Prerpints.org logo
Preprint
Review

Advances in Somatic Embryogenesis of Banana

Altmetrics

Downloads

158

Views

48

Comments

0

A peer-reviewed article of this preprint also exists.

Submitted:

29 May 2023

Posted:

31 May 2023

You are already at the latest version

Alerts
Abstract
Bananas and plantains (Musa spp.) cultivation holds significant global economic importance, but it faces numerous challenges, which may include diverse abiotic and biotic factors such as drought and various diseases caused by fungi, viruses, and bacteria. The genetic and asexual nature of cultivated banana varieties makes them unattractive for improvement via traditional breeding. To overcome these constraints, modern biotechnological approaches like genetic modification and genome editing have become essential for banana improvement. However, these techniques rely on somatic embryogenesis, which has only been successfully achieved in a limited number of banana cultivars. Therefore, developing new strategies for improving somatic embryogenesis in banana is crucial. This review article focuses on advancements in banana somatic embryogenesis, highlighting the progress, the various stages involved, cryopreservation techniques, and the molecular mechanisms underlying the process. Furthermore, the article discusses the factors that could influence somatic embryogenesis and explores the prospects for improving the process, especially in recalcitrant banana cultivars. By addressing these challenges and exploring potential solutions, researchers aim to unlock the full potential of somatic embryogenesis as a tool for banana improvement, ultimately benefiting the global banana industry.
Keywords: 
Subject: Biology and Life Sciences  -   Plant Sciences

1. Introduction

Bananas and plantains (Musa spp.) are important staple food crops grown in over 136 countries in the tropics, with global production of 170 million tons annually (FAOSTAT2021). It is a valuable food security and cash crop as it can be cultivated in diverse environments and produces fruits throughout the year in these favorable weather conditions. Smallholder farmers mainly cultivate banana for domestic consumption and local or regional markets; only about 15% of production enters international markets. Hundreds of cultivars of bananas are grown and consumed worldwide, but large-scale farmers mainly grow the Cavendish type of dessert bananas for commercialization in local and international markets. Other dessert banana varieties, such as Sukali Ndiizi, Gros Michel, Rasthali, and Lakatan, are also produced in several countries worldwide, where they are sold for economic and social benefits. Plantain is grown largely in Central and West Africa, and East African Highland banana (EAHB) is cultivated in East Africa, especially in the Great Lakes region of Africa, and is a source of livelihood for millions of people [1,2].
Banana production is affected by several diseases and pests, declining soil fertility, narrow genetic diversity in germplasm, and inadequate availability of clean planting material among smallholder farmers. In addition, changes in climate and weather are also significantly impacting banana yields, particularly in the regions where the crop is grown with minimal or no irrigation. These factors reduce its production and threaten livelihoods. These cultivation & production constraints are exacerbated by the triploid nature of cultivated banana varieties, which is a challenge to breeding programs and compromises the goal of expanding the genetic diversity of the crop [1,3]. Thus, banana improvement via modern biotechnology is important in ensuring their existence and improved yield. Biotechnical approaches to banana improvement include in-vitro regeneration techniques involving organogenesis and somatic embryogenesis, which are prerequisites for further improvement via genetic engineering [4].
Organogenesis is a morphogenic response involving the development of plant organs, such as shoot buds and roots, either directly from the explant or callus [5]. Thus, organogenesis is the basis for the in-vitro propagation of bananas. Micropropagation of banana is useful in providing disease-free and true-to-type planting materials for farmers and has been shown to improve yield significantly [6].
Somatic embryogenesis is a unique phenomenon in plants involving the development of embryos from somatic cells [7]. Like in most plant species, somatic embryogenesis in banana is induced by exogenous hormones in specific tissues, such as meristems and immature male flowers. Embryogenic calli from these tissues easily proliferate in cell suspension cultures, forming millions of cells, which can be regenerated into complete plantlets. The embryogenic cells are also ideal explants for genetic engineering, including transgenesis and gene editing [8]. Thus, somatic embryogenesis is critical to banana improvement through modern breeding. However, somatic embryogenesis is highly dependent on genotype and very laborious. Only a few cultivars of bananas have so far responded to somatic embryogenesis, with most elite cultivars, including most EAHBs, remaining recalcitrant [9]. Notably, low frequencies of somatic embryogenesis are observed in the few varieties amenable to the process. Therefore, there is a need to explore new approaches for inducing somatic embryogenesis in recalcitrant elite cultivars and improve the success rates of somatic embryogenesis in cultivars that show poor response to the process. Accumulating evidence shows that somatic embryogenesis in banana is modulated by certain genes, including transcription factors, induced by specific hormones and environmental conditions [10,11]. This review discusses advances in somatic embryogenesis in banana with emphasis on the progress, molecular basis underlying the process, and the potential application of this knowledge to optimize somatic embryogenesis in recalcitrant banana cultivars.

2. Progress in somatic embryogenesis of banana

The earliest records of somatic embryogenesis in banana can be traced to the 1980s when Cronauer and Krikorian obtained somatic embryos from cell suspensions of split young shoots [12]. Based on their findings, somatic embryos were only obtained in a medium containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) instead of 2,4-dichlorophenoxyacetic acid (2,4-D) commonly used today. A year later, Escalant and Teision, for the first time, reported successful plant regeneration from somatic embryos derived from the callus of zygotic embryos of diploid banana using 2,4-D [13]. The same year, Novak and colleagues regenerated hundreds of plants from somatic embryos derived from dicamba-induced callus of leaf sheaths and rhizome tissues [14]. Progress on somatic embryogenesis and the establishment of embryogenic cell suspension cultures proceeded swiftly, and breakthroughs in 1991 were game-changing. Specifically, the first report on the use of multiple meristems (also known as scalps) as explant for establishing somatic embryogenesis and embryogenic cell suspension (ECS) [15]. In the same year, Ma described an elaborated method for establishing ECS from embryogenic calli derived from immature male flowers [16]. Its noteworthy that scalps and immature male flowers have been the most responsive and extensively used explants for establishing ECS. In 1993, Panis et al. obtained and regenerated protoplasts from ECS derived from scalps [17]. Other notable reports on the optimization of somatic embryogenesis and plant regeneration in the 1990s and early 2000 were the establishment of temporary immersion cultures and bioreactor systems to increase the efficiency of mass propagation of plants from ECS [18,19], and cryopreservation of banana ECS for long-term storage [20].
Notably, research in somatic embryogenesis of banana has not seen major discoveries in the last two decades. New reports have mainly focused on optimizing the previous protocols and processes in various banana and plantain cultivars. For example, Xu et al. reported an efficient protocol for developing and regenerating ECS derived from scalps of banana cv. Cavendish Williams [21]. Wong et al. enhanced plant regeneration from ECS by incorporating a liquid-based embryo development medium [22]. Further, Strosse et al. conducted large-scale experiments to assess the embryogenic potential of scalps from 18 different banana cultivars belonging to five genome groups, including wild diploid (AA), Cavendish (AAA), highland (AAA-EAH), plantain (AAB), and cooking types (ABB) [23]. They confirmed that the scalp is suitable explant for somatic embryo induction in banana, but success was not achieved with the highland banana cultivars. However, successful induction of somatic embryogenesis and ECS from immature male flowers of one cultivar of EAHB has been reported [24]. In 2007, Wei et al. demonstrated that picloram could be used as an alternative to 2,4-D for the induction of somatic embryos in male flower explants of banana. The researchers also showed that a low concentration of thidiazuron (TDZ, 0.2 mg/l) could increase the frequency of somatic embryo germination [25]. Torres et al. revealed that meristematic domes of axillary sprouted buds from a diploid banana Calcutta 4 responded better than scalps for somatic embryogenesis [26]. An efficient somatic embryo induction and regeneration protocol was optimized for the plantain cultivar ‘Gonja manjaya’ using scalps, where up to 30,000 plantlets were generated from 1 ml settled cell volume of ECS [27]. In 2014, Remakanthan et al. reported unique findings in which direct somatic embryogenesis was induced from split shoot tips of banana cultivar ‘Grand naine’ in just 15 days, without an intervening callus phase [28]. According to the study, combining 4.14 μM picloram and 0.22 μM benzyl aminopurine (BAP) led to 100% embryo induction; however, these findings have not been reproduced by any other group so far.
Researchers have analyzed the genetic stability of plants regenerated from somatic embryos. Morais-Lino et al. employed simple sequence repeat markers to analyze the genetic variations among banana plants regenerated from somatic embryos and found no genetic variation [29]. Also, ISSR analysis of somatic embryo-derived banana plants did not detect soma-clonal variation among the plants, further confirming that somatic embryogenesis can serve as a tool for the large-scale regeneration of genetically stable plants [30].

3. Stages of somatic embryogenesis and regeneration in banana

Regeneration of banana plantlets via somatic embryogenesis involves several stages, including induction of embryogenic callus, initiation of ECS, embryo development, maturation, and embryo germination into complete plantlets, which are then acclimatized and potted (Figure 1a-j).

3.1. Induction of embryogenic callus

Somatic embryogenesis in banana can be induced from various explants, including mature/immature zygotic embryos, immature male flowers, female flowers, multiple meristems (scalps), rhizome slices, and corm tissues [8]. However, scalps (Figure 1a) and immature male flowers (Figure 1b) have been the most responsive, with consistent results across diverse banana genotypes. The plant growth hormones and media used for somatic embryo induction vary with the explant. Most studies have reported success for immature male flower explants when the MA1 medium is used; whereas the ZZ medium was found to be appropriate for scalps (Table 1) [16]. Variations in the type and concentrations of auxins have also been reported, particularly in the substitution of 2,4-D alone or with either picloram or dicamba for callus induction [25,31,32]. However, such variations have not been reproducible in most laboratories and banana genotypes. Regarding culture conditions, somatic embryogenesis in banana occurs better in the dark at 26-28 °C and mostly emerges after six weeks of culture, reaching the highest frequency at three months from the time of explant initiation in the callus induction medium. The ideal embryogenic callus (Figure 1c) for initiating ECS is typically obtained after four to five months of culture, particularly when immature male flowers are used as explants. The culture usually begins to turn brown after six months due to medium depletion and accumulation of phenolics. Thus, the timely selection of the ideal callus for transferring to a liquid medium is important for the successful initiation of ECS [33].

3.2. Initiation and maintenance of cell suspension cultures

Generation of ECS from embryogenic calli has contributed significantly to banana improvement. ECS are generated by culturing embryogenic calli in a liquid medium and maintaining the culture indefinitely in a rotary shaker. The components of the liquid media vary with the explant and medium used for callus induction. For example, calli induced from scalp explants proliferate easily in the same medium used for callus initiation minus a gelling agent (ZZ, Table 1) [23]. On the other hand, significant changes in the cell suspension medium are made for callus induced from immature male flowers. Specifically, the 2,4-D concentration is reduced from 4 mg/l to 1 mg/l and other auxins such as indole-3-acetic acid (IAA) and naphthaleneacetic acid (NAA) are excluded from the liquid medium (MA2, Table 1) [16]. Initiation of ECS involves two stages; in the first stage, a friable embryogenic callus is dropped into a culture vessel (such as 50 or 100 ml conical flask) containing 3 to 10 ml medium, depending on the size of the callus complex. The liquid culture is then maintained at 26-28 °C in a shaker (95-100 rpm) in the dark for about two weeks or until the cells begin to proliferate rapidly. In the second stage, the suspension culture is transferred into a bigger vessel (such as a 250 ml conical flask), after which the culture medium is adjusted to about 50 ml (Figure 1d). The ECS in the bigger flask is maintained in the dark indefinitely at 26-28 °C. Half of the liquid medium is usually replaced with fresh medium every 7 to 20 days, depending on the rate of cell proliferation. The ECS cultures are usually well proliferated and ready for other uses at three months post-initiation and remain useful for at least nine months [33].

3.3. Development, maturation, and germination of somatic embryos

Embryogenic cells from cell suspension cultures need to develop into somatic embryos (Figure 1e) and then mature (Figure 1f) before they can germinate into shoots (Figure 1g) and then into plantlets (Figure 1h). This process typically takes four to six months, depending on the quality of ECS. The medium for embryo development, maturation, and germination also varies depending on the origin of the ECS [33]. Usually, ECS from scalp explants require a much simpler medium for development, maturation, and germination than those from immature male flowers. Specifically, ECS derived from scalp explants can develop, mature, and regenerate into plants in semi-solid half-strength MS medium without growth hormones (RD1, Table 1). However, a trace amount of BAP (0.25 mg/l) can be added to the RD1 medium to increase germination frequency after three months of embryo maturation [23,33]. Meanwhile, ECS derived from immature male flowers require richer media supplemented with several growth hormones and antioxidants to mature and regenerate into plants (MA3 and MA4, Table 1) [33]. After germination, banana plants can be multiplied further in a proliferation medium (PM, Table 1, Figure 1i) supplemented with BAP [27]. Notably, banana shoots maintained in PM supplemented with BAP may not develop proper roots and must be transferred to a rooting medium (RM, Table 1) for complete plant regeneration [27].

3.4. Post flask

Acclimatization is the final step before the plants can be introduced to the farm or greenhouses for further growth and development. Ex-vitro banana plants are more accessible to acclimate than those from other plant species and do not require special substrates such as peat moss or vermiculite. Well-rooted plants (Figure 1i) can be transferred to normal soil in small pots after washing off the medium. However, the plants must be maintained in a humid environment for at least three weeks to ensure a high survival rate [36]. Acclimatized plants are usually transferred to bigger pots (Figure 1j) for further use.

3.5. Cryopreservation of cell suspension cultures

It takes about 12-18 months to develop banana ECS. Besides, periodic refreshing of the culture medium is time-consuming, labour-intensive, and involves using chemicals and equipment, making it expensive to maintain ECS. Long-term culture and frequent handling of ECS also expose them to the risk of somaclonal variation, contamination, and reduced and eventual loss of morphogenic potential. Cryopreservation of banana ECS is, thus, a vital technique in laboratories that generate and maintain ECS. However, studies on the cryopreservation of banana ECS are limited. The most widely applied protocol for cryopreservation of banana ECS was described by Panis et al. [20]. The protocol was initially optimized for cryopreserving ideal ECS, which are homogenous, containing isodiametric cells with multiple small vacuoles, large nuclei, and tiny protein and starch granules. The procedure was later optimized to include less ideal ECS, including heterogeneous cell suspensions containing cells with large vacuoles and starch granules [37]. Generally, the protocol involves first subjecting ECS to cryoprotection using dimethyl sulphoxide at 0 °C, then slowly freezing them to -40 °C at the rate of 1°C per minute before dipping them in liquid nitrogen for storage. After storage, the cells are thawed rapidly in a water bath at 40 °C. A protocol for cryopreservation of banana ECS via vitrification has also been reported [38]. As an alternative to cryopreservation, banana ECS can also be maintained for longer periods under slow growth conditions [39]. Notably, cryopreserved ECS has been shown to regenerate into normal plants with typical morphology and yield [40].

4. Factors influencing somatic embryogenesis

Generating ECS of most elite banana varieties remains a challenge because most of these varieties are irresponsive to the available protocols. In the past, most somatic embryogenesis protocols for plants were developed based on a trial-and-error approach; however, increasing reports on somatic embryogenesis and recent advances in molecular biology uncover molecular mechanisms underpinning the process, providing novel research directions. Here, we summarize the factors to consider when optimizing somatic embryogenesis in banana.

4.1. Explant selection and manipulation

Most banana varieties have not responded to somatic embryogenesis using the most responsive explants like scalps and immature male flowers. Thus, the focus should shift to explant selection and manipulation before callus initiation. Factors such as age, lushness, and position within the plant affect the response of an explant to somatic embryogenesis. For instance, the response of immature male flowers to somatic embryo induction is better when the male buds are collected within three weeks after emergence, which also varies with the genotype [33,41]. Also, the position of immature flowers on the male bud has been shown to affect somatic embryogenesis [42]. These factors could be attributed to the effect of various environmental and age-related factors on the expression of genes associated with somatic embryogenesis. Explant manipulation before in-vitro culture may also affect somatic embryo induction and frequency. For instance, wounding of explants before culture can activate specific genes involved in somatic embryogenesis and improve explant response to the process [43]. Therefore, future studies should explore the effects of explant-related factors on somatic embryogenesis to improve the frequency of somatic embryogenesis in recalcitrant banana varieties.

4.2. Plant growth hormones for embryogenesis

Somatic embryogenesis is induced by supplementing the medium with different combinations of exogenous auxins and cytokinins. Typically, higher auxin concentration relative to cytokinin is needed for the induction and proliferation of somatic embryos in banana [8]. Despite the obvious participation of plant growth hormones in somatic embryo induction, knowing the type, quantities, and combinations to apply for a specific banana genotype remains challenging, especially because endogenous levels of hormones vary between genotypes. Thus, current studies still rely on the trial-and-error approach to optimize somatic embryogenesis. However, this is changing with recent molecular studies, which continue to uncover the intricacy of hormone signaling in plants. For example, two enzymes, indole-3-pyruvate monooxygenase and adenylate isopentenyl transferase, involved in endogenous auxin synthesis, were found to accumulate more in embryogenic than non-embryogenic callus of banana, suggesting that endogenous auxin levels influence somatic embryogenesis [44]. Thus, a further study designed to incorporate tryptophan in the medium, a precursor of IAA, reported enhanced somatic embryo induction and germination frequencies in banana cultivars that were initially recalcitrant to somatic embryogenesis. Also, the addition of NAA and gibberellic acid (GA), which are associated with the synthesis of enzymes that induce somatic embryo germination in banana, enhanced the germination of somatic embryos [45]. These findings suggest that formulating the right hormone regime could improve somatic embryogenesis in recalcitrant banana cultivars. However, further studies are needed to determine the exact hormone type and quantities for a specific genotype.

4.3. Culture medium additives

Plant tissue culture medium additives such as amino acids and antioxidants also play a significant role in somatic embryo induction and germination. Amino acids provide organic nitrogen and serve as precursors for endogenous synthesis of proteins and growth hormones that facilitate somatic embryogenesis. Proline and glutamine are associated with embryo maturation, which improves embryo germination [42]. Tryptophan promotes embryogenesis by boosting endogenous auxin synthesis [45]. Antioxidants such as ascorbic acid, citric acid, and cysteine are crucial in reducing phenolic accumulation and explant necrosis [46,47]. Ethylene accumulation is associated with reduced somatic embryogenesis; thus, the addition of ethylene inhibitors such as salicylic acid can enhance somatic embryo induction [48]. Recently, the addition of calcium chloride in the callus induction medium significantly improved the frequency of somatic embryogenesis in recalcitrant banana cultivars [49]. Calcium related proteins, including calcium-dependent protein kinase and the calcium-binding mitochondrial carrier protein, were upregulated in embryogenic callus more than non-embryogenic callus, suggesting that calcium potentially plays a crucial role is somatic embryogenesis [49]. However, further molecular analysis should be conducted to determine the precise role of these medium supplements on somatic embryogenesis.

4.4. Growth room conditions

Conditions under which plant tissue cultures are maintained significantly affect the outcome of an experiment and, therefore, should be considered when optimizing somatic embryogenesis in banana. These conditions mainly include temperature, light, and humidity but can be extended to include medium conditions such as gelling strength, pH, and frequency of sub-culture/medium change. Temperature regulates many physiological processes in plants and thus plays a crucial role in somatic embryogenesis. The optimum temperature for somatic embryogenesis can vary with genotype and the embryogenesis stage. For instance, enhanced somatic embryo induction was observed at a lower temperature of 17 °C than at 28 °C in radiata pine [50]. In contrast, a higher temperature of 30 °C is optimum for somatic embryo induction in cotton, while 25 °C is optimal for embryo maturation and germination [51].
Light requirement during somatic embryogenesis also varies with genotype and stage of embryogenesis[52,53]. In banana, somatic embryogenesis has been reported to occur better in darkness; however, further studies should be conducted to determine whether this is true for all genotypes. Studies have also revealed that somatic embryo induction varies with light quality. For instance, blue light improved the frequency of somatic embryo induction in Agave tequilana[54]. Moreover, high ratio of red to far-red was found to promote somatic embryogenesis in Araujia sericifera [55], suggesting that light quality may play a significant role in somatic embryogenesis.
Other factors such as humidity, gelling strength, pH, and frequency of sub-culture also affect somatic embryogenesis. For example, Strosse et al. 2003 reported that a relative humidity of more than 70% is optimum for somatic embryogenesis in banana[23].

5. Molecular basis of somatic embryogenesis

Molecular studies have revealed specific genes and signaling pathways that regulate plant somatic embryogenesis. These genes comprise transcriptional factors, cell cycle proteins, and stress/defense-related proteins that modulate endogenous hormone synthesis and other signaling pathways [56]. Transcriptional factors play a critical role in the transcriptional regulation of somatic embryogenesis, and recent reports have implicated the roles of WUSCHEL (WUS), BABYBOOM (BBM), LEAFY COTYLEDON (LEC), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) in enhancing somatic embryo formation efficiencies [57].
WUS is a homeodomain TF modulating the formation of apical shoot meristem, which is crucial in somatic embryogenesis [58]. WUS expression is induced by auxins and mediates cell switch from a vegetative to an embryogenic state in A. thaliana [59]. Upregulation of WUS genes has been reported during somatic embryogenesis in several plant species, including Medicago truncatula [60], Elaeis guineensis [61], and Betula platyphylla [62]. Recently, overexpression of GhWUS from Gossypium hirsutum in A. thaliana promoted somatic embryogenesis [63]. A banana homolog of WUS (MaWUS2) is upregulated during the late stage of ECS, revealing that it potentially plays a crucial role in ECS proliferation in banana [10].
BBM gene encodes AP2/ERF domain transcription factor implicated in multiple plant morphogenic responses [64,65]. It also interacts with other TFs, such as LEC1/2, ABI3, and FUS3 network to induce somatic embryogenesis [66]. BBM has been implicated in somatic embryogenesis in many plant species, including A. thaliana [67], Capsicum annuum [68], Glycine max [64], and Zea mays [69]. Shivani et al. demonstrated that a banana homolog of BBM (MaBBM2) is highly expressed at all stages of ECS development relative to NECS, suggesting that it could play an essential role in somatic embryo induction and proliferation in banana [10].
The SERK family of TFs can serve as a marker of somatic embryogenesis in several plant species [70,71,72]. Initially identified in Daucus carota, SERK genes are upregulated during somatic embryo formation up to the globular stage, indicating their essential role in the initiation and early stages of somatic embryogenesis [72]. Analysis of MaSERK1 expression in banana showed that it is upregulated in the embryogenic callus of immature male flowers compared with the non-embryogenic callus. Its expression is also higher during ECS proliferation, suggesting that it may be vital in the formation and proliferation of embryogenic callus in banana [73].
The LEC family of TFs plays significant roles in somatic embryogenesis and has been demonstrated to induce somatic embryo formation in heterologous systems [74,75,76,77]. However, the transcript level of its homolog (MaLEC2) in banana is higher in NECS, and it remains unclear whether MaLEC2 plays a negative regulatory role in banana somatic embryogenesis [10].
Besides TFs, other genes that regulate somatic embryogenesis have been identified in banana. Kumaravel et al. identified many genes involved in somatic embryo induction, development, and germination. Specifically, calcium-binding mitochondrial carrier protein and calcium-dependent protein kinase (CDPK) were upregulated 34.2 folds in embryogenic callus relative to non-embryogenic callus of banana cv. Rasthali [49]. CDPK facilitates calcium transfer into the cell to promote cell differentiation and proliferation during somatic embryogenesis [78,79]. Shivani et al. studied the differential expression patterns of 16 genes during somatic embryogenesis and ECS proliferation in banana and identified MaPIN1 as a positive regulator and MaCRE2 and MaCRE3 as either negative regulators or insignificant in banana somatic embryogenesis [11]. Furthermore, molecular analysis of genes expressed during somatic embryogenesis in banana identified tryptophan aminotransferase relate 2 (TAR2), an enzyme involved in auxin biosynthesis, as an important facilitator of the transition from early to late stages of somatic embryo development [80]. Defense-related proteins have also been implicated in somatic embryo induction in banana, including cysteine proteinase inhibitor, 5-epi-aristolochene synthase, and E3 ubiquitin-protein ligase[49]. Cysteine-rich peptides facilitate the supply of amino acids and nitrogen in germ tissues during germination and have been used as molecular markers for somatic embryo maturation[81,82]. Notably, higher levels of chaperon Heat shock 70 protein were observed in EC than in NEC, suggesting that defense and stress-related proteins could also play an essential role in the somatic embryogenesis of banana [49]. In contrast, upregulation of enzymes related to ethylene biosynthesis (such as 1-aminocyclopropane-1-carboxylate oxidase) was observed in NEC of banana, implying that ethylene biosynthesis could suppress somatic embryo induction [49].
Apart from DNA sequence variations, epigenetic factors have recently emerged as significant contributors to somatic embryogenesis [83]. This effect is mainly associated with the degree of chromatin remodeling during somatic embryogenesis and could be influenced by explant choice and other environmental factors, including medium components and culture conditions [84]. Although the effect of epigenetic factors on somatic embryogenesis has not been demonstrated in Musa spp., insights from studies involving other plant species could inform future related studies in banana. Studies have shown that DNA methylation influences somatic embryogenesis [85]. Specifically, lower global DNA methylation has been reported in embryogenic cultures, while the reverse has been observed in non-embryogenic cultures of several plant species [86,87,88]. A study on A. thaliana demonstrated that de novo DNA methylation and its maintenance can affect somatic embryogenesis [89]. Notably, alterations in the pattern of global DNA methylation, especially during long-term subcultures in exogenous hormones, can affect the embryogenic potential of pro-embryogenic masses [90]. Many experiments have been conducted to demonstrate that DNA methylation indeed affects somatic embryogenesis. For example, the application of the demethylating agent 5-azacitidine (5AzaC) inhibited somatic embryogenesis in A. thaliana and other plant species [89,91,92]. Posttranslational modification of histone has also been implicated in somatic embryo formation [93]. Histone deacetylation modulates cell cycle reprogramming during the initial stages of somatic embryogenesis, which could be associated with the presence of exogenous auxins [94]. Also, the expression of polycomb repressive complex 2 (PRC2), a gene family involved in lysine 27 methylation in histone H3 is negatively associated with somatic embryogenesis and is explant specific [95].

6. Future prospects for improving somatic embryogenesis

The main obstacle hindering banana improvement via modern biotechnology is the recalcitrance of elite cultivars to somatic embryogenesis. Although manipulation of explants, medium components, and culture conditions can improve the frequency of somatic embryogenesis, it is improbable that such approaches will work for the most recalcitrant cultivars like the EAHBs. Besides, optimizing somatic embryogenesis via medium variation is time-consuming and less precise since it relies on trial-and-error approaches. Studies have also revealed that although many genes that mediate somatic embryogenesis are shared across diverse plant species, some are limited to certain plant species [55]. Thus, future breakthroughs in somatic embryogenesis, especially for the recalcitrant banana cultivars, will likely involve the heterologous expression of morphogenic genes. So far, studies on the heterologous expression of morphogenic genes have mainly focused on TFs, including LEC, WUS, and BBM. Although not yet reported in Musa spp., overexpression of these TFs can significantly promote somatic embryogenesis and transformation efficiencies in diverse plant species, including dicotyledonous and monocotyledonous plants.
For example, LEC 2 overexpression induced somatic embryogenesis in the seedlings of A. thaliana, confirming its potential role in auxin signaling [72]. In coffee, overexpressing WUS induced callus formation and somatic embryogenesis by 400 folds [94]. Heterologous expression of AtWUS from A. thaliana in cotton enhanced callus differentiation and positively modulated other TFs, including FUS3, LEC1, and LEC2 [95]. In addition, ectopic expression of BBM in A. thaliana revealed that it is mainly upregulated in plant tissues undergoing differentiation and morphogenesis [96]. Heterologous expression of BBM from Brassica napus and A. thaliana in Nicotiana tabacum promoted cell differentiation. Still, they exhibited different responses depending on the plant species, suggesting that components of its signaling pathway may vary between species [97]. Notably, somatic embryogenesis without hormone supplementation in the culture medium was observed in transgenic lines of A. thaliana and Theobroma cacao overexpressing the TcBBM gene from T. cacao. However, subsequent suppression of embryo development was observed in the T. cacao transgenic lines, implying that overexpression of BBM genes can adversely affect some genotypes' later stages of somatic embryogenesis [98]. Recent monocot studies have shown that ectopic expression of morphogenic genes can expand the genotype range for somatic embryogenesis and transformation. For instance, overexpression of maize BBM and WUS genes enhanced somatic embryogenesis and transformation of recalcitrant maize, sorghum, and rice varieties [94,99–101]. Genome-wide analysis of TFs during somatic embryogenesis in banana revealed that MaWUS2 and MaBBM2 could play a crucial role in somatic embryogenesis, suggesting that their overexpression has a great potential in revolutionizing somatic embryogenesis and transformation of recalcitrant banana varieties [10].

7. Conclusion

Somatic embryogenesis and the establishment of ECS in banana are highly dependent on genotype, which has limited banana genetic engineering for decades. Moreover, the process is time-consuming, and the maintenance of ECS are expensive and laborious. As a result, only a few banana varieties have seen significant advancements through this method, while elite cultivars like the EAHBs have been left behind. Somatic embryogenesis in banana can be optimized by manipulating explants, culture medium components, and growth room conditions. However, overcoming the challenges posed by highly recalcitrant cultivars, particularly EAHB requires additional approaches. Fortunately, ongoing molecular studies provide new insights into the process and will likely contribute to future breakthroughs. Notably, recent investigations on the heterologous expression of morphogenic genes in monocots offer promising insights. This approach is anticipated to play a critical role in overcoming the genotype barrier associated with somatic embryogenesis and ultimately enabling the genome engineering of banana.
By leveraging these molecular advancements, researchers can work towards improving the efficiency and effectiveness of somatic embryogenesis in bananas. It could lead to the successful somatic embryogenesis of elite banana cultivars like the EAHBs, which were previously challenging to incorporate into biotechnology programs. The enhanced somatic embryogenesis has the potential to revolutionize banana improvement efforts through modern biotechnology and open new possibilities for improving the traits and characteristics of this globally important crop.

Authors Contributions

LT conceived the idea, and all authors wrote, edited, and reviewed the manuscript. JNT prepared the figure.

Acknowledgement

This study was supported by the United States Agency for International Development (USAID).

Conflict of interest

The authors declare that they have no competing interests.

References

  1. Dale, J.; Paul, J.-Y.; Dugdale, B.; Harding, R. Modifying Bananas: From Transgenics to Organics? Sustainability 2017, 9, 333. [Google Scholar] [CrossRef]
  2. Ainembabazi, J.H.; Tripathi, L.; Rusike, J.; Abdoulaye, T.; Manyong, V. Ex-Ante Economic Impact Assessment of Genetically Modified Banana Resistant to Xanthomonas Wilt in the Great Lakes Region of Africa. PLOS ONE 2015, 10, e0138998. [Google Scholar] [CrossRef]
  3. Ploetz, R.C.; Evans, E.A. The Future of Global Banana Production; 2015; ISBN 978-1-119-10775-0.
  4. Arvanitoyannis, I.S.; Mavromatis, A.G.; Grammatikaki-Avgeli, G.; Sakellariou, M. Banana: Cultivars, Biotechnological Approaches and Genetic Transformation. International Journal of Food Science & Technology 2008, 43, 1871–1879. [Google Scholar] [CrossRef]
  5. Sluis, A.; Hake, S. Organogenesis in Plants: Initiation and Elaboration of Leaves. Trends in Genetics 2015, 31, 300–306. [Google Scholar] [CrossRef]
  6. Suman, S. PLANT TISSUE CULTURE : A PROMISING TOOL OF QUALITY MATERIAL PRODUCTION WITH SPECIAL REFERENCE TO MICROPROPAGATION OF BANANA. 2017, 17, 1–26.
  7. Thorpe, T.A.; Stasolla, C. Somatic Embryogenesis. In Current Trends in the Embryology of Angiosperms; Bhojwani, S.S., Soh, W.-Y., Eds.; Springer Netherlands: Dordrecht, 2001; ISBN 978-94-017-1203-3. [Google Scholar]
  8. Escobedo-GraciaMedrano, R.M.; Enríquez-Valencia, A.J.; Youssef, M.; López-Gómez, P.; Cruz-Cárdenas, C.I.; Ku-Cauich, J.R. Somatic Embryogenesis in Banana, Musa Ssp. In Somatic Embryogenesis: Fundamental Aspects and Applications; Loyola-Vargas, V.M., Ochoa-Alejo, N., Eds.; Springer International Publishing: Cham, 2016; ISBN 978-3-319-33705-0. [Google Scholar]
  9. Uma, S.; Kumaravel, M.; Backiyarani, S.; Saraswathi, M.S.; Durai, P.; Karthic, R. Somatic Embryogenesis as a Tool for Reproduction of Genetically Stable Plants in Banana and Confirmatory Field Trials. Plant Cell Tiss Organ Cult 2021, 147, 181–188. [Google Scholar] [CrossRef]
  10. Shivani; Awasthi, P. ; Sharma, V.; Kaur, N.; Kaur, N.; Pandey, P.; Tiwari, S. Genome-Wide Analysis of Transcription Factors during Somatic Embryogenesis in Banana (Musa Spp.) Cv. Grand Naine. PLOS ONE 2017, 12, e0182242. [Google Scholar] [CrossRef]
  11. Shivani; Kaur, N. ; Awasthi, P.; Tiwari, S. Identification and Expression Analysis of Genes Involved in Somatic Embryogenesis of Banana. Acta Physiol Plant 2018, 40, 139. [Google Scholar] [CrossRef]
  12. Cronauer, S.; Krikorian, A.D. Somatic Embryos from Cultured Tissues of Triploid Plantains (Musa ‘ABB’). Plant Cell Reports 1983, 2, 289–291. [Google Scholar] [CrossRef]
  13. Cronauer-Mitra, S.S.; Krikorian, A.D. Plant Regeneration via Somatic Embryogenesis in the Seeded Diploid Banana Musa Ornata Roxb. Plant Cell Reports 1988, 7, 23–25. [Google Scholar] [CrossRef]
  14. Novak, F.J.; Afza, R.; Van Duren, M.; Perea-Dallos, M.; Conger, B.V.; Xiaolang, T. Somatic Embryogenesis and Plant Regeneration in Suspension Cultures of Dessert (AA and AAA) and Cooking (ABB) Bananas (Musa Spp.). Nat Biotechnol 1989, 7, 154–159. [Google Scholar] [CrossRef]
  15. Plant Regeneration in Cell Suspension Cultures of the Cooking Banana Cv. Bluggoes’ (Musa Spp. ABB Group). Available online: https://www.musalit.org/seeMore.php?id=1635 (accessed on 12 September 2022).
  16. Ma, S.S. Somatic Embryogenesis and Plant Regeneration from Cell Suspension Culture of Banana. In Proceedings of the Proceedings of symposium on tissue culture of horticultural crops, Taipei, Taiwan; pp. 19888181–188.
  17. Panis, B.; Van Wauwe, A.; Swennen, R. Plant Regeneration through Direct Somatic Embryogenesis from Protoplasts of Banana (Musa Spp.). Plant Cell Reports 1993, 12, 403–407. [Google Scholar] [CrossRef] [PubMed]
  18. Escalant, J.-V.; Teisson, C.; Cote, F. Amplified Somatic Embryogenesis from Male Flowers of Triploid Banana and Plantain Cultivars (Musa Spp.). In Vitro Cell.Dev.Biol.–Plant 1994, 30, 181–186. [Google Scholar] [CrossRef]
  19. Gómez Kosky, R.; de Feria Silva, M.; Posada Pérez, L.; Gilliard, T.; Bernal Martínez, F.; Reyes Vega, M.; Chávez Milian, M.; Quiala Mendoza, E. Somatic Embryogenesis of the Banana Hybrid Cultivar FHIA-18 (AAAB) in Liquid Medium and Scaled-up in a Bioreactor. Plant Cell, Tissue and Organ Culture 2002, 68, 21–26. [Google Scholar] [CrossRef]
  20. Cryopreservation of Musa Suspension Cultures and Subsequent Regeneration of Plants. Available online: https://www.musalit.org/seeMore.php?id=7653 (accessed on 22 September 2022).
  21. Xu, C.; Panis, B.; Strosse, H.; Li, H.; Xiao, H.; Fan, H.; Swennen, R. Establishment of Embryogenic Cell Suspensions and Plant Regeneration of the Dessert Banana ‘Williams’ (Musa AAA Group). The Journal of Horticultural Science and Biotechnology 2005, 80, 551–556. [Google Scholar] [CrossRef]
  22. Wong, W.C.; Jalil, M.; Ong-Abdullah, M.; Othman, R.Y.; Khalid, N. Enhancement of Banana Plant Regeneration by Incorporating a Liquid-Based Embryo Development Medium for Embryogenic Cell Suspension. The Journal of Horticultural Science and Biotechnology 2006, 81, 385–390. [Google Scholar] [CrossRef]
  23. Strosse, H.; Schoofs, H.; Panis, B.; Andre, E.; Reyniers, K.; Swennen, R. Development of Embryogenic Cell Suspensions from Shoot Meristematic Tissue in Bananas and Plantains (Musa Spp.). Plant Science 2006, 170, 104–112. [Google Scholar] [CrossRef]
  24. Namanya, P.; Magambo, S.M.; Mutumba, G.; Tushemereirwe, W. Somatic Embryogenesis from Immature Male Inflorescences of East African Highland Banana CV ‘ Nakyetengu’. African Crop Science Journal 2004, 12, 43–49. [Google Scholar] [CrossRef]
  25. Yue-rong, W.E.I.; Hu, Y.; Bing-zhi, H.; Xia, H.; Xue-lin, H.; Ji-shui, Q.I.U.; Lin-bing, X.U. Effects of Picloram, ABA and TDZ on Somatic Embryogenesis of Banana. Acta Horticulturae Sinica 2007, 34, 81. [Google Scholar]
  26. Torres, J.L.; Kosky, R.J.; Perez, N.M.; Alvarez, D.R.; Cabrera, A.R.; Jova, M.C.; Pino, A.S.; Vega, V.M.; Pérez, M.B.; Roux, N. New Explant for Somatic Embryogenesis Induction and Plant Regeneration from Diploid Banana (‘Calcutta 4’, Musa AA). Biotechnologia Vegetal 2012, 12, 25–31. [Google Scholar]
  27. Tripathi, J.N.; Muwonge, A.; Tripathi, L. Efficient Regeneration and Transformation of Plantain Cv. “Gonja Manjaya” (Musa Spp. AAB) Using Embryogenic Cell Suspensions. In Vitro Cell.Dev.Biol.-Plant 2012, 48, 216–224. [Google Scholar] [CrossRef]
  28. Remakanthan, A.; Menon, T.G.; Soniya, E.V. Somatic Embryogenesis in Banana (Musa Acuminata AAA Cv. Grand Naine): Effect of Explant and Culture Conditions. In Vitro Cell.Dev.Biol.-Plant 2014, 50, 127–136. [Google Scholar] [CrossRef]
  29. Morais-Lino, L.S.; Santos-Serejo, J.A.; Amorim, E.P.; de Santana, J.R.F.; Pasqual, M.; de Oliveira e Silva, S. Somatic Embryogenesis, Cell Suspension, and Genetic Stability of Banana Cultivars. In Vitro Cell.Dev.Biol.-Plant 2016, 52, 99–106. [Google Scholar] [CrossRef]
  30. Natarajan, N.; Sundararajan, S.; Ramalingam, S.; Chellakan, P.S. Efficient and Rapid In-Vitro Plantlet Regeneration via Somatic Embryogenesis in Ornamental Bananas (Musa Spp.). Biologia 2020, 75, 317–326. [Google Scholar] [CrossRef]
  31. Escobedo-GraciaMedrano, R.M.; Maldonado-Borges, J.I.; Burgos-Tan, M.J.; Valadez-González, N.; Ku-Cauich, J.R. Using Flow Cytometry and Cytological Analyses to Assess the Genetic Stability of Somatic Embryo-Derived Plantlets from Embryogenic Musa Acuminata Colla (AA) Ssp. Malaccensis Cell Suspension Cultures. Plant Cell Tiss Organ Cult 2014, 116, 175–185. [Google Scholar] [CrossRef]
  32. Novak, F.J.; Afza, R.; Van Duren, M.; Perea-Dallos, M.; Conger, B.V.; Xiaolang, T. Somatic Embryogenesis and Plant Regeneration in Suspension Cultures of Dessert (AA and AAA) and Cooking (ABB) Bananas (Musa Spp.). Nat Biotechnol 1989, 7, 154–159. [Google Scholar] [CrossRef]
  33. Banana_and_plantain_embryogenic_cell_suspensions_975.Pdf.
  34. Murashige, T.; Skoog, F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia plantarum 1962, 15, 473–497. [Google Scholar] [CrossRef]
  35. Schenk, R.U.; Hildebrandt, A.C. Medium and Techniques for Induction and Growth of Monocotyledonous and Dicotyledonous Plant Cell Cultures. Can. J. Bot. 1972, 50, 199–204. [Google Scholar] [CrossRef]
  36. Ahmed, S.; Sharma, A. Studies on Hardening and Acclimatization of Micro-Propagated Plantlets of Banana Cv. Grand Naine. 2014, 9, 965–967. [Google Scholar]
  37. Panis, B.; Schoofs, H.; Remy, S.; Sági, L.; Swennen, R. Cryopreservation of Banana Embryogenic Cell Suspensions: An Aid for Genetic Transformation. Cryopreservation of tropical plant germplasm: Current research progress and application, 2000; 103–109. [Google Scholar]
  38. YanNa, L.; YiMing, W.; GuiBing, H.; HouBin, C.; ChunXiang, X. Plant regeneration via somatic embryogenesis after cryopreservation of embryogenic cell suspensions of banana (Musa spp. AAA) by vitrification and the genetic stability of regenerated plant. Acta Horticulturae Sinica 2010, 37, 899–905. [Google Scholar]
  39. Kulkarni, V.M.; Ganapathi, T.R. A Simple Procedure for Slow Growth Maintenance of Banana (Musa Spp.) Embryogenic Cell Suspension Cultures at Low Temperature. Current Science 2009, 96, 1372–1377. [Google Scholar]
  40. Cote, F.; Goue, O.; Domergue, R.; Panis, B.; Jenny, C. In-Field Behaviour of Banana Plants (Musa AA Sp) Obtained after Regeneration of Cryopreserved Embryogenic Cell Suspensions. Cryo Letters 2000, 21, 19–24. [Google Scholar] [PubMed]
  41. Youssef, M.; James, A.; Mayo-Mosqueda, A.; Ku-Cauich, J.R.; Grijalva-Arango, R.; Escobedo-GM, R.M. Influence of Genotype and Age of Explant Source on the Capacity for Somatic Embryogenesis of Two Cavendish Banana Cultivars (Musa Acuminata Colla, AAA). African Journal of Biotechnology 2010, 9, 2216–2223. [Google Scholar] [CrossRef]
  42. Nandhakumar, N.; Kumar, K.; Sudhakar, D.; Soorianathasundaram, K. Plant Regeneration, Developmental Pattern and Genetic Fidelity of Somatic Embryogenesis Derived Musa Spp. Journal of Genetic Engineering and Biotechnology 2018, 16, 587–598. [Google Scholar] [CrossRef] [PubMed]
  43. Iwase, A.; Mita, K.; Nonaka, S.; Ikeuchi, M.; Koizuka, C.; Ohnuma, M.; Ezura, H.; Imamura, J.; Sugimoto, K. WIND1-Based Acquisition of Regeneration Competency in Arabidopsis and Rapeseed. J Plant Res 2015, 128, 389–397. [Google Scholar] [CrossRef] [PubMed]
  44. Kumaravel, M.; Uma, S.; Backiyarani, S.; Saraswathi, M.S.; Vaganan, M.M.; Muthusamy, M.; Sajith, K.P. Differential Proteome Analysis during Early Somatic Embryogenesis in Musa Spp. AAA Cv. Grand Naine. Plant Cell Rep 2017, 36, 163–178. [Google Scholar] [CrossRef]
  45. Kumaravel, M.; Backiyarani, S.; Saraswathi, M.S.; Arun, K.; Uma, S. Induction of Somatic Embryogenesis (SE) in Recalcitrant Musa Spp. by Media Manipulation Based on SE’s Molecular Mechanism. Acta Hortic. 2020; 119–127. [Google Scholar] [CrossRef]
  46. Ko, W.H.; Su, C.C.; Chen, C.L.; Chao, C.P. Control of Lethal Browning of Tissue Culture Plantlets of Cavendish Banana Cv. Formosana with Ascorbic Acid. Plant Cell Tiss Organ Cult 2009, 96, 137–141. [Google Scholar] [CrossRef]
  47. Ngomuo, M.; Mneney, E.; Ndakidemi, P. Control of Lethal Browning by Using Ascorbic Acid on Shoot Tip Cultures of a Local Musa Spp. (Banana) Cv. Mzuzu in Tanzania. African Journal of Biotechnology 2014, 13. [Google Scholar] [CrossRef]
  48. Leslie, C.A.; Romani, R.J. Salicylic Acid: A New Inhibitor of Ethylene Biosynthesis. Plant Cell Reports 1986, 5, 144–146. [Google Scholar] [CrossRef]
  49. Marimuthu, K.; Subbaraya, U.; Suthanthiram, B.; Marimuthu, S.S. Molecular Analysis of Somatic Embryogenesis through Proteomic Approach and Optimization of Protocol in Recalcitrant Musa Spp. Physiologia plantarum 2019, 167, 282–301. [Google Scholar] [CrossRef]
  50. García-Mendiguren, O.; Montalbán, I.A.; Goicoa, T.; Ugarte, M.D.; Moncaleán, P. Environmental Conditions at the Initial Stages of Pinus Radiata Somatic Embryogenesis Affect the Production of Somatic Embryos. Trees 2016, 30, 949–958. [Google Scholar] [CrossRef]
  51. Firoozabady, E.; DeBoer, D.L. Plant Regeneration via Somatic Embryogenesis in Many Cultivars of Cotton (Gossypium Hirsutum L.). In Vitro Cell Dev Biol - Plant 1993, 29, 166–173. [Google Scholar] [CrossRef]
  52. Kim, Y.W.; Moon, H.K. Enhancement of Somatic Embryogenesis and Plant Regeneration in Japanese Red Pine (Pinus Densiflora). Plant Biotechnol Rep 2014, 8, 259–266. [Google Scholar] [CrossRef]
  53. Mengxi, L.; Zhigang, X.; Yang, Y.; Yijie, F. Effects of Different Spectral Lights on Oncidium PLBs Induction, Proliferation, and Plant Regeneration. Plant Cell Tiss Organ Cult 2011, 106, 1–10. [Google Scholar] [CrossRef]
  54. Rodríguez-Sahagún, A.; Acevedo-Hernández, G.; Rodríguez-Domínguez, J.M.; Rodríguez-Garay, B.; Cervantes-Martínez, J.; Castellanos-Hernández, O.A. Effect of Light Quality and Culture Medium on Somatic Embryogenesis of Agave Tequilana Weber Var. Azul. Plant Cell Tiss Organ Cult 2011, 104, 271–275. [Google Scholar] [CrossRef]
  55. Torné, J.M.; Moysset, L.; Santos, M.; Simón, E. Effects of Light Quality on Somatic Embryogenesis in Araujia Sericifera. Physiologia Plantarum 2001, 111, 405–411. [Google Scholar] [CrossRef] [PubMed]
  56. Gulzar, B.; Mujib, A.; Malik, M.Q.; Sayeed, R.; Mamgain, J.; Ejaz, B. Genes, Proteins and Other Networks Regulating Somatic Embryogenesis in Plants. J Genet Eng Biotechnol 2020, 18, 31. [Google Scholar] [CrossRef]
  57. Méndez-Hernández, H.A.; Ledezma-Rodríguez, M.; Avilez-Montalvo, R.N.; Juárez-Gómez, Y.L.; Skeete, A.; Avilez-Montalvo, J.; De-la-Peña, C.; Loyola-Vargas, V.M. Signaling Overview of Plant Somatic Embryogenesis. Frontiers in Plant Science 2019, 10. [Google Scholar] [CrossRef]
  58. Jha, P.; Ochatt, S.J.; Kumar, V. WUSCHEL: A Master Regulator in Plant Growth Signaling. Plant Cell Rep 2020, 39, 431–444. [Google Scholar] [CrossRef]
  59. Zuo, J.; Niu, Q.-W.; Frugis, G.; Chua, N.-H. The WUSCHEL Gene Promotes Vegetative-to-Embryonic Transition in Arabidopsis. The Plant Journal 2002, 30, 349–359. [Google Scholar] [CrossRef]
  60. Kadri, A.; Grenier De March, G.; Guerineau, F.; Cosson, V.; Ratet, P. WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago Truncatula Gaertn. Plants 2021, 10, 715. [Google Scholar] [CrossRef]
  61. Zhang, A.; Li, Y.; Yarra, R.; Li, R.; Cao, H.; Jin, L. Genome-Wide Identification of WUSCHEL-Related Homeobox Gene Family and Their Expression Analysis During Somatic Embryogenesis in Oil Palm (Elaeis Guineensis). Tropical Plant Biol. 2022, 15, 55–64. [Google Scholar] [CrossRef]
  62. Lou, H.; Huang, Y.; Wang, W.; Cai, Z.; Cai, H.; Liu, Z.; Sun, L.; Xu, Q. Overexpression of the AtWUSCHEL Gene Promotes Somatic Embryogenesis and Lateral Branch Formation in Birch (Betula Platyphylla Suk.). Plant Cell Tiss Organ Cult 2022, 150, 371–383. [Google Scholar] [CrossRef]
  63. Xiao, Y.; Chen, Y.; Ding, Y.; Wu, J.; Wang, P.; Yu, Y.; Wei, X.; Wang, Y.; Zhang, C.; Li, F.; et al. Effects of GhWUS from Upland Cotton (Gossypium Hirsutum L.) on Somatic Embryogenesis and Shoot Regeneration. Plant Science 2018, 270, 157–165. [Google Scholar] [CrossRef]
  64. El Ouakfaoui, S.; Schnell, J.; Abdeen, A.; Colville, A.; Labbé, H.; Han, S.; Baum, B.; Laberge, S.; Miki, B. Control of Somatic Embryogenesis and Embryo Development by AP2 Transcription Factors. Plant Mol Biol 2010, 74, 313–326. [Google Scholar] [CrossRef]
  65. Passarinho, P.; Ketelaar, T.; Xing, M.; van Arkel, J.; Maliepaard, C.; Hendriks, M.W.; Joosen, R.; Lammers, M.; Herdies, L.; den Boer, B.; et al. BABY BOOM Target Genes Provide Diverse Entry Points into Cell Proliferation and Cell Growth Pathways. Plant Mol Biol 2008, 68, 225–237. [Google Scholar] [CrossRef] [PubMed]
  66. Horstman, A.; Li, M.; Heidmann, I.; Weemen, M.; Chen, B.; Muino, J.M.; Angenent, G.C.; Boutilier, K. The BABY BOOM Transcription Factor Activates the LEC1-ABI3-FUS3-LEC2 Network to Induce Somatic Embryogenesis. Plant Physiology 2017, 175, 848–857. [Google Scholar] [CrossRef] [PubMed]
  67. Boutilier, K.; Offringa, R.; Sharma, V.K.; Kieft, H.; Ouellet, T.; Zhang, L.; Hattori, J.; Liu, C.-M.; van Lammeren, A.A.M.; Miki, B.L.A.; et al. Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to Embryonic Growth. The Plant Cell 2002, 14, 1737–1749. [Google Scholar] [CrossRef]
  68. Irikova, T.; Grozeva, S.; Denev, I. Identification of BABY BOOM and LEAFY COTYLEDON Genes in Sweet Pepper (Capsicum Annuum L.) Genome by Their Partial Gene Sequences. Plant Growth Regul 2012, 67, 191–198. [Google Scholar] [CrossRef]
  69. Salvo, S.A.G.D.; Hirsch, C.N.; Buell, C.R.; Kaeppler, S.M.; Kaeppler, H.F. Whole Transcriptome Profiling of Maize during Early Somatic Embryogenesis Reveals Altered Expression of Stress Factors and Embryogenesis-Related Genes. PLOS ONE 2014, 9, e111407. [Google Scholar] [CrossRef]
  70. Hecht, V.; Vielle-Calzada, J.-P.; Hartog, M.V.; Schmidt, E.D.L.; Boutilier, K.; Grossniklaus, U.; de Vries, S.C. The Arabidopsis Somatic Embryogenesis Receptor Kinase 1 Gene Is Expressed in Developing Ovules and Embryos and Enhances Embryogenic Competence in Culture. Plant Physiology 2001, 127, 803–816. [Google Scholar] [CrossRef]
  71. Somleva, M.N.; Schmidt, E.D.L.; de Vries, S.C. Embryogenic Cells in Dactylis Glomerata L. (Poaceae) Explants Identified by Cell Tracking and by SERK Expression. Plant Cell Reports 2000, 19, 718–726. [Google Scholar] [CrossRef] [PubMed]
  72. Schmidt, E.D.; Guzzo, F.; Toonen, M.A.; de Vries, S.C. A Leucine-Rich Repeat Containing Receptor-like Kinase Marks Somatic Plant Cells Competent to Form Embryos. Development 1997, 124, 2049–2062. [Google Scholar] [CrossRef]
  73. Huang, X.; Lu, X.-Y.; Zhao, J.-T.; Chen, J.-K.; Dai, X.-M.; Xiao, W.; Chen, Y.-P.; Chen, Y.-F.; Huang, X.-L. MaSERK1 Gene Expression Associated with Somatic Embryogenic Competence and Disease Resistance Response in Banana (Musa Spp.). Plant Mol Biol Rep 2010, 28, 309–316. [Google Scholar] [CrossRef]
  74. Stone, S.L.; Kwong, L.W.; Yee, K.M.; Pelletier, J.; Lepiniec, L.; Fischer, R.L.; Goldberg, R.B.; Harada, J.J. LEAFY COTYLEDON2 Encodes a B3 Domain Transcription Factor That Induces Embryo Development. Proceedings of the National Academy of Sciences 2001, 98, 11806–11811. [Google Scholar] [CrossRef]
  75. Zhang, Y.; Clemens, A.; Maximova, S.N.; Guiltinan, M.J. The Theobroma Cacao B3 Domain Transcription Factor TcLEC2plays a Duel Role in Control of Embryo Development and Maturation. BMC Plant Biology 2014, 14, 106. [Google Scholar] [CrossRef] [PubMed]
  76. Kim, H.U.; Jung, S.-J.; Lee, K.-R.; Kim, E.H.; Lee, S.-M.; Roh, K.H.; Kim, J.-B. Ectopic Overexpression of Castor Bean LEAFY COTYLEDON2 (LEC2) in Arabidopsis Triggers the Expression of Genes That Encode Regulators of Seed Maturation and Oil Body Proteins in Vegetative Tissues. FEBS Open Bio 2014, 4, 25–32. [Google Scholar] [CrossRef]
  77. Guo, F.; Liu, C.; Xia, H.; Bi, Y.; Zhao, C.; Zhao, S.; Hou, L.; Li, F.; Wang, X. Induced Expression of AtLEC1 and AtLEC2 Differentially Promotes Somatic Embryogenesis in Transgenic Tobacco Plants. PLOS ONE 2013, 8, e71714. [Google Scholar] [CrossRef]
  78. Ghasemi, Y.; Mashayekhi, K.; Ghasemnezhad, A.; MOVAHEDI, S. The Effects of Calcium and Magnesium on Carrot (Dacus Carrota Cv. Nants) Petiole Somatic Embryogenesis. 2009.
  79. Kiselev, K.V.; Turlenko, A.V.; Zhuravlev, Y.N. CDPK Gene Expression in Somatic Embryos of Panax Ginseng Expressing RolC. Plant Cell, Tissue and Organ Culture (PCTOC) 2009, 99, 141–149. [Google Scholar] [CrossRef]
  80. Enríquez-Valencia, A.J.; Vázquez-Flota, F.A.; Ku-Cauich, J.R.; Escobedo-GraciaMedrano, R.M. Differentially Expressed Genes during the Transition from Early to Late Development Phases in Somatic Embryo of Banana (Musa Spp. AAB Group, Silk Subgroup) Cv. Manzano. Plant Cell Tiss Organ Cult 2019, 136, 289–302. [Google Scholar] [CrossRef]
  81. Xie, Z.; Saha, N.; Chlan, C. Antimicrobial Activity of a Cys-Rich Peptide Derived from a Centrosema Virginianum Vicilin. American Journal of Plant Sciences 2016, 7, 92–107. [Google Scholar] [CrossRef]
  82. dos Santos, A.L.W.; Elbl, P.; Navarro, B.V.; de Oliveira, L.F.; Salvato, F.; Balbuena, T.S.; Floh, E.I.S. Quantitative Proteomic Analysis of Araucaria Angustifolia (Bertol.) Kuntze Cell Lines with Contrasting Embryogenic Potential. Journal of Proteomics 2016, 130, 180–189. [Google Scholar] [CrossRef] [PubMed]
  83. Nic-Can, G.I.; De la Peña, C. Epigenetic Advances on Somatic Embryogenesis of Agronomical and Important Crops. In Epigenetics in Plants of Agronomic Importance: Fundamentals and Applications: Transcriptional Regulation and Chromatin Remodelling in Plants; Alvarez-Venegas, R., De la Peña, C., Casas-Mollano, J.A., Eds.; Springer International Publishing: Cham, 2014; ISBN 978-3-319-07971-4. [Google Scholar]
  84. Kumar, V.; Van Staden, J. New Insights into Plant Somatic Embryogenesis: An Epigenetic View. Acta Physiol Plant 2017, 39, 194. [Google Scholar] [CrossRef]
  85. Méndez-Hernández, H.A.; Ledezma-Rodríguez, M.; Avilez-Montalvo, R.N.; Juárez-Gómez, Y.L.; Skeete, A.; Avilez-Montalvo, J.; De-la-Peña, C.; Loyola-Vargas, V.M. Signaling Overview of Plant Somatic Embryogenesis. Front Plant Sci 2019, 10, 77. [Google Scholar] [CrossRef] [PubMed]
  86. Bravo, S.; Bertín, A.; Turner, A.; Sepúlveda, F.; Jopia, P.; Parra, M.J.; Castillo, R.; Hasbún, R. Differences in DNA Methylation, DNA Structure and Embryogenesis-Related Gene Expression between Embryogenic and Non Embryogenic Lines of Pinus Radiata D. Don. Plant Cell Tiss Organ Cult 2017, 130, 521–529. [Google Scholar] [CrossRef]
  87. Xu, M.; Li, X.; Korban, S.S. DNA-Methylation Alterations and Exchanges during in Vitro Cellular Differentiation in Rose (Rosa Hybrida L.). Theor Appl Genet 2004, 109, 899–910. [Google Scholar] [CrossRef]
  88. Noceda, C.; Salaj, T.; Pérez, M.; Viejo, M.; Cañal, M.J.; Salaj, J.; Rodriguez, R. DNA Demethylation and Decrease on Free Polyamines Is Associated with the Embryogenic Capacity of Pinus Nigra Arn. Cell Culture. Trees 2009, 23, 1285. [Google Scholar] [CrossRef]
  89. Grzybkowska, D.; Morończyk, J.; Wójcikowska, B.; Gaj, M.D. Azacitidine (5-AzaC)-Treatment and Mutations in DNA Methylase Genes Affect Embryogenic Response and Expression of the Genes That Are Involved in Somatic Embryogenesis in Arabidopsis. Plant Growth Regul 2018, 85, 243–256. [Google Scholar] [CrossRef]
  90. Fraga, H.P.F.; Vieira, L.N.; Heringer, A.S.; Puttkammer, C.C.; Silveira, V.; Guerra, M.P. DNA Methylation and Proteome Profiles of Araucaria Angustifolia (Bertol.) Kuntze Embryogenic Cultures as Affected by Plant Growth Regulators Supplementation. Plant Cell Tiss Organ Cult 2016, 125, 353–374. [Google Scholar] [CrossRef]
  91. Yamamoto, N.; Kobayashi, H.; Togashi, T.; Mori, Y.; Kikuchi, K.; Kuriyama, K.; Tokuji, Y. Formation of Embryogenic Cell Clumps from Carrot Epidermal Cells Is Suppressed by 5-Azacytidine, a DNA Methylation Inhibitor. Journal of Plant Physiology 2005, 162, 47–54. [Google Scholar] [CrossRef]
  92. Santos, D.; Fevereiro, P. Loss of DNA Methylation Affects Somatic Embryogenesis in Medicago Truncatula. Plant Cell, Tissue and Organ Culture 2002, 70, 155–161. [Google Scholar] [CrossRef]
  93. De-la-Peña, C.; Nic-Can, G.; Galaz-Ávalos, R.; Avilez-Montalvo, R.; Loyola-Vargas, V. The Role of Chromatin Modifications in Somatic Embryogenesis in Plants. Frontiers in Plant Science 2015, 6. [Google Scholar] [CrossRef] [PubMed]
  94. Lee, K.; Park, O.-S.; Jung, S.-J.; Seo, P.J. Histone Deacetylation-Mediated Cellular Dedifferentiation in Arabidopsis. Journal of Plant Physiology 2016, 191, 95–100. [Google Scholar] [CrossRef] [PubMed]
  95. Mozgová, I.; Muñoz-Viana, R.; Hennig, L. PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of Arabidopsis Thaliana. PLOS Genetics 2017, 13, e1006562. [Google Scholar] [CrossRef] [PubMed]
Preprints 74971 g001
Figure 1. Stages of somatic embryogenesis and regeneration in banana. (a) Multiple meristems (scalps), (b) male bud, (c) Friable embryogenic ideal callus, (d) Embryogenic cell suspension, (e) Embryo development, (f) Embryo maturation, (g) White embryo germination in the dark, (h) White shoots turned into green in the light, (i) Fully developed green shoots with roots, (j) well-developed plants in the screen house.
Figure 1. Stages of somatic embryogenesis and regeneration in banana. (a) Multiple meristems (scalps), (b) male bud, (c) Friable embryogenic ideal callus, (d) Embryogenic cell suspension, (e) Embryo development, (f) Embryo maturation, (g) White embryo germination in the dark, (h) White shoots turned into green in the light, (i) Fully developed green shoots with roots, (j) well-developed plants in the screen house.
Preprints 74971 g001
Table 1. Various media used for banana somatic embryogenesis and regeneration.
Table 1. Various media used for banana somatic embryogenesis and regeneration.
Medium Components Reference
Ma1 Full-strength MS salts, MS vitamins, 3% sucrose, 1 mg/l biotin, 4 mg/l 2,4-D,1 mg/l NAA, 1 mg/l IAA, 3 g/l gelrite, pH 5.8 [16,27]
Ma2 Full-strength MS salts plus vitamins, 3% sucrose, 1 mg/l biotin, 1 mg/l 2,4-D, 100 mg/l glutamine, 100 mg/l malt extract, pH 5.8 [16,23]
Ma3 SH basal salts, MS vitamins, 60 mg/l ascorbic acid, 100 mg/l glutamine, 250 mg/l proline, 100 mg/l malt extract, 400 mg/l cysteine, 60 mg/l citric acid, 0.2 mg/l NAA, 0.2 mg/l 2-ip, 0.2 mg/l kinetin, 4.5% sucrose, 3 g/l gelrite, pH 5.8 [16,23]
Ma4 Full-strength MS salts, 3% sucrose, 2 mg/l IAA, 0.5 mg/l BAP, Morel vitamins, pH 5.8 [16,23]
RD1 ½ strength MS macro salts, MS micro salts, 3% sucrose, 10 mg/l Ascorbic acid [16,23]
RD2 ½ strength MS macros salts, MS micro salts, 3% sucrose, 10 mg/l Ascorbic acid, 0.25 mg/l BAP, pH 5.8 [23]
ZZ ½ strength MS macro salts, MS micro salts, 3% sucrose, 10 mg/l Ascorbic acid, 1mg/l 2,4-D, 0.2 mg/l zeatin, 3g/l gelrite, pH 5.8 [23,27]
PM Full-strength MS salts plus vitamins, 10 mg/l ascorbic acid, 3% sucrose, 2.5 mg/l BAP, 2.4 g/l gelrite, pH 5.8 [27]
RM Full-strength MS salts plus vitamins, 10 mg/l ascorbic acid, 3% sucrose, 1 mg/l IBA, 2.4 g/l gelrite, pH 5.8 [27]
MS; Murashige and Skoog [34], SH; Schenk & Hildebrandt Medium [35].
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
Prerpints.org logo

Preprints.org is a free preprint server supported by MDPI in Basel, Switzerland.

facebook logotwitter logolinkedin logo
MDPI Initiatives
Important Links
Subscribe

Disclaimer

Terms of Use

Privacy Policy

© 2024 MDPI (Basel, Switzerland) unless otherwise stated