, primarily found in Northern Europe, has an iteroparous life cycle, meaning it undergoes vegetative growth after reproduction. In contrast, annual and biennial beets are semelparous, meaning they die after reproducing. All of these species are long-day plants [33,34,35,36,37].

3. Genetic Mechanisms of Transition to Flowering in Sugar Beet

The genetic control of the transition to flowering in sugar beet occurs through complex gene networks, in which the key factors are the allelic states of the genes themselves, the epigenetic regulation of their expression (histone modification and chromatin remodeling), and the mutual influence of genes on each other in a system of gene networks (cis- and trans- regulation, repression or induction, up- or downstream genes, and epistatic interaction) as well as external factors such as temperature and daylight hours (Figure 2).

3.1. Genes Regulating Transition to Flowering in Sugar Beet

The study of sugar beet flowering genetics heavily relies on the understanding of genetic systems in Arabidopsis. Such an approach includes the study of the expression of individual orthologous genes using primers for conserved regions [38,43,44]. Whole-genome-sequencing technologies have become widespread, allowing for the comprehensive study of the sugar beet transcriptome [42,45,46,47]. These studies compare the genotypes of plants grown under conditions that stimulate the transition to flowering, such as long days, vernalization, and exogenous GA. For example, Hébrard et al. (2016) identified 169 differentially expressed genes (DEGs) associated with bolting resistance and involved in perceiving environmental conditions, hormonal signaling, and controlling flowering [42]. When comparing vernalized and non-vernalized plants, Mutasa-Göttgens et al. (2012) found 570 differentially expressed mRNAs and 292 lncRNAs. These genes are involved in various processes, such as histone modification, flowering control, circadian rhythms, and the synthesis of flavonols and flavonoids [46]. Zhao et al. (2023) revealed 7471 DEGs in response to vernalization, which were enriched by the GO terms “response to stimulus”, “response to hormone”, and “shoot system development”, while KEGG annotation showed that 16, 13, and 11 DEGs were involved in gibberellic acid (GA), abscisic acid (ABA), and jasmonic acid (JA) biosynthesis [48].

In Arabidopsis, in addition to the meristem identity genes and genes that act as floral integrators, the genes involved in flowering induction may participate in the following pathways: (i) the autonomous pathway, (ii) the vernalization pathway, (iii) the photoperiod pathway, (iv) the light quality pathway, and (v) the gibberellin pathway [49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64]. The same classification is used when the genes for flowering induction in sugar beet are studied.

The central role in the genetic system of the transition to flowering in sugar beet is considered to be played by the B locus [65]. Based on BAC library screening and chromosome walking, researchers successfully cloned a gene, BvBTC1 (BOLTING TIME CONTROL 1), from the B gene locus; this gene belongs to the PSEUDO RESPONSE REGULATOR (PRR) gene family [41]. BvBTC1 is the primary gene responsible for life cycle adaptation, determining whether an organism undergoes annual or biennial development. Plants carrying the dominant BvBTC1 haplotype typically follow an annual development pattern. On the other hand, plants carrying the recessive Bvbtc1 haplotype exhibit reduced expression of the BvBTC1 protein and have a two-year life cycle. These plants require a long period of cold before they can bolt and flower. Heterozygotes, which have one dominant and one recessive haplotype, behave like annuals under favorable conditions but bolt a few days later than annual homozygotes. Mutasa-Gottgens et al. (2010) demonstrated that under long-day conditions without vernalization, homozygotes for the dominant haplotype exhibit a higher number of flowering instances compared to heterozygotes. In fact, heterozygotes may even revert back to the vegetative phase, suggesting the possibility of incomplete dominance [66]. The life cycle control governed by BvBTC1 involves integrating photoperiods and vernalization signals [28].

BvBTC1, as a member of the PRR gene family (more details in [59,60,61]), shares similarities with the Arabidopsis gene PRR7, which is the closest homolog to the photoperiodism sensitivity gene PPD1 in cereals. BvBTC1 encodes a protein that contains the receiver response regulator domain (REC, more details in [67]) as well as the CONSTANS, CONSTANS-Like, and TOC1 domains (abbreviated as CCT, more details in [62]) responsible for sensitivity to photoperiods). Another PRR family gene, known as BvPRR7, was found in the sugar beet genome. It exhibited diurnal expression rhythms in beets and had an impact on the expression of the master clock gene CCA1 when overexpressed. Additionally, in Arabidopsis, its expression increased when exposed to cold [68].

The dominant haplotype is widely distributed in wild beet (B. vulgaris ssp. maritima). The absence of vernalization in Mediterranean populations leads to early bolting, causing an annual life cycle. Haplotype comparison of BvBTC1 in annual and biennial genotypes revealed that an insertional mutation in the BvBTC1 promoter region disrupts the sequences of light-sensitive motifs. These results uncover the molecular processes involved in the domestication of biennial beet and its production [41,69]. Based on the polymorphism of the BvBTC1 gene, it is possible to differentiate between the annual and biennial phenotypes of sugar beet [25,41,69,70].

Another gene associated with bolting, BvBBX19 (DOUBLE B-BOX TYPE ZINC FINGER), was cloned from the B2 locus. In the gene network, BvBBX19 acts as an upstream regulator of BvBTC1 and has an epistatic effect on the vernalization requirement in beet [40]. An annual habit is characterized by plants that carry both functional haplotypes BvBTC1 and BvBBX19 simultaneously. Both BvBBX19 and BvBTC are homologous to the Arabidopsis CO protein, which activates the expression of the FT gene. However, unlike CO, BvBBX19 carries two zinc finger domains (B-box) but lacks a CCT domain. In contrast, the BvBTC protein carries a CCT domain [71].

In sugar beet, two FLOWERING LOCUS T (FT) genes, BvFT1 and BvFT2, homologous to a floral integrator gene, AtFT, in Arabidopsis [52,64], are part of the phosphatidylethanolamine-binding protein (PEBP) gene family. They are located downstream of BvBTC1 and BvBBX19 in the gene network, and their expression is controlled by the products of these genes [40,72]. BvFT1 and BvFT2 are antagonist genes. BvFT2 promotes flowering and is necessary for flower development, similar to its Arabidopsis-orthologous gene FT. On the other hand, BvFT1 acts as a repressor of flowering, unlike Arabidopsis FT. After vernalization, BvFT1 and BvFT2 are down- and up-regulated, respectively, and they are both hypermethylated in bolting-sensitive genotypes [41,42,48]. It is likely that the functional proteins BvBTC and BvBBX19 form a heterodimer with both CCT and B-box domains. This heterodimer has the ability to increase the expression level of the flowering inducer gene BvFT2 and inhibit the expression of the flowering repressor gene BvFT1. This ensures an annual type of development. However, when BvBTC and BvBBX19 have dysfunctional mutations, this ability is lost. This can lead to either a biennial phenotype or a general loss of reproductive ability [40,71].

Among orthologs of floral integrators, BvSOC1 was found to be up-regulated in the apical meristem after vernalization [48], while BvAGL24 showed a higher relative expression level in bolting-sensitive genotypes in response to cold [42]. An ortholog of the Arabidopsis floral meristem identity gene BvFUL exhibited increased expression following vernalization and was also found to be highly expressed in the bolting-resistant genotypes of sugar beet compared to the sensitive ones [42,45].

Although in Arabidopsis, a repressor gene, FLOWERING LOCUS C (FLC), involved in both autonomous (i) and vernalization (ii) pathways plays the central role in regulating flowering [52,64], its ortholog in sugar beet, the BvFL1 gene (also referred to as BvFLC or BvFL), is not a major regulator of the vernalization response. The data obtained indicate that there is a response of BvFL1 expression to vernalization and its association with resistance to bolting. In transgenic Arabidopsis plants, the expression of BvFL1 decreases in response to vernalization [73]. The expression of BvFL1 in sugar beet plants decreases in response to vernalization in bolting-sensitive genotypes but increases in resistant ones. During vernalization, the content of BvFL1 mRNA decreases in bolting-sensitive genotypes, while it accumulates in bolting-resistant genotypes; late-flowering genotypes showed a slow accumulation of mRNA [42,74]. An association was found between nucleotide polymorphisms in BvFL1 and both early bolting and winter hardiness [75]. According to Trap-Gentil et al. (2011), BvFL1 exhibited hypermethylation at cytosine positions CG, CHG, or CHH before vernalization. In bolting-sensitive plants, methylation decreased during vernalization, while in resistant plants, only a temporary decrease was observed by the third week of vernalization [74]. BvFLC demonstrated a higher level of expression in the apical meristem of bolting-resistant genotypes after 9 weeks of vernalization, while in bolting-insensitive plants, it showed a higher level of methylation [42]. On the other hand, Pi et al. (2020) found no significant changes in the expression levels of sugar beet after vernalization [38]. In the study conducted by Vogt et al. (2014), the overexpression of BvFL1 resulted in the moderate delay in bolting. However, RNA silencing did not have any effect on flowering time after vernalization [76]. The overexpression or silencing of the BvFL1 gene did not impact the expression of BvBTC1, BvFT1, and BvFT2, which are central genes in the Arabidopsis model [42]. BvSVP, another sugar beet ortholog of a central floral repressor SVP in Arabidopsis (more details in [77]), showed higher expression in sensitive genotypes in response to vernalization [42].

Besides BvFT1, BvFT2, BvBTC, and BvBBX19, some additional genes involved in the vernalization pathway (ii) of flowering were discovered in sugar beet. Trap-Gentil et al. (2011) discovered that the increase in BvVIN3 mRNA amount in bolting-resistant late-flowering genotypes is slightly delayed compared to resistant early-flowering and sensitive genotypes in response to vernalization. In addition, BvVIN3 was found to be hypermethylated to a greater extent in the bolting-resistant genotypes compared to the sensitive genotypes during vernalization [74]. The analysis of differentially expressed genes revealed 34 genes that encode proteins containing the B3 domain. Most of these genes were activated through vernalization, including BvVRN1, BvVRN1-like, BvVAL1, and BvVAL2. Additionally, the differential expression of the BvVAL1-like gene MSTRG.26204.1, which encodes a long non-coding RNA, was shown [46].

Several genes, whose orthologs in Arabidopsis play roles in the autonomous pathway (i) regulation of flowering, were identified in sugar beet, namely, BvFY, BvFCA, BvFLD, BvFLK, BvFVE1, BvFVE2, BvLD, and BvLDL1 [38,42,78]. The overexpression of the BvFLK transgene fully compensates for the flk mutation in late-flowering Arabidopsis. This leads to an accelerated flowering process through the repression of the FLC protein, highlighting the structural and functional conservation of BvFLK [78]. BvFVE was characterized by a higher level of methylation in bolting-sensitive genotypes compared to resistant ones after vernalization [42]. However, when BvFVE1 was overexpressed in late-flowering Arabidopsis with a mutant fve gene, it did not have a compensatory effect. Unlike FVE in Arabidopsis, BvFVE1 is regulated by the circadian clock. Since beet has a second closely related homolog of FVE called BvFVE2, it is possible that BvFVE1 and BvFVE2 have undergone subfunctionalization. It can be hypothesized that BvFVE2 serves as a functional ortholog of FVE [78].

Among the photoperiod pathway (iii) genes, BvBOA, BvTOC1, BvCO, BvCOL, BvLHY, BvGI, and BvELF3 were found to be differentially regulated in sugar beet in response to vernalization in sugar beet [38]. It was found that an ortholog of the Arabidopsis gene CONSTANS (CO) modulated by the circadian clock and day length, BvCOL (CONSTANS-LIKE), does not play an important role in regulating flowering. Although its expression is higher in plants sensitive to bolting compared to resistant ones, its expression pattern differs from that of Arabidopsis [42,79]. The potential RNA polymerase II transcription mediator subunit that plays a role in determining flowering time and circadian rhythms was shown to be differentially methylated and expressed between resistant and sensitive genotypes of sugar beet [42].

The light-quality pathway (iv) genes, whose orthologs in Arabidopsis constitute the miR156-SPL module that regulates flowering time depending on the red-to-far-red-light ratio [80], were also discovered in sugar beet. The overexpression of the sugar beet gene miR156 in transgenic sea beet plants effectively reduces the expression of SPL4 and SPL9 genes. This leads to suppressed flowering of sea beet when exposed to long daylight conditions. Additionally, if the decrease in SPL4 expression is solely caused by miR156 overexpression, it is likely that the decrease in SPL9 expression is also influenced by other factors related to the photoperiodic pathway [44].

The genes of the gibberellin pathway (v) responsible for hormonal status and participating in hormone signaling are reviewed below in the corresponding section, considering the hormonal regulation of the transition to flowering.

The process of transitioning to flowering is complex due to epigenetic interactions. One key mechanism of plant adaptation to changing environmental conditions is the alteration of gene expression. In general, bolting-sensitive genotypes exhibit higher levels of genome methylation compared to resistant genotypes both before and during vernalization [42,74]. Trap-Gentil et al. (2011) showed that treating vernalized plants with hydroxyurea, a methylation inhibitor, decreased the percentage of plants that bolted and increased the time it took for them to flower [74]. Hébrard et al. (2013) identified 39 differentially methylated regions (DMRs), most of which were CpG islands, in the apical meristem. Of these DMRs, 33 varied depending on the duration of the cold period, and 14 varied between genotypes [81]. Hébrard et al. (2016) identified 111 DMRs between bolting-resistant and bolting-sensitive genotypes after 9 weeks of vernalization; out of these 111 sequences, 14 sequences showed both differential methylation and expression. Nine out of twenty-two candidate genes involved in regulating bolting in sugar beet exhibited differences in expression and/or methylation between resistant and sensitive genotypes [42]. Gutschker et al. (2022) discovered that cold conditions cause a significant reduction in sugar beet DNA methylation levels. This effect is more pronounced at the CHH motif and less so at CHG. However, this study did not evaluate the genotypes’ resistance to flowering [82]. It has been observed that vernalization leads to a decrease in the transcription of chromomethyltransferase CMT2, which is responsible for methylation in the context of CHG. Additionally, there is a significant increase in the expression of genes involved in DNA demethylation, such as RELEASE OF SILENCING 1 (ROS1), PIE1, and SWC4. These genes are part of the SWR1 complex, which incorporates the histone variant H2A.Z into the histone octamer, thereby recruiting the ROS1 protein [82].

DNA methyltransferase (DNMT) activity varied between vernalized and devernalized plants, with the isoform MET-1 (which modifies cytosines in the context of CpG) accumulating in higher levels in vernalized plants compared to devernalized plants [74]. Bolting-resistant and bolting-sensitive genotypes were shown to exhibit different patterns of methylation and gene expression in response to cold treatment. In resistant genotypes, the gene BvRTP, which is likely a retrotransposon, showed hypermethylation before and during the third week of vernalization. In sensitive genotypes, hypermethylation was only observed during the third week of vernalization, and its expression decreased. The gene BvRNMTa, which is involved in RNA methylation, was hypermethylated before vernalization in bolting-sensitive accessions and became hypomethylated in both resistant and non-resistant accessions during vernalization. In contrast, BvRNMTb was hypermethylated in both genotypes before and during vernalization and showed higher levels of methylation after vernalization in sensitive genotypes only. These findings may indicate an antagonistic relationship between these two genes [42,81].

Based on the analysis of the co-expression and/or methylation of multiple genes in sugar beet leaves, various models have been proposed to explain the response to vernalization in bolting-resistant and sensitive genotypes. These models are partially summarized in Figure 2. The model constructed by Pi et al. (2021) comprises two modules of genes [38]. The first module includes four genes (BvELF3, BvGI, BvTOC1, and BvBOA) from the photoperiod pathway, three genes (BvFVE1, BvFLD, and BvFCA) from the autonomous pathway, and BvBTC1. All of these genes were observed to be positive regulators of each other, except for FVE1, which showed a negative correlation with ELF3 expression. In the second module, BvFT1 and BvFT2 were found to be associated with BvLHY, BvGATA22, and BvFVE2. BvGATA22 exhibited negative feedback with respect to the flowering activator BvFT2 and positive feedback with respect to the flowering inhibitor BvFT1. The expression of BvFT1 was also positively correlated with the expression of BvLHY and BvFVE2 [38]. Hébrard et al. (2016) developed the model based on the integrated analysis of DEGs and DMRs. At the center of the model is a “core” consisting of Bvbtc1-BvFT1-BvFT2. Above this core is a flowering repressor called BvFL1, which is activated by BvRNMT and inhibited by BvFVE and long-term vernalization (9 weeks). BvFT2, a flowering activator, is positively regulated by BvCOL1, and it also positively regulates the expression of BvAGL24 and BvFUL. BvCOL1 and Bvbtc1 are also positively regulated by the length of a photoperiod. In genotypes that are not resistant to flowering, the BvRNMT, BvFVE, BvFL1, BvFT1, and BvFT2 genes are hypermethylated [42].

Hébrard et al. (2016) showed that the sugar beet mitochondrial genome is involved in the vernalization process. Two mitochondrial genes, ORF104 and COX2, were found to be differentially expressed between bolting-resistant and sensitive sugar beet genotypes. Analysis of the methylation and expression of the mitochondrial genome identified three genes (ORF152, ORF102b, and ORF192) that showed lower expression in resistant genotypes compared to susceptible genotypes, potentially due to methylation [42].

In addition to molecular genetic approaches such as genome and transcriptome analysis, EMS mutagenesis (TILLING and EcoTILLING methods) is commonly used to study the effects of genetic mutations on flowering times and vernalization requirements in sugar beet. This is followed by the analysis of segregating populations and the mapping of loci [83]. Büttner et al. (2010) identified two loci, B3 and B5, that influence flowering time and are not associated with the BvBTC1 locus [84]. Abou-Elwafa et al. (2012) identified the B4 locus, located 11 cM away from the B locus on chromosome II, as the determinant for vernalization requirements [85]. Further analysis of the population resulting from crossing annual sea beet and biennial sugar beet revealed that the B4 locus inhibits flowering under exposure to short daylight hours and delays the timing of flowering. This suggests that the gene is regulated by circadian rhythms and that its function is influenced by photoperiodism [86].

Several genes that regulate flowering in sugar beet have been discovered through the identification of naturally occurring allelic variants in genetic collections. Two unlinked loci, LB and LB2, were identified on chromosome II. When in a recessive state, these loci contribute to a late-flowering phenotype [87,88]. Kuroda et al. (2023) discovered two sugar beet lines that can flower under 24 h of light without vernalization. Through the genetic analysis of backcrosses with biennial beet, they identified the dominant B_LOND gene, which enables accelerated selection by producing seeds in just four months [89]. Pfeiffer et al. (2014) identified QTL BR1 on chromosome IX, which is associated with resistance to post-winter bolting [90]. Tränkner et al. (2016) identified the Arabidopsis homologous gene CPSF73-I as the most likely candidate gene for BR1, with a 2 bp deletion. The presence of the BvCPSF73-Ia allele in the BETA 1773 line, which exhibits a low percentage of flowering after wintering, results in the production of a shortened protein [91]. However, the null allele BvCPSF73-Ia can be partially compensated by the second gene, BvCPSF73-Ib. This gene is located at position 954 bp. The BETA 1773 allele for post-winter bolting resistance may have incomplete penetrance due to a higher expression level compared to BvCPSF73-Ia [91]. Pfeiffer et al. (2017) identified three QTLs (DTBnat1 and DTBart1 on chromosome III, DTBnat2 and DTBart2 on chromosome V, and DTBnat3 on chromosome IX) that influenced the timing of the transition to bolting [92]. A tandemly duplicated locus, Bv_22330_orky, was identified on chromosome VI in beet; within the intron of this locus, SNP183 was found to be associated with bolting [93]. Bv_22330_orky is a homologue of the matrix metalloproteinase MMP gene in Arabidopsis, which is known to cause late flowering and early senescence [93]. Kuroda et al. (2023) identified a major quantitative trait locus (QTL) called qB6 that is closely linked to this single-nucleotide polymorphism (SNP). This QTL is associated with resistance to flowering and may be related to either BvFL1 or Bv_22330_orky, according to the authors’ hypothesis [94]. Ravi et al. (2021) discovered two SNPs in the sugar beet genome that are linked to a reduced likelihood of flowering [95]. The first SNP, SNP_36780842, is located on chromosome I in the 3’ UTR of a gene similar to the chaperone-J-domain superfamily genes that regulate flowering. The second SNP, SNP_48607347, is found on chromosome II in exon 3 of the xylose isomerase gene, which likely plays a role in modulating internal sugar levels, an important signaling mechanism during the transition to flowering [95]. Kuroda et al. (2023) identified several QTLs associated with flowering, including qB1 on chromosome I near QTL SNP_36780842. They also discovered minor QTLs, namely, qB8 on chromosome VIII and qB9 on chromosome IX [94].

Proteomics approaches are crucial for studying the mechanisms of flowering. Liang et al. (2018) discovered the role of phenylpropanoids, which are phenylalanine derivatives including active lignins, flavonols, isoflavonols, and anthocyanins, in vernalization. A comparison of the proteomes of vernalized and non-vernalized sugar beet plants grown from root crops showed variations in the abundance of disulfide-rich peptides (DRPs) at different stages of development. Specifically, there were 84 DRPs at week 17 (the germination stage), 195 DRPs at week 19 (the bolting stage in vernalized plants), and 82 DRPs at the 20th week (the flowering stage in vernalized plants). In vernalized plants, specific groups of genes are affected at different stages of growth. During germination and flowering, the expression of genes related to “plant hormone signal transmission”, “nitrogen metabolism”, “glutathione metabolism”, and “biosynthesis of diterpenoids” is suppressed. However, during the stage of bolting, there is an increase in the expression of genes related to “protein processing in the endoplasmic reticulum”. The “phenylpropanoid synthesis” group was enriched in both groups of plants. At the germination stage, PER52 proteins were found in non-vernalized plants, while BGLU42 protein was found in vernalized plants. At week 19, PER27 was found in non-vernalized plants, and CAD9, BGLU17, and OMT1 proteins were found in vernalized plants [96].

3.2. Hormonal Control of the Transition to Flowering in Sugar Beet

The transition to flowering is influenced by the intricate hormonal balance within a plant. Researchers are focusing on identifying sugar beet genes that are associated with hormonal status in Arabidopsis (gibberellin pathway (v)), as flowering in plants is influenced by their hormonal status. Chiurugwi et al. (2012) discovered that during vernalization, the expression of the gibberellin oxidase gene BvGA20ox2 increases and the expression of BvGA20ox1 decreases in the leaves and apical meristem [97]. Mutasa-Göttgens et al. (2009, 2012) also observed an increase in the expression of BvGA20ox1 during the postvernalization period [45,98]. Mutasa-Göttgens et al. (2012) detected differential expression of BvRAV1 (Related to ABA-insensitive 3/viviparous1) in the sugar beet apex. They found that gene expression increased in response to vernalization and decreased in response to exogenous exposure to GA [45]. Liang et al. (2017) developed a model that suggests the expression of BvFT1 is suppressed under long-day conditions after vernalization (Figure 2). This suppression is due to the coordinated expression of BvFT2, BvRAV1-like, and the lncRNA-encoding gene AGL15X2, homologous to the Arabidopsis COOLAIR gene [39]. Pi et al. (2020) found that the BvRAV1, BvRGA, and BvGATA22 genes in sugar beet show differential expression in response to vernalization. The expression of BvGATA22 was found to be regulated by cytokinins [99,100]. The expression of BvABFs and BvMYC2s was repressed by vernalization, implying the inhibition of ABA and JA signaling. Moreover, the down-regulation of BvDELLAs was observed, indicating that GA signaling is important in vernalization. The model developed by Zhao et al. (2023) implies there are three blocks of genes regulated by the hormones GA, ABA, and JA, directly or indirectly affecting the BvFT1-BvFT2-BvSOC module (Figure 2). Apparently, a transition to flowering is due to competitive cross-talk between ABA and GA, inhibiting and inducing bolting, respectively [48].

Smit (1983) developed a model of the bolting process in sugar beet, suggesting the presence of two substances: V, related to vernalization, and F, associated with flowering hormones. Substance V undergoes two processes: process I, known as vernalization, where V synthesis takes place at both high and low temperatures, and process II, called devernalization, where V destruction only occurs at high temperatures. At low temperatures, V accumulates. Substance F is synthesized through process III and occurs after vernalization, when temperatures increase and daylight hours lengthen. The rate of F synthesis is directly influenced by three factors: the length of daylight hours, the amount of accumulated substance V, and temperature. A mathematical model of the following form was suggested as a result. The equation dF/dt = kθVkp represents a mathematical relationship where F and V represent the content of substances F and V, kθ is the temperature coefficient, and kp is the photophase coefficient [101].

Koda et al. (2001) discovered that the application of exogenous JA results in the thickening of both the main and lateral roots. Additionally, it inhibits bolting caused by GA₃ treatment and vernalization. The levels of JA in the apical leaves of field plants increase during the summer, reaching their peak in August and then decreasing in September [102]. Zhao et al. (2023) discovered that 32 phytohormone compounds were significantly altered after a 16-week vernalization; in particular, the content of ABA and JA decreased more than sixteen and three times. In addition, GA₁₉ accumulated after vernalization, while the content of GA₅₃ and GA₂₀ decreased. GA₃-induced bolting in sugar beet plants was retarded via a treatment of exogenous ABA and methyl jasmonate (MeJA) [48].

Short-term exposure of sugar beet plants to cold stress leads to rapid rearrangement of their metabolisms [103]. The upregulation of ABA synthesis usually occurs at the beginning of cold stress and decreases due to the inhibition of biosynthesis during prolonged exposure to low temperatures. Interestingly, the CYSTATIN 6 CYSB gene, which plays a role in plant adaptation to cold, was found to be among DEGs and within DMRs between the bolting-resistant and sensitive genotypes [42].

Mutasa-Gottgens et al. (2010) conducted a study to investigate the effects of GA treatment, BvBTC1 allelic status, day length, and vernalization on bolting and flowering. Their findings suggest that the B locus pathway or the gibberellin pathway can initiate bolting regardless of photoperiod. In annual plants (with the genotypes BB and Bb), stem growth requires long days, and the GA pathway is not a limiting factor under these conditions. In biennial sugar beet plants (genotype bb), the B locus controls the requirement for vernalization, while the GA pathway regulates post-vernalization development. Vernalization is crucial for activating processes that influence the GA-dependent growth of the shoot apex, resulting in bolting and ultimately flowering. In some cases, the processes of bolting and flowering may not be related. This can be seen in plants that undergo bolting but do not produce flowers, instead maintaining vegetative rosettes. The developed experimental model suggests that carriers of the dominant allele B are stimulated to bolt by long days, while carriers of the recessive allele b require vernalization to activate the GA pathway for bolting. The transition to flowering in both genotypes is induced by long days [66].

The transgenic sugar beet line with the bean gene PcGA2ox1, which is responsible for breaking down biologically active forms of GA, exhibited a dwarf phenotype and sterility, requiring an additional 20 days for the transition to bolting. Male fertility was restored via the spraying of GA₃. Additionally, the introduction of the Arabidopsis transgene gai, which is a variant of the DELLA protein that lacks the DELLA domain and is less sensitive to GA, caused a delay in bolting in sugar beet plants for 11–14 days while maintaining fertility [98]. Liang et al. (2018) discovered that vernalized plants exhibited higher levels of gibberellins and indolylacetic acid (IAA) during the germination stage, specifically at the 17th week of development from a beet root. These levels were found to be correlated with the presence of the auxin signaling protein GH3.1 (A0A0J8CC67) and the gibberellin signaling protein GA3OX1 (A0A0J8CVX5). The GA3OX1 protein (A0A0J8CVX5) is likely involved in converting GA₂₀ to GA₁ when vernalization is not transpiring. IAA and GA₃ likely stimulate sugar beet development during the germination stage from the second-year sugar beet root in a threshold manner [96]. The expression of the BvRAV1-like gene increased by 2.5 times after vernalization and an additional 3 times after treating sugar beet plants with gibberellins. Furthermore, in the absence of vernalization, gibberellin treatment decreased the expression of this gene [45]. Mutasa-Gottgens et al. (2012) identified 19 genes that were differentially expressed under exogenous GA treatment [45].

In sugar beet, the process of the vernalization of the shoot apex is not a necessary requirement for it to exhibit a response to a floral stimulus. It is probable that the floral stimulus is synthesized within the leaves at low temperatures and subsequently transported to the apex in response to long days. Furthermore, the induction of floral stimulation in sugar beet does not necessitate the initiation of leaves from a vernalized meristem. Nevertheless, it is worth noting that only young leaves possess the capacity to exhibit vernalization responsiveness and, as a result, have the capability to provide a floral stimulus in conditions of extended daylight. Older foliage has limited responsiveness to vernalization; however, the probability of it generating floral inhibitors remains minimal [104].

Following the sensing of cold temperatures by immature or young leaves with actively dividing cells, plants need to maintain their vernalization memory through mitotic divisions in order to successfully transition to the flowering stage. The stable effect of vernalization can be maintained via cellular division occurring under warm environmental conditions. Subsequent investigations have provided additional evidence indicating that the process of vernalization memorization is intricately related to the epigenetic modulation of the main vernalization genes [105]. Complex studies are necessary to integrate the hormonal and epigenetic regulation of the floral transition in order to explain the nature of the floral stimulus and provide molecular models of vernalization memory in sugar beet.

4. Bolting of Sugar Beet in the Field and Prevention Methods

4.1. Prerequisites for Sugar Beet Bolting in the Field

The flowering of sugar beet can be triggered by various external factors or a combination of them. These factors include early spring sowing and vernalization (exposure to temperatures between 0 and +10 °C for 1–6 weeks, with the most effective range being +5 to +10 °C, although the effect diminishes at temperatures below +2.5 °C [106]), particularly in lowland areas during the growth of cotyledon leaves and the emergence of true leaves [107]. Additionally, the duration and quality of day light, as well as its intensity, can also influence flowering [108,109]. Vernalization is detected by the shoot apex, while photoperiod and light intensity are primarily detected by mature leaves [110]. Additionally, germinating seeds and seeds attached to the mother plant have the ability to respond to vernalization conditions [101,111].

One of the main causes of beet bolting is planting too early in the spring, especially during prolonged cold periods with sudden drops in temperature and no rainfall. In this case, the seeds remain in the ground for a period of up to 40 days, allowing them to undergo a vernalization stage. Vernalization is the process of acquiring or accelerating the ability to bolt and flower through prolonged exposure to low temperatures that mimic winter conditions. In 1974, the Vinnitsa region of Ukraine experienced a dry and cold spring. On March 28, fields in this region were sown with beet. However, due to poor seedlings, some of them had to be replanted on May 13. According to a relevant study [112], during the harvesting period, 27% of flowering beets were found in the early-sowing area, while there were no bolting sugar beets in the reseeded areas.

According to the All-Union Scientific Research Institute of Sugar Beet’s laboratory of physiology, as crops move northward, flowering increases due to a decrease in temperature, longer daylight hours, and the saturation of light with long-wave rays known as the far-red spectrum. This phenomenon, known as the “shade avoidance syndrome,” causes stems to grow rapidly towards a light source (

Table 2. The influence of different temperatures on the development of sugar beet [3].

Temperature, °C	Number of Plants, % of Total Number
	Formed a Vegetative Rosette	Entered the Reproductive Stage	Bolted	Flowered	Formed Seeds
20–23	100	0	0	0	0
15–18	90	10	10	0	0
8–12	0	100	100	75	25

) [3].

In a comparison of the cultivation of the same beet cultivars in the Vologda region (with 20 h and 5 min of daylight in June) and in Kyrgyzstan (with 15 h and 10 min of daylight in June), the resulting flowering values were 10.2% and 0.01%, respectively [8].

Several researchers have found a correlation between plant growth and development and bolting. Factors such as soil fertility, watering, and the application of fertilizers, particularly nitrogen fertilizers after vernalization, have been associated with an increased proportion of bolting plants. Additionally, lower plant densities have been shown to result in more bolting plants [101,112,113].

The bolting process is influenced by various factors, including planting density, fertilization, soil fertility, humidity, temperature, and light. It is important to note that in addition to vernalization, there is also the phenomenon of devernalization, also known as “reversal”. This means that a relatively short period of high temperatures can cancel out or lessen the impact of previous low temperatures [101,114]. In years when spring frost occurs after emergence, followed by a rapid warm period, a lower percentage of bolters is obtained compared to years without frost but with prolonged low temperatures [16]. Wood and Scott (1974) demonstrated that there is a positive correlation between the percentage of bolters and the number of days with a minimum air temperature below +7 °C within 4–6 weeks after planting. They also found a negative correlation between the percentage of bolters and the number of days with a maximum temperature exceeding +13 °C [115]. Similarly, Jaggard et al. (1983) discovered a positive correlation between the percentage of flowering plants in Great Britain and the number of days with a maximum air temperature below +12 °C [21].

Monogerm cultivars and hybrids of beet exhibit higher levels of flowering compared to multi-seeded varieties [112]. The monogerm tetraploid forms are the most sensitive to bolting, followed by monogerm diploids and multigerm tetraploids. Multigerm diploid cultivars are less sensitive to flowering because of improved selection methods [18].

4.2. Preventing Bolting in Field-Grown Sugar Beet Using Agrotechnical Methods

The primary agrotechnical methods for controlling sugar beet bolting include sowing at the optimal time, following agricultural cultivation practices, and using resistant cultivars and hybrids suitable for the recommended sowing region [112]. Even the most productive hybrid genotype will exhibit unsatisfactory traits if poorly prepared seeds are used and if they are grown in violation of agrotechnical requirements [116].

The timely destruction of individual early-flowering sugar beet plants in fields, from the bolting to the budding stage, is an effective and cost-efficient method for preventing the spread of weedy beet, a harmful weed [117].

There are patents for chemical seed treatments for preventing sugar beet bolting. This method involves treating seeds with a solution of chlorocholine chloride, which is particularly beneficial in northern regions. This treatment delays the initial formation of seedlings, resulting in beet development in the spring at higher air temperatures. As a result, the number of bolters decreases 1.6 to 4.2 times, leading to increased yields [118]. Another option for improving the quality of root crops, specifically sugar beet, is the use of paclobutrazol. This plant growth regulator and gibberellic acid inhibitor has been shown to reduce bolting percentages and increase sugar content, depending on the sugar beet genotype [119,120].

An additional approach is to use mathematical models to predict the percentage of sugar beet plants that will bolt in a field. This helps determine the optimal time for sowing. Milford et al. (2010) proposed an equation for calculating weighted vernalization hours. However, this model does not consider days with temperatures above 23 °C, which can lead to devernalization processes [108]. This model determined that the best time to sow sugar beets in the UK, with a maximum 5% risk of bolting plants, is after March 22 [97]. This model is useful for conducting experiments in climate chambers in the UK. A model like this would also be useful for different climatic conditions and latitudes. The vernalization weight coefficient was calculated based on long-term observations of British selection genotypes in the UK. Therefore, recalculating this indicator is necessary for other conditions. Additionally, this model necessitates the incorporation of new coefficients, such as daylight hours, for both field forecasts and experiments conducted in an artificial climate.

4.3. Breeding for Bolting Resistance

4.3.1. Conventional Breeding for Bolting Resistance

One important issue in sugar beet cultivation is the reliance on vernalization and long photoperiods to determine resistance to flowering in different geographical zones. It is crucial to conduct breeding work for resistance to flowering in the specific environmental and agricultural conditions for which a cultivar is being developed [17].

In breeding practice, various methods are employed to select non-bolting material. These methods include pre-winter and very early sowing, the selection of vernalized seedlings under long day conditions, sowing in western and northwestern regions with long-vernalized seeds, the negative selection of early-ripening plantings, and the performance of tests in polar day conditions. Selection within a population is more effective [18].

Breeders in Northwest Europe have traditionally relied on early sowings in the field to select for resistance to bolting. However, this approach has limitations, as early sowings may not always be feasible and the percentage of bolting plants is often too small for accurately assessing effectiveness [17]. Bell conducted a study in 1939 on the impact of low temperature and continuous light on sugar beet seedlings. The study revealed that different genotypes have varying requirements for photothermal treatment in order to bolt and flower. It was also found that plants resistant to bolting, selected from groups where most plants had bolted, produced offspring with a high level of resistance [121,122]. This finding was subsequently confirmed by other researchers [123,124,125]. Bell’s methods and principles were widely adopted and expanded upon by sugar beet breeders in Northwest Europe.

Logvinov et al. (2021) developed and implemented new reliable methods for assessing and selecting bolt-resistant initial breeding materials and commercial hybrids in the Krasnodar Territory. These methods were applied during winter and early-spring sowings. Experiments showed that monogerm genotypes exhibited a greater degree of bolting compared to multigerm plants. Further studies have shown that breeding work performed to evaluate and develop breeding material resistant to bolting can be effectively conducted using the method of provocative early-spring sowing with germinated seeds maintained at +9 °C or treated with an aqueous solution of the herbicide Burefen FD-11 (5 mL/L) or pre-soaked in water and kept at a temperature of +3 °C for 20 days [16,126].

However, although the potential yield advantage of autumn sowing is more than 25%, this benefit can be negated by summer droughts and various diseases such as rhizomania, beet nematode infection, powdery mildew, and downy mildew. Combining high yield and quality traits with exceptional resistance to bolting, diseases, frost, and drought necessitates the use of repeated selection methods [127].

The method of sowing vernalized germinated seeds in a greenhouse with additional lighting can be utilized to isolate bolting-resistant forms from the beet population [128]. The use of greenhouses is a proven method. Many genotypes can flower when grown under short-day conditions, as long as they are exposed to long-day conditions for subsequent growth. This approach was widely utilized in Sweden during the 1960s. If it was not possible to sow outdoors early enough, the material was sown in a heated greenhouse without additional lighting in late January. When the plants were 3–4 weeks old, ventilation systems were connected to maintain a temperature of +3–8 °C. Then, when the conditions allowed, typically in early April, the plants were transferred to the fields. Populations resulting from this selection have demonstrated strong resistance to bolting in Northwest Europe. With smaller amounts of material, seeds can be planted in the fall in a greenhouse with natural conditions for 4–5 weeks. They can then be moved to a heated greenhouse with continuous lighting, where the more vulnerable genotypes will sprout quickly. After removing all the plants that show signs of bolting, the remaining plants are transferred to the first greenhouse for a period of 12–14 weeks. If the initial treatment is conducted in a timely manner during the autumn, the second treatment will be finished on schedule. This will allow the plants to be transplanted into the field for propagation at the usual time in the spring [17].

Kornienko et al. (1983) proposed a method of enhancing the bolting trait by introducing a mixture of the herbicides eptam and lenatsil into the soil. This method increases the manifestation of the bolting trait by 20% and allows for the selection of non-bolting forms [129]. It is suggested that growers treat their seeds with a treflan solution and store them at +8–10 °C for winter sowing. In the spring, plants that bolt should be removed, while non-bolting plants should be kept until the end of the growing season. These non-bolting plants can then be used as starting material with resistance to bolting [130]. Finally, a mechanical method for selecting bolt-resistant forms has been developed using a multi-level rejection system for cultivating stubborn varieties [131].

Assessing the genetic diversity of sugar beet helps us find new sources of resistance to bolting. Kutnyakhova et al. (2016) conducted an evaluation of sugar beet hybrids from 2012 to 2014. The study, carried out by Lion Seeds and the Mazlumov Scientific Research Institute of Sugar Beet, found that half of the accessions exhibited bolting. Specifically, the Humber hybrid had a bolting rate of 1%, while Granat, Simbol, Shannon, and Etalon had a bolting rate of 0.4% [132]. Burenin and Piskunova (2016) found that accessions from Sweden exhibited the highest resistance to bolting in their assessment of the VIR collection. The success of German breeders in developing monogerm cultivars that are resistant to bolting was also acknowledged [133].

4.3.2. Marker-Assisted and Genomic Selection for Bolting Resistance

The haplotype diversity of the BvBTC1 gene has been extensively studied. The “a”, “b”, “c”, and “l” haplotypes are associated with a biennial phenotype, while the “d”–“k”, “m”–“o” haplotypes are mainly found in annual phenotypes. The “m” haplotype is only found in sea beet from northern latitudes [25,41,69,70]. Haplotypes “a”–“c” and “l” differ from others due to a large insertion (~28 kb) in the promoter region of the recessive haplotypes of the biennial habit btc1. Additionally, haplotypes “a”–“c” and “l” lack low-complexity regions that are necessary for the interaction between BTC1 and BvBBX19 proteins [41,69,70]. The majority of cultivated beets carry the recessive Bvbtc1a haplotype, while sea beets primarily have BvBTC1 haplotypes from the “annual” class. The authors of the studies also found that the Bvbtc1a genotype is present in sugar beet accessions that are not resistant to bolting, while resistant accessions contain haplotypes such as “g” and “o” in Japanese cultivars [25,69]. Hussain et al. (2020) discovered new variants of BvBTC1 in a bolting-sensitive accession. These variants include single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) found in exon 10 [134]. Buttner et al. (2010) developed the codominant marker GJ1001c16 to differentiate between the dominant and recessive BvBTC1 haplotypes. This marker has been extensively tested in various studies using segregated populations to explore alternative vernalization genes [45,85,92,135]. Hoft et al. (2018) developed a CAPS marker, CAU4206, which is located on a single-nucleotide polymorphism (SNP) in exon 9. The “A” variant of this marker is associated with the biennial habit haplotype Bvbtc1l, which was obtained through EMS mutagenesis [70]. Kuroda et al. (2019) developed a marker to distinguish between haplotypes “a” (biennial development type) and “g” and “o” (annual development type) [25].

Hoft et al. (2018) discovered allelic diversity in different beet species for the BvBBX19 gene. In sugar beet, haplotypes “a”, “e”, “d”, and “f” were found. For the BvFT1 gene, haplotypes “a”, “b”, “g”, and “h” were identified in sugar beet. Lastly, sugar beet contained haplotypes “a” and “d” for the BvFT2 gene. Most cultivated sugar beet accessions with a biennial habit carried combinations of the haplotypes btc1a, BvBBX19a, BvFT1a, and BvFT2a or BvFT1a and BvFT2d [69]. Since these gene sequences are publicly available [25,41,69,70], it is possible to create new molecular markers for their haplotypes and study their phenotypic manifestations in segregated populations. These markers can then be used in breeding through association genetics methods. The publicly available sugar beet genome sequences and their corresponding gene annotations, such as “Beta vulgaris EL10_1.0”, available at A Comprehensive Website for Beta vulgaris Genome Sequence and Annotations [136,137]; “Beta vulgaris Resource” (University of Natural Resources and Life Sciences, Vienna, Austria) [138,139,140,141]; and “Sugar Beet Microsatellite Database” (Indian Agricultural Statistics Research Institute) [142,143], are invaluable for various purposes such as searching for target and candidate genes, mapping sequences, designing primers, and conducting in silico analyses of newly obtained genome and transcriptome data.

Among the genomic selection markers, the TaqMan marker for SNP18 stands out. The T allele is associated with resistance to bolting, while the C allele is associated with sensitivity [93]. Additionally, there are two HRM markers: SNP/SNP_36780842, where the G allele is linked to resistance to bolting and the C allele is linked to sensitivity, and SNP21/SNP_48607347, where the C allele is associated with resistance to bolting and the A allele is associated with sensitivity [95].

4.3.3. Mutagenesis and Genetic Engineering for Bolting Resistance

The expansion of allelic diversity in sugar beet includes genes that determine vernalization requirements, sensitivity to long daylight hours, and resistance to bolting. It is possible to create new alleles and haplotypes or introduce new genes into the beet genome through mutagenesis, genetic engineering, and genome editing.

Hohmann et al. (2005) used EMS mutagenesis within the TILLING strategy to create a collection of sugar beet lines based on the early-flowering line 930190. Based on experimental data from mutant lines, the B2, B3, B4, and B5 loci were identified [70,83,84,85]. Additionally, an interaction model was developed between the BvBTC1 and BvBBX19 proteins [40,71], resulting in the discovery of a new allele, BvBBX19h [71]. Frerichmann et al. (2013) detected associations between a nucleotide polymorphism in BvFL1 and winter bolting and winter hardiness when the EcoTILLING method was applied [75].

Another method for enhancing sugar beet is through the creation of genetically modified plants. Van Roggen et al. (1997) found that overexpression of the pumpkin GA20-oxidase transgene and the use of antisense constructs of genes of GA20-oxidase and GA3-β-hydroxylase resulted in the downregulation of GA₁ and GA₄ in sugar beet [144]. Mutasa-Gottgens et al. (2009) conducted a study to examine the impact of hormonal status on bolting and flowering in sugar beet. They obtained transgenic lines of sugar beet with the bean hormonal metabolism genes PcGA2ox1 and Arabidopsis gai, which resulted in a significant delay in flowering [98].

Genome editing enables the creation of new alleles and haplotypes using existing genes, without the presence of transgenes in resulting plants. Only one report has been published on the use of CRISPR/Cas9 editing to confer resistance to Beet Curly Top Iran Virus in sugar beet [145]. Genome editing shows great promise as a tool for creating new forms of sugar beet resistant to bolting. This is because allelic variants that result in a biennial phenotype requiring vernalization are caused by impaired functionality of proteins involved in the transition to bolting and flowering. By using genome editing to obtain non-functional alleles and haplotypes, it is possible to develop sugar beet varieties that are resistant to bolting.

5. Bolting as a Tool for Accelerating Breeding and Seed Production

If flowering occurs in the first year of growth when cultivating sugar beet for root crops, it is considered unacceptable. However, during seed production, first-year flowering is intentionally encouraged. The study of flowering regulatory mechanisms has led specialists to reconsider their approaches to sugar beet breeding. They have been able to develop practical methods, such as obtaining seeds in one year through artificial vernalization, including in artificial climate laboratories.

It is widely recognized that young sugar beet plants can be induced to bolt in nearly 100% of cases through photothermal treatment. This treatment involves subjecting the plants to temperatures of 5–8 °C and providing them with 16 h of daylight for a period of 8–14 weeks, depending on the specific genotype and objectives. This knowledge can be used as the basis for achieving a generation from seed to seed in just one year, which accelerates breeding progress [17,121,122]. It is important to consider a key point when designing such approaches: vernalization prepares a plant for flowering but does not directly cause it. There is often a noticeable time gap between the cold treatment, bolting, and flowering [146].

To induce the bolting and flowering of various sugar beet genotypes, a photothermal method is usually employed. This method involves subjecting plants to extended vernalization under long daylight conditions in climate-controlled chambers or specialized greenhouses. This method can be used to obtain seeds within one year via speed-breeding technologies. Questions regarding the particular timing, temperature, and light conditions of vernalization are relevant as vernalization protocols vary (

Table 3. Vernalization protocols for assessing genotypes for bolting/flowering resistance, vernalization requirements, and annual and biennial development patterns in greenhouses and climate chambers.

Pre-Vernalization	Vernalization	Acclimatization	Post-Vernalization	Bolting Phenotyping	Reference
20 °C 119 days 22 h of light 315 μmol m−2 s−1	4 °C 90 days 22 h of light 315 μmol m−2 s−1	–	20 °C 22 h of light 315 μmol m−2 s−1	Plants that did not bolt 16 weeks after vernalization were classified as “never bolting”	[69]
32 days	38 days 5 °C 16 h of light fluorescent illumination	5 days outdoors in the shade (25–30 April 2014)	Plants were transplanted into a field (Memuro, Hokkaido) and grown from 30 April to 29 July 2014. Average temperature: May—15C June—20C July—23C Day length increases from 13 h 50 m to 15 h 01 m on 16 July and decreased to 14 h 37 m on 29 July	Most bolted plants had already appeared by 29 July	[25]
20 °C 135 days	4 °C 12 weeks	12 °C 3 days	20 °C 102 days	Every second day, the onset of bolting was recorded (BBCH scale code: 51) according to the method reported by Meier et al.: (1) Annual plants that bolted within 135 days; (2) Biennial plants that only bolted after cold treatment; (3) Plants that did not bolt until the end of the experiment after 325 days	[71]
18 °C 2 weeks 12 h of light 200 µmol m−2 s−1	6 °C 15 weeks 12 h of light 200 µmol m−2 s−1	stepwise temperature increases from 6 °C to 18 °C 2 weeks 12 h of light 200 µmol m−2 s−1	18 °C 18 h of light 200 µmol m−2 s−1	–	[72]
20 °C 16 h of light	4 °C 3 months 16 h of light	6 weeks temperature was increased from 4 to 25 °C during the light cycle and from 4 to 15 °C during the dark cycle 16 h of light	–	Plants were phenotyped for the occurrence and time of bolting three times per week until 6 months after vernalization	[41]
22 °C 16 h of light 220 µmol m−2 s−1 for 15 h + tungsten light for 1 h (far-red)	6 °C 8 weeks in annuals 18 weeks in biennials 10 µmol m−2 s−1	15 °C 1 week	22 °C 16 h of light 220 µmol m−2 s−1 for 15 h + tungsten light for 1 h (far-red)	Plants initiated bolting within 2–3 weeks and flowered within 6 weeks of vernalization	[79]
24 °C 30 days 24 h of light high-pressure sodium and metal halide lamps	5 °C 90 days 24 h of light high-pressure sodium and metal halide lamps	no gradual temperature transition	24 °C 24 h of light high-pressure sodium and metal halide lamps	–	[73]
24 °C 24 days 16 h of light 200 µmol m−2 s−1	4 °C 16 weeks 16 h of light 200 µmol m−2 s−1	–	24 °C 16 h of light 200 µmol m−2 s−1	After transferring vernalized seedlings to room temperature, the elongation of the stems was observed daily	[38]
16 h 6 weeks 300 µmol m−2 s−1	5 °C 3 months 8 h of light 300 µmol m−2 s−1	–	16 h 6 weeks 300 µmol m−2 s−1	–	[78]
22 °C 8 weeks 16 h of light 700 μmol m−2 s−1	4 °C 18 weeks 16 h of light	–	22 °C 6 weeks 16 h of light 1000 μmol m−2 s−1	After 9 weeks at 4 °C BI = 100% and average BD < 25 d considered as sensitive to bolting BI = 60% and average BD > 30 d considered as resistant to bolting	[74,81]
20 °C 16 h of light 900 μmol m−2 s−1 400–700 nm 32 days if bolted 94 days if bolting did not occur; then, additional 22 days 22 h 1200 μmol m−2 s−1	5 °C 16 weeks 22 h of light 200 μmol m−2 s−1	8 °C 1 week 22 h of light 200 μmol m−2 s−1	20 °C 22 h of light 1200 μmol m−2 s−1	Additional vernalization was applied for 26 weeks for those plants that did nor bolt after the first round of vernalization and were then kept under post-vernalization conditions	[92]
15 °C 14 h of light 14–15 days (two true leaves)	3/7/11/15 °C 0/14/28/42/49 days 14 h of light	10 °C 1 week 14 h of light	10 °C/15 °C/25 °C 90 days 14/18/24 h of light	Proportion bolting was counted for 100–160 days after chilling Temperature and duration of chilling as well as photophase and temperature of postvernalization were the factors of different experiments	[101]
20 °C 16 h of light 4/7 weeks	5 °C 16 weeks 22 h of light	8 °C 1 week 22 h of light	Planted in field nurseries	Bolting was scored when stem elongation was visible.	[91]

), and they concern the following: (i) the stage of plant development at which cold treatment begins (pre-vernalization period), ranging from seeds to four-month-old plants; (ii) vernalization temperature, ranging from 0 °C to 10 °C; (iii) the duration of vernalization, ranging from 8 to 18 weeks; (iv) the length of daylight hours, ranging from 16 h to 24 h; and (v) additional influencing factors, namely, the intensity of lighting and the types of lamps used.

To compare genotypes and the effectiveness of vernalization protocols, two parameters are used: the bolting index, which measures the percentage of plants that have formed a bolt, and the bolting delay, which measures the number of days from the end of vernalization to bolting. The bolting delay can reach up to 50 days. The developmental stage at which vernalization starts affects the bolting index. The plants of sugar beet are usually exposed to vernalization when they are 2 weeks to one month old or when they have reached the phase of 5–6 pairs of true leaves [25,38,72,73,78,147]. However, in certain studies, vernalization may begin when the plants are 4–4.5 months old [69,71]. Vernalization can happen at any stage of development and occurs with different levels of intensity. It is interesting to note that the vernalization of seeds can occur while they are still on the mother plant [148]. Wellensiek and Verkerk (1954) discovered that vernalization for 14–16 weeks under 24 h day conditions is more effective than pre-treating seeds at +1 °C in the dark and then vernalizing them for 8–10 weeks [149]. This is likely because when plants are grown in light, they develop green leaf mass earlier and start accumulating daylight hours sooner. Various protocols recommend different options for the pre-vernalization period. These include 24 days at 24 °C with a 16 h light day [38], one month followed by transplantation in a field with an average air temperature of about 15 °C for the next month [25], 8 weeks at 22 °C with a 16 h light day [74], 120 days at 20 °C with a 22 h light day [69], and 135 days at 20 °C with a 16 h light day [71].

A higher bolting index was observed when vernalization was conducted at 4 °C compared to 7 °C. This trend was consistent across various genotypes [74]. In studies comparing vernalization durations ranging from 6 to 18 weeks, it has been found that vernalization duration is directly proportional to the flowering index and inversely proportional to the period before bolting [38,74,149]. Although data and studies suggest that the optimal duration of vernalization is typically 10–12 weeks at 4 °C with a 16–22 h light day [69,71,147], it is important to note that this timeframe can vary depending on the genotype. If vernalization is prolonged, a plant may not have sufficient time to develop green mass. While this may increase the bolting index, it can also decrease the number of seeds produced per plant [149]. Additionally, to reduce plant stress when transitioning from temperatures above 20 °C to temperatures below 5 °C, it is recommended to implement an acclimation period of 3–5 days at 12 °C [25,71].

The bolting of sugar beet is stimulated by various factors, including temperature variations and changes in photoperiod. Far-red light has a substantial impact on the transition to flowering [150,151,152,153]. Cameras equipped with speed-breeding technology allow for the adjustment of light using this spectrum at various intensities.

Traditionally, researchers have studied the effects of photoperiods using tungsten incandescent lamps. According to Curth (1960), there is no need to increase the luminous flux above 100–200 lux for growing sugar beet, as this does not affect the percentage of flowering. These conditions also allowed for equal growth rates among plants. The most effective light sources had a peak luminous flux density in the blue or orange-red area of the spectrum, while sources with a peak in the green areas were less effective [124]. Lane et al. (1965) conducted a study comparing red light (600–700 nm) with far-red light (700–800 nm) to extend daylight hours. In their experiment, half of the spectrum (600–770 nm) was red light, and the other half was far-red light. In a study on annual sugar beet, it was found that supplementation with far-red light was more effective in promoting flowering plants compared to red light [154].

At the start of the flowering process, plants use their leaves to detect photoperiods. This is carried out through the photoreceptor phytochrome, which is a blue-green pigment. The receptors are sensitive to light in the red and far-red regions of the spectrum. They can be activated by either the former or the latter, enabling them to measure the ratio of red to far-red light and initiate responses. In the first state, the phytochrome is configured to detect radiation at 660 nm (the inactive form of Pr, which detects red). Once detected, the phytochrome is transferred to the second state, the active form of Pfr, which detects far-red. In this state, it waits for far-red radiation with a wavelength of 730 nm (which is not suitable for photosynthesis). When far-red light is detected, the phytochrome returns to its initial state of “red detection,” known as Pr. It is widely acknowledged that far-red light does not have a serious impact on photosynthesis. However, this spectrum provides plants with crucial information about their environment [155].

Curth (1960) and Lane et al. (1965) found that beet plants have spectral exposure peaks in the blue and red to far-red regions of the spectrum, indicating a sensitivity to high-intensity light [124,154].

In order to fully and successfully determine and test methods for obtaining sugar beet seeds within a one-year “seed to seed” cycle, it is necessary to create and utilize artificial climate chambers. These chambers allow for the artificial creation of any temperature and light conditions, which can stimulate and accelerate the necessary processes in the studied plant.

The development of artificial climate chambers that can control various environmental conditions for plant growth, such as light, temperature, and humidity, characterizes a groundbreaking plant-breeding technology known as speed breeding. The manipulation of environmental conditions can greatly accelerate plant growth and allow for multiple generations per year. This technique also promotes faster flowering and seed ripening compared to traditional greenhouse or field conditions. Accelerated breeding can increase the number of generations per year for various crops. For example, spring wheat, durum wheat, barley, chickpea, and peas can be bred such that up to six generations per year are produced, while rapeseed can be bred such that up to four generations can be produced per year. This is great progress compared to the 2–3 generations that can be achieved under normal greenhouse conditions. Accelerated plant development can be achieved through the use of fully enclosed growth chambers in controlled environments with varying temperatures. This method is particularly useful for research purposes such as phenotyping, high-throughput genotyping, genome editing, and genomic selection. The use of supplemental lighting in greenhouses enables rapid crop cycling and the flexibility to accommodate larger crop improvement programs. Fast growth chambers not only have value in terms of breeding and biotechnology but also provide cost savings through the use of LED lighting [156]. Modern speed-breeding technologies allow for the induction of flowering in sugar beet via its subjection to a series of temperature and photoperiodic stresses, all applied within a single chamber [157].

6. Conclusions

The transition to flowering in sugar beet is a complex process regulated by multiple mechanisms at different levels. Despite multiple studies and proposed models that explain the molecular bases of floral induction in sugar beet, many questions remain unanswered. Moreover, as shown in the present review, inconsistencies between different studies may emerge due to either the different methods or plant materials used. The following is, in our opinion, a long but far from exhaustive list of relevant issues that need to be addressed in future investigations.

• Fundamental scientific issues:

  (a)

  The development of comprehensive flowering regulatory networks for sugar beet based on the analysis of DEGs, DMRs, proteomes, and hormones in various tissues and organs. This analysis should involve comparing leaves of different ages and the apical meristem before, after, and during vernalization and/or long-day exposure in genotypes that are tolerant and sensitive to bolting.

  (b)

  The development of models for the co-expression of genes involved in cold stress (short-term chilling), vernalization (long-term chilling), and devernalization.

  (c)

  The study of recently discovered genes that regulate the floral transition using novel plant materials, including (i) lines with dysfunctional genes developed through RNA silencing or CRISPR/Cas9 knockout; (ii) RILs and NILs with different alleles/haplotypes; and (iii) diverse collections of cultivated and wild beet species.

• Practical issues:

  (a)

  The study of the allelic diversity of bolting-regulating genes in collections from various climatic zones.

  (b)

  The expansion of the allele/haplotype diversity of bolting-regulating genes can be achieved through various methods, such as genome editing, mutagenesis, and wide hybridization.

  (c)

  The improvement of the conventional, marker-assisted, and genomic selection of bolting-resistant genotypes.

  (d)

  The development of bolting-resistant cultivars and hybrids.

  (e)

  The improvement of agricultural techniques for preventing bolting.

• Speed-breeding issues:

  (a)

  The optimization of a set of conditions to accelerate the transition to the reproductive stage, namely, temperature, light intensity, duration, and spectral composition during different phases (pre-vernalization, vernalization, acclimatization, and post-vernalization).

  (b)

  The optimization of chemical treatments, such as those involving mineral nutrition, epimutagens, and hormones, to accelerate the transition to the reproductive stage.

  (c)

  The optimization of artificial conditions for the development of a sufficient amount of viable seeds.

  (d)

  The possibility of the vernalization of embryos in vitro using the embryo rescue technique.

  (e)

  The possibility of positive selection for bolting in segregating populations under speed-breeding conditions.

In summary, the bolting of sugar beet has two aspects. Bolting can have negative effects when it occurs in a field during cold weather and long daylight hours. Agrotechnical practices, such as selecting appropriate cultivars, following recommended sowing dates, and treating seeds, can prevent bolting. Breeders working on developing bolting-resistant sugar beet cultivars should have a thorough understanding of this plant’s flowering physiology. This knowledge will help them to establish analytical frameworks for implementing negative selection against bolting. Molecular geneticists studying the genetic mechanisms of bolting and flowering in sugar beet provide valuable support to breeders. The study of allelic diversity in flowering genes enables researchers to create functional markers for marker-assisted selection. By genotyping and phenotyping genetic collections and cultivar panels, SNP-based genomic markers can be developed. At the same time, bolting may be useful for breeding and genetic studies. Speed-breeding technology will accelerate the development of cultivars and the creation of panels of isogenic lines of sugar beet through classical crossings and genome-editing methods. Panels like these will aid in studying the impact of genes, alleles, and haplotypes on the sugar beet life cycle and bolting requirements. The collaborative efforts of molecular biologists, biotechnologists, physiologists, and breeders will enable the development of sugar beet cultivars and hybrids that are resistant to bolting. Additionally, these efforts will accelerate breeding and seed production processes and facilitate the study of the fundamental principles underlying the transition of plants from the vegetative phase to the generative phase of development.