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Review

Morphogenetic Factors as a Tool for Enhancing Plant Regeneration Capacity During In Vitro Transformation

by
Semyon D. Bakulin
1,
Sokrat G. Monakhos
1 and
Sergey A. Bruskin
2,*
1
Department of Molecular Breeding, Cell and Seed Technology, Russian State Agrarian University—Moscow Timiryazev Agricultural Academy, 127550 Moscow, Russia
2
Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(17), 8583; https://doi.org/10.3390/ijms26178583
Submission received: 22 July 2025 / Revised: 28 August 2025 / Accepted: 1 September 2025 / Published: 3 September 2025
(This article belongs to the Section Molecular Plant Sciences)
Browse Figure
Versions Notes

Abstract

Morphogenetic factors (MTFs) are specialized plant genes and transcription factors that play pivotal roles in embryogenesis and organogenesis. This review focuses on their functions in plant development regulation and their applications in plant biotechnology and modern breeding. Common challenges in transformation and regeneration were discussed, along with successful case studies demonstrating improved regeneration capacity and transgene stability in rice (Oryza sativa), soybean (Glycine max), rapeseed (Brassica napus), tomato (Solanum lycopersicum) and other less common crops and plant model organisms. These improvements were achieved through the utilization of key developmental MTFs such as WUCHEL, BABY BOOM, GRF-GIF, etc. The principles of designing genetic constructs with MTFs are explored, including promoter selection and regulatory elements, as well as their synergistic effects with phytohormones like auxins and cytokinins for optimizing in vitro morphogenesis. Current limitations in MTF expression and strategies to overcome them are analyzed. The article highlights recent advances, including MTFs potential for developing stress-resistant, high-yielding cultivars. Key discussion points include the discovery of novel morphogens, their application to recalcitrant species, and prospects for expanding the range of easily transformable and regenerable crops. Future directions involve developing universal transformation protocols and integrating morphogens with precision genome editing technologies, offering new opportunities for agriculture and global food security.

Graphical Abstract

1. Introduction

Plants genetic transformation represents a broad group of methodologies widely used today in both fundamental research and practical breeding applications. The integration of tools such as CRISPR/Cas with plant transformation methodologies allows for the accurate and targeted modification of plant genomes, including those of economically valuable crop varieties. This approach allows targeted modification of specific traits or trait groups while minimizing potential negative effects on other valuable agricultural characteristics [1].
Successful transformation examples date back to the 1980s, with the first transgenic plants of soybean (Glycine max L.) [2], cotton (Gossypium hirsutum L.) [3], rice (Oryza sativa L.) [4], tomato (Solanum lycopersicum L.) [5], and rapeseed (Brassica napus L.) [6]. Transgenic varieties of other key crops followed in the 1990s: maize (Zea mays L.) [7], wheat (Triticum aestivum L.) [8], sorghum (Sorghum bicolor (L.) Moench) [9], and sugarcane (Saccharum officinarum L.) [10].
Parallel developments occurred in plant tissue culture techniques in vitro culture techniques, particularly in optimizing regeneration of transformed tissues. Fundamental work by F. Skoog and C. Miller [11] established the crucial role of phytohormones in regulating plant tissue growth and development. Subsequent studies by various researchers explored transformation protocols and phytohormonal regulation in different plant species [12,13,14]. Despite progress, challenges persist in achieving efficient transformation and regeneration for most agricultural cultivars [15,16,17,18]. The complex and multifactorial process of transformation shows a strong genotype dependence, with many protocols failing to work when applied to new varieties, despite working reliably with long-established model cultivars [19,20,21].
Beyond classical phytohormonal approaches, emerging strategies employ transcription factors that activate morphogenesis pathways (morphogenesis regulators/morphogens) [13].

2. Morphogenetic Factors: Definition and Classification

MTFs typically refer to plant genes whose ectopic expression can initiate morphogenesis. These genes most often encode transcription factors—the “master switches” of development. To date, a broad spectrum of such genes with diverse functions and origins has been discovered [22]. The principal classes of MTFs include the following groups.

2.1. WOX Factors (WUSCHEL-Related Homeobox)

A family of homeodomain transcription factors responsible for maintaining meristem activity. The key representative is the WUSCHEL (WUS) gene, first identified in Arabidopsis thaliana (L.) Heynh. [23]. It is essential for shoot apical meristem function: WUS stimulates meristem cell proliferation while preventing their differentiation, thereby sustaining meristem activity. Mutations in WUS lead to meristem depletion and shoot growth arrest [24]. Studies show that WUS overexpression can induce embryoid formation directly on vegetative organs in adult plants [25]. For example, introducing WUS into Arabidopsis leaves triggered somatic embryogenesis, enabling plant regeneration without phytohormonal regulators [26,27]. Today, WUS from Arabidopsis and rapeseed is increasingly used to enhance regeneration capacity across recalcitrant species like coffee (Coffea canephora Pierre ex A.Froehner) [26], orchids (Phalaenopsis Blume) [27], banana (Musa acuminata Colla) [28], cotton [29], maize [30] sorghum [31].
WOX genes regulate meristem cell differentiation, root meristem growth, and somatic embryogenesis, improving regeneration efficiency [32,33]. For instance: WOX13 is critical for callus cell differentiation in A. thaliana [34]; WOX4 participates in wood formation, while WOX11 and WOX12 mediate salt and water stress responses [35]; WOX11/12 determine root meristem cell fate in A. thaliana [36]; WOX5 promotes root initiation in other plants [37,38].

2.2. BBM (BABY BOOM)

This APETALA2-like (AP2-like) transcription factor was discovered during studies of somatic embryogenesis in rapeseed [39]. The constitutive expression of rapeseed BnBBM in Arabidopsis induced massive somatic embryoid formation on vegetative tissues, such as leaves and shoot apices, etc. These embryoids developed into intact plants, even without the use of phytohormones [39]. This demonstrated that a single gene could activate the full embryonic program in somatic cells. Subsequent studies has confirmed the universality of BBM: similar effects were observed in tobacco (Nicotiana tabacum L.) [40], soybean [41], cacao (Theobroma cacao L.) [42]. Functionally, BBM and related AIL (AINTEGUMENTA-like) factors maintain embryo and young organ meristem activity by stimulating cell division and initiating embryogenesis.

2.3. PLT Factors (PLETHORA)

PLT5 in Arabidopsis contributes to root meristem formation. Overexpression enhances callus and shoot regeneration from stem wounds [43]. Like BBM, PLT actors induce embryogenesis but primarily influence root pole development, making them promising regeneration enhancers.

2.4. GRF-GIF Gene Family

This paired module consists of a growth-stimulating transcription factor (GRF) and a stabilizing cofactor—GRF-interacting factor (GIF). The co-expression of GRF-GIF fusion proteins significantly enhances regeneration. For example, GRF4-GIF1 significantly increased wheat regeneration efficiency by 8-fold, allowing for genotype-independent transformation [44]. In soybean, the expression of GmGRF-GIF allowed the transformation of previously resistant cultivars [45]. Heterologous AtGRF5 expression enhanced the recovery of transgenic melons (Cucumis melo L.) [46]. Unlike WUS/BBM, which induces direct embryogenesis, this strategy promotes the general growth of meristems to facilitate regeneration.

2.5. Other Morphogenetic Factors

LEC1/LEC2 (LEAFY COTYLEDON) are embryo maturation factors that rejuvenate cells and promote somatic embryogenesis. The effect of this MTF has been observed in tobacco, during the process of dedifferentiation from immature pollen into embryogenic cells, as well as in Arabidopsis, during somatic embryogenesis [47,48] and embryo development [49]. SERK1 (Somatic Embryogenesis Receptor Kinase 1) is a receptor gene marking embryogenic competence; its overexpression leads to an increase in the yield of somatic embryos [25]. ESR1 (Enhancer of Shoot Regeneration 1) is a factor that enhances Arabidopsis shoot formation in vitro [50,51] sometimes in synergy with WUS [52]. WIND1 (WOUND INDUCED DEDIFFERENTIATION1) is a wound-induced factor initiating dedifferentiation via cascaded morphogen expression in Arabidopsis [53,54,55] and tobacco [55]. RKD (RWP-RK domain) is a gene family that is involved in nitrogen response [56] and gametophyte development [57], as well as the development of symbiotic root nodules in the Fabaceae family [58] and embryogenesis in monocot plants [59]. DOF (DNA-binding One Zinc Finger) are root/shoot meristem regulators (e.g., TaDOF3.4, TaDOF5.6 in wheat) [60].
Some Agrobacterium (syn. Rhizobium) oncogenes exhibit morphogenesis effects: ipt (isopentenyltransferase) (from Ti-plasmids) encodes cytokinin synthase, inducing shoot branching [55,61,62]; rolB/rolC (root loci from A. rhizogenes) alter hormonal balance to stimulate root or shoot meristem activity [13].
REF1 (REGENERATION FACTOR1) Peptides. These wound-responsive peptides bind PORK1 (PEPR1/2 Ortholog Receptor-Like Kinase1) receptors to activate WIND1 and regeneration, as demonstrated in tomato, wheat, maize, and soybean [63].
MTFs often cause developmental abnormalities if constitutively expressed [13]. Thus, their activity must be tightly controlled [24]. Key MTFs used in plant biotechnology are summarized in Table 1.

3. Case Studies of Morphogenetic Factors’ Utilization in Crop Transformation and In Vitro Regeneration

The use of MTFs is becoming more and more common in transformation and regeneration protocols for various plant species. However, most of these experiments remain basic or even theoretical [15]. These factors are especially valuable for difficult-to-transform crops with low efficiency at the species or variety level, as well as for important commercial plants. Extensive studies on the use of morphogens have been conducted with rice, rapeseed, soybeans, and tomatoes. The detailed applications of various MTFs are presented in Table 2.

3.1. Rice

Among Poaceae, rice was the first crop successfully transformed [100,101]. Most high-efficiency transformation reports (up to 90%) involve O. sativa subsp. Japonica cultivars [20,102], typically using scutellum-derived callus [103]. By contrast, Indica subspecies (e.g., O. sativa subsp. indica) exhibit poor regeneration due to low morphogenesis-related gene activity [20], hypersensitivity to phytohormone ratios, and phenolic compound accumulation [104].
Co-overexpression of maize WUS and rice BBM enhances somatic embryogenesis and meristematic activity, dramatically improving regeneration rates. Indica rice, OsBBM + ZmWUS2 yielded transgenic shoots in 43% of cases (vs. ~3% in controls), while ZmBBM + ZmWUS2 achieved 27% efficiency [15]. The regulatory module uORF-HsfA1a-WOX11 fine-tunes WOX11 expression, enabling root system modifications without affecting shoot traits [72].
The GRF4-GIF1 module significantly doubles the regeneration efficiency in the ‘Kitaake’ cultivar, producing fertile plants without any developmental abnormalities, unlike constitutive WUS/BBM expression [44]. AtGRF5 orthologs also boost transformation rates in monocots, including rice [20].
For recalcitrant black rice (O. sativa ‘Cempo Ireng’), ectopic expression of OsRKD3 induced somatic embryogenesis by activating a gene network (AP2/ERF, MYB, COL) and hormonal regulators, restoring near-zygotic totipotency [59].

3.2. Rapeseed

Rapeseed was first transformed by Moloney [6]. Today rapeseed cultivars like ‘Westar’ [65,105] and ‘ZS11’ [106,107] achieve 50–90% transformation efficiency. However, winter cultivars requiring vernalization (like ‘Express617’) remain highly recalcitrant [65].
WUS and BBM are widely used to improve plant regeneration. While standard methods result in less than 1% transgenic shoot formation in ‘Westar’, overexpression of BnBBM stimulates the development of somatic embryos in the absence of exogenous phytohormones [39]. Sugar beet WUS enabled transgenic shoot regeneration in winter rapeseed, bypassing callus formation. However, prolonged WUS expression may cause abnormalities [15]. AtGRF5 increased shoot regeneration to 19.6% (vs. 3% in controls) without developmental defects [20].
No publications are known regarding the use of other MTFs for rapeseed transformation. For other Brassicaceae species like Arabidopsis combining factors (e.g., WUS + STM) is promising: their co-expression generated meristems and floral structures in vitro [67]. WIND1, RKD, PLT, and ESR1 are also candidates for rapeseed [51,65,77].

3.3. Soybean

A notoriously difficult-to-transform crop, soybean was first modified via biolistics [108], with Agrobacterium-based methods lagging due to complexity [109]. Challenges include low efficiency, genotype dependence, and laborious protocols via biolistics [108] and cotyledonary nodes from mature seeds [110]. Reliable methods exist only for lab cultivars (e.g., ‘Williams 82′), while elite varieties remain recalcitrant: ‘Heinong44′ [111], ‘Shennong 9′, ‘Bert’ [112], ‘BR-16′, ‘BR-19′ and others [113].
GmGRF3-GIF1 increased regeneration up to 5.5–13.8% (vs. 2–5% in controls) in previously untransformable cultivars, reaching 16.7% with GRF3 mutations [45]. AtGRF5 achieved 50% shoot regeneration in ‘Jack’ and ‘CD215′ [20]. BBM + WUS triggered somatic embryogenesis, and BnBBM enhanced transformation [43]. WIND1, LEC2, and ipt are also promising, as demonstrated in tobacco [53,68,77], tomato [77], poplar [114].

3.4. Tomato

Easier to transform than soybean or rice, tomato protocols date to 1986 [115,116]. Yet genotype-specific issues persist: low WUS/CLV (CLAVATA) expression [117], phytohormone sensitivity [118], Agrobacterium resistance [119], somaclonal variation [120], and selective agent toxicity [121].
PLT5 and WUS enabled in planta transformation with 13% regeneration (vs. 0% in controls). It has also been observed in snapdragons (Antirrhinum majus L.) through Agrobacterium injection. Enhanced transformation and shoot regeneration of turnip (Brassica rapa L.), as well as the formation of transgenic calli and somatic embryos in sweet pepper (Capsicum annuum L.) have been observed [43]. The peptide REF1 also induced callus-to-shoot morphogenesis [63].
Several examples are known of using other MTFs for transformation in species of the Solanaceae family. Tobacco (Nicotiana) experiences enhanced regenerative capabilities through the action of WIND1 [53], LEC2, and ipt [77], while pepper (Capsicum) species rely on BBM for regeneration [25,73]. The use of these morphogens in tomato may also enhance its in vitro regenerative capacity.

3.5. Other Crops

There is known data on the use of MTFs to induce regeneration processes after transformation for other common or more exotic crops or model plant species.
Some of the most challenging crops are fruit trees, such as Citrus L. species. For certain Citrus species, MTFs have been used to overcome regeneration bottlenecks. For instance, the gene L1L (a homolog of LEC1), when overexpressed in sweet orange (Citrus × sinensis (L.) Osbeck) and tangerine (Citrus reticulata Blanco), induced embryoid formation as early as one month after Agrobacterium-mediated transformation [83]. Interestingly, in Citrus, some MTFs can also enhance transformation efficiency itself. For example, in lemon (Citrus limon (L.) Osbeck) and sweet orange, overexpression of ZmKN1 under the 35S promoter increased Agrobacterium-mediated transformation efficiency by 3–15 times [84]. In the study by Debernardi et al. [44], the use of GRF-GIF was demonstrated to enhance regeneration in citron (Citrus medica L.) by 4.7-fold. It is known that Citrus species are considered quite challenging subjects, both for transformation and general in vitro cultivation, such as in micropropagation [122,123,124].
Other fruit tree crops are also challenging for gene editing. However, even for these species, there are some—albeit limited—successful examples of improving regeneration capacity through morphogens.
For example, in the case of the apple plant (Malus domestica L.), which is known to be a difficult system for in vitro transformation, preliminary transformation of leaf explants with the MdBBM1 gene resulted in an increased frequency of transgenic shoot regeneration of approximately 31%, which is significantly higher than the standard range of 5–10% for this species. The study by Chen et al. [85] demonstrated that BBM expression in apples accelerates regeneration initiation and increases the number of regenerant plants without compromising their development. The BBM cassette was subsequently removed, yielding transgenic plantlets free of morphogenic genes. This was the first successful application of a BBM-type gene in a fruit tree.
Grapevine (Vitis vinifera L.) is another highly relevant crop for gene editing. In one study, the regeneration efficiency of the ‘Cabernet Sauvignon’ cultivar from embryogenic culture was increased to 42.85% using VvBBM [86]. Additionally, the use of GRF-GIF in grapevine improved shoot regeneration efficiency by more than 4-fold [44].
Among other berry crops, the application of morphogens has so far only been reported in strawberry (Fragaria vesca L.). For example, the grape-derived VvGRF4-GRF1 enhanced strawberry transformation efficiency by more than 2-fold, reaching 40% [87]. For commercially important berry crops such as raspberry (Rubus subg. Idaeobatus (Focke) Focke), blackberry (Rubus subg. Rubus L.), bilberry (Vaccinium myrtillus L.), and blueberry (Vaccinium corymbosum L.), there are no known studies yet on the use of MTFs. However, this approach could hold significant promise for genomic selection in these species.
Among woody plants, the successful application of MTFs in cacao (Theobroma cacao L.) is particularly noteworthy. In this species, the endogenous gene TcBBM was used to enhance embryogenesis, eliminating the need for exogenous phytohormones in the culture media [42].
Palms (Arecaceae Bercht. & J.Presl.) present significant challenges for gene editing. Genetic engineering studies on date palm (Phoenix dactylifera L.) are rare and involve complex, time-consuming techniques, such as biolistics and protoplast culture [125,126]. For example, only one gene, PdSERK1, has been identified [88] as playing an active role in the embryogenesis of this plant. Indirect studies have also been conducted on the coconut palm (Cocos nucifera L.) using genes such as WUS, BBM, and SERK to examine the effects of 5-Azacytidine on explants, as well as its influence on gene expression and somatic embryogenesis [89].
For another economically important monocot, banana (Musa L.), direct data on the use of WUS/BBM are not yet available. However, research efforts are focused on developing somatic embryogenesis systems where these genes could potentially be applied. While direct introduction of BBM or WUS into banana has not been reported, progress is being made through optimization of somatic embryogenesis conditions. It is hypothesized that, similar to rice and maize, BBM/WUS expression could induce embryogenic callus formation from banana meristems, accelerating the production of transgenic lines [18,44].
Some tropical cereal crops have been influenced by MTFs during in vitro transformation. For instance, in the aforementioned study by Lowe et al. [15], transgenic sorghum and sugarcane plants were successfully obtained using maize-derived ZmBBM and ZmWUS2 in the transformation vectors. The yield of transgenic plants increased from 0–5% to 25–50%. These MTFs promoted somatic embryogenesis in explants. Interestingly, the use of morphogenetic approaches to induce in vitro regeneration is currently more common in monocots, which are generally more challenging to edit compared to dicots. For example, in two cultivars of switchgrass (Panicum virgatum L.), another challenging monocot species, a successful case of overexpressing maize-derived ZmBBM and ZmWUS2 has been reported. This approach increased the transformation efficiency of switchgrass to 6% and improved regeneration efficiency to over 40% [90].
For cassava (Manihot esculenta Crantz), a crucial tropical crop, researchers have successfully employed grape-derived VvGRF4-GIF1 and Arabidopsis AtGRF5. Their overexpression induced shoot regeneration, increasing efficiency to 50%, though complete elimination of exogenous phytohormones from culture media was not achieved [91]. In cucurbits, including cucumber (Cucumis sativus L.), regeneration frequency was enhanced through AtGRF5 overexpression [92]. Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) transformation using the ClGRF4-GIF1 chimera further demonstrated the broad applicability of this approach [93]. For sugar beet (Beta vulgaris L.), AtGRF5 overexpression in callus cells significantly improved both shoot regeneration and transformation rates [20].
Studies have also been conducted on the application of MTFs in the genetic modification of forest trees. Of particular significance are species that are prized for their timber, their ability to withstand abiotic stress, their resistance to disease, and their potential for reforestation. One such species is eucalyptus (Eucalyptus L’Hér.). The treatment of E. urophylla × E. grandis ‘DH32-29′ hybrid tissues with WUS proteins enhanced the success of transformation and regeneration processes [94].
Several examples of morphogen application have been demonstrated in Populus species and cultivars. In P. tomentosa, heterologous expression of the BrBBM gene from turnip led to the formation of somatic embryos from callus cultures. Calli from leaf explants were constitutively expressed with the i gene, and these calli then spontaneously formed embryoid structures and produced shoots [95,96]. To prevent morphological abnormalities, a gene excision system was employed, where the BBM cassette was removed from the genome through heat shock-induced site-specific recombination after embryogenesis initiation.
In a study by Pan et al. [96], the co-expression of PtWUS and PtWOX11 increased callus formation, shoot regeneration, and root formation in poplar. Similar results were achieved earlier through the expression of WOX11/WOX12a in poplar [97]. In a study by Pan et al. (96), the co-expression of PtWUS and PtWOX11 increased callus formation, shoot regeneration, and root formation in poplar. Similar results were achieved earlier through the expression of WOX11/WOX12a in poplar (97).
In gymnosperm experiments, the LEC1 gene family has been studied most extensively. For Norway spruce (Picea abies (L.) H. Karst.), the overexpression of PaHAP3A, an angiosperm LEC1 homolog, during zygotic embryo development led to the formation of ectopic somatic embryos. This result is consistent with data from angiosperms, where some cells (e.g., embryonic or meristematic cells) are more prone to embryogenic induction. Additionally, studies have shown that the overexpression of genes that maintain meristems can enhance the natural embryogenic capacity of conifers [98]. However, inducing organogenesis or shoot formation de novo in mature conifer tissue using foreign WUS/BBM has proven challenging, with no such studies published [127]. Data on the classical WUS and BBM applications in conifers is lacking due to the high in vitro complexity of these species.
For other valuable species such as oak (Quercus L.) and birch (Betula L.), as well as other forest trees, no specific publications on the use of MTFs have been identified. These species are also known to be difficult to regenerate in vitro, and research is ongoing to identify endogenous factors that may enhance their morphogenic potential [128].
MTFs could also be applied to ornamental plants. Many ornamental species are resistant to transformation and in vitro regeneration, including roses, carnations, and orchids [25]. Orchids are particularly attractive targets for improving genome editing protocols. However, current successes are limited to optimizing CRISPR/Cas systems, promoters, and transformation methods (including protoplast techniques) rather than in vitro regeneration and morphogenesis. This techniques applicated to little number of orchid species like Dendrobium Sw. [129,130] and Phalaenopsis [131]. Applying morphogenic factors could accelerate transgenic plant production in Orchidaceae Juss. Challenging status of orchids in genetic transformation is proved by exist of work with PEG-transformation of different orchids species protoplasts like Cymbidium Sw., Phalaenopsis, Paphiopedilum Pfitzer, Dendrobium, Arundina (D.Don) Hochr. [132]. No literature exists on morphogen use for aquatic ornamental plants.
No reports exist on MTF application in transgenic medicinal plant production. This approach could be promising, as most medicinal plants exhibit poor in vitro regeneration due to high phenolic compound and secondary metabolite content in their tissues, which hinders regeneration processes [133,134,135].
The potential effectiveness of morphogens in spore-bearing and gymnosperm plants presents a particularly interesting field for speculation. Current literature contains no reports on artificial overexpression or knockout of morphogens in mosses, lycophytes, ferns, cycads, Gnetales or Ginkgo L. Only data on key morphogen identification in some representatives of these groups, such as bryophytes [99] and certain ferns [136], are available.

4. Engineering Principles of Morphogenetic Factor Expression Systems

Incorporating morphogenetic factors into transformation constructs requires careful attention to their configuration and expression control. Unregulated constitutive expression of these powerful regulators can lead to developmental abnormalities and regenerant mortality [13]. Recent years have seen the development of several strategies for creating safe and effective MTF-containing constructs (Table 3).

4.1. Transient Expression (Non-Intergrated)

One approach involves ensuring short-term gene expression without genomic integration by placing the gene cassette outside the T-DNA boundaries [13]. In Agrobacterium-mediated transformations, such cassettes typically do not integrate (due to the termination of the T-DNA boundary) but remain temporarily active. For example, constructs containing WUS2 and BBM positioned outside the left T-DNA border in maize allowed the formation of embryogenic structures without stable transgene integration, enabling the transformation of previously resistant genotypes [15].

4.2. Chemically Inducible Promoters

Chemical induction systems (e.g., dexamethasone-, tetracycline-, or estrogen-responsive promoters) allow precise temporal control. The MTF remains inactive until induced, minimizing pleiotropic effects. For instance, Arabidopsis constructs with *35S::BBM-GR* (glucocorticoid-receptor fusion) enabled dexamethasone-inducible embryoid formation on hypocotyls [39,40]. While underutilized in routine transformation due to protocol complexity and variable inducer penetration, these systems show experimental efficacy [13].

4.3. Tissue-Specific Promoters

Spatial restriction using promoters active only in target tissues (e.g., callus or embryoids) reduces off-target effects. Corteva’s maize system employed the PLTP (plastid lipid transfer protein) promoter—active during early embryogenesis but silent in seedlings—to drive WUS/BBM expression, yielding normal regenerants [15]. Such precision enhances viable transgenic plant recovery.

4.4. Gene Excision Systems

Several primary strategies have been established and should be explicitly discussed:
1. Site-specific recombinases: Heat- or stress-inducible Cre/loxP systems in maize are commonly used in MTF pipelines to excise Wus2/BBM after embryogenesis. This approach mitigates pleiotropy and preserves efficiency [15]. Similarly, a FLP/FRT approach was used to remove BBM modules in Populus tomentosa after a brief heat shock, restoring normal growth [119]. In hexaploid wheat, the moCRE/loxP system excised both morphogenic and marker cassettes in the “QuickWheat” system, yielding high-quality events without MTFs in the final product [137]. Double-T-DNA and segregation strategies: Delivering MTFs on a separate T-DNA molecule allows for simple genetic segregation to remove them from the trait locus in T1 families. This is particularly useful when the goal is gene editing rather than stable MTF expression [15]. For workflows and trade-offs across crops, please see the targeted methodological descriptions in the maize and sorghum MTF papers (see the review in [13] for details). (iii) Non-integrating or transient delivery. Non-integrating WUS2 vectors can induce embryogenesis without a stable insertion, significantly reducing the burden of cassette cleanup in Zea mays [138]. “Altruistic” helper constructs that only express MTFs in a subset of cells can also catalyze regeneration, while being excluded from most recovered events in Sorghum bicolor (methodological proof-of-concept; primary report) [31]. (iv) Activation instead of integration. CRISPR-Combo platforms combine editing with transient activation of endogenous regeneration genes (e.g., WUS and WOX11). This approach shortens culture time and reduces the need for integrating exogenous MTF cassettes [96]. This approach complements excision systems and is compatible with transgene-free editing strategies.

4.5. Combining Multiple Morphogenetic Factors

Co-expression of synergistic regulators (e.g., WUS + BBM) requires balanced expression—achieved via separate promoters or self-cleaving peptides (e.g., WUS-P2A-BBM). While maize transformation techniques can benefit from this combination [43], Arabidopsis exhibits meristem overproliferation, underscoring species-specific optimization needs.
Practical recipes increasingly lean on limited, early exposure to MTFs (hours–days), tissue-preferred promoters, and excision triggers (heat, desiccation) to exit morphogenic states promptly. In wheat, TaWOX5—rather than WUS2/BBM—overcame genotype dependence with fewer adverse phenotypes [72], and GRF–GIF chimeras boosted regeneration across dicots and monocots while minimizing pleiotropy relative to classic embryogenic MTFs [44]. These “lighter-touch” regulators are attractive when the application tolerates smaller gains in efficiency in exchange for simpler cleanup.

5. Limitations, Contradictions, and Failure Modes of Morphogenetic-Factor-Assisted Regeneration

MTFs, such as members of the WUSCHEL/WOX family and BABY BOOM (BBM/BBML), can dramatically increase transformation and regeneration rates. However, their misexpression has been shown to cause pleiotropic defects. Classic gain-of-function studies have shown that ectopic expression of BBM in Arabidopsis thaliana and Brassica induces hormone-independent somatic embryos and calli, as well as abnormal, neoplastic growth and changes in leaf/flower morphology [39]. Similarly, chemical-induced overexpression of WUS in A. thaliana triggers high-frequency somatic embryogenesis in various tissues, with phenotypes that are closely linked to dosage and timing [30]. In crop plants, constitutive expression of WUS often disrupts development. For example, overexpression of AtWUS in cotton enhances embryogenesis, but also induces ectopic organogenesis and abnormal structures [29]. These findings highlight both the potential benefits and risks of MTFs in crop plants.
The precision of MTF dosage, tissue specificity, and exposure time are therefore crucial. In maize and related monocots, the co-delivery of ZmWUS2 and ZmBBM has increased transformation efficiency and expanded the genotype scope. However, persistent expression produced stunted or sterile plants if MTFs were not transcriptionally limited or excised prior to maturity [15]. In sorghum, Wus2-mediated direct somatic embryogenesis has boosted both transformation and CRISPR editing rates, but the study also emphasized the need to restrict MTF expression to the early regeneration stages to avoid harmful carry-over into whole plant development [139]. At the other end of the spectrum, the wound-responsive factor WOX13 can inhibit shoot organogenesis. In A. thaliana, loss-of-function of WOX13 has increased shoot regeneration, while its misexpression has impeded it. This underscores the importance of considering developmental context and the specific gene family member involved [34]. Together, these primary studies help to explain the contradictory outcomes reported across different species and plant tissues when MTFs are not properly regulated.
The cellular context also plays a significant role in determining the outcome of MTF activity. WUS (WOX5) can reprogram root meristem cells to become shoot-like, but this only occurs within specific hormonal environments and developmental stages, highlighting how mis-targeted expression can lead to abnormal outcomes [69]. In practical terms, limiting MTF expression to specific promoters that are active during early development or in response to wounding (such as those used in maize systems) is key to reducing the risk of pleiotropic effects while still maintaining the benefits of regeneration [15]. Guidance for implementing this approach across different taxa can be found in the focused review by Gordon-Kamm et al. [13], which also provides information on promoter and induction strategies.
Beyond visible morphology, culture- and MTF-driven regeneration can induce persistent epigenetic changes. Methylome maps of regenerated Oryza sativa showed widespread, stable loss of DNA methylation across generations, which is linked to deregulated gene expression and is consistent with somaclonal variation [140]. Tissue culture steps leave specific epigenomic “footprints” that differ depending on the treatment, warning that even apparently normal regenerants may have latent epigenetic shifts [141]. In clonally propagated Elaeis guineensis, the “mantled” somaclonal variant was traced back to hypomethylation of the transposon-rich Karma element in MANTLED. These epimutations are responsible for significant field-level defects [142]. These data support the need for explicit, standardized stability testing, including methylation assays, transgene expression trajectories, and multi-generation phenotyping, in pipelines that utilize MTFs.
Finally, “negative results” and contradictions often arise from the interaction between genotype, explant state, and cassette architecture. In wheat (Triticum aestivum), MTF-enhanced protocols have produced significant efficiency gains, but dwarfism or reduced fertility has been observed unless the morphogenic cassette is removed or tightly controlled; excision-based “QuickWheat” variants restore normal growth while maintaining the throughput benefits [137]. Even within the same species, the choice of ortholog (e.g., WOX5 vs. WUS2) and promoter sequence can lead to different outcomes, from success to failure. These findings emphasize the importance of reporting not only success stories but also the frequency and range of MTF-related abnormalities and the specific mitigation strategies used (promoter, dosage, induction, excision triggers).

6. The Interplay of Exogenous Plant Growth Regulators and Endogenous Morphogenetic Factors During In Vitro Regeneration

The application of MTFs can be combined with phytohormone-mediated regeneration control. In many cases, morphogenesis genes reduce dependence on exogenous phytohormones. For instance, as noted earlier, BBM expression in Arabidopsis plants led to somatic embryo formation without requiring auxin or cytokinin supplementation in the medium [13]. Similarly, WUS, by inducing meristem cell division, can partially substitute cytokinin function [77]. Experimental protocols often demonstrate that when strong MTFs are present, external hormone concentrations can be reduced. For example, maize transformation media containing WUS/BBM utilize lower 2,4-D levels than standard protocols to avoid excessive callus formation [15,69].
On the other hand, even when MTFs are incorporated into genetic constructs, phytohormones often remain essential for successful regeneration. Many protocols still include traditional steps: callus induction on 2,4-D-containing medium (where auxin promotes dedifferentiation while morphogenesis gene expression directs callus toward embryogenesis), followed by transfer to cytokinin medium for shoot regeneration. The use of MTFs allows reduced hormone concentrations or shorter induction periods, though complete hormone elimination remains rare except in specific cases [39].
Another approach combines endogenous and exogenous hormonal stimulation. For example, the Agrobacterium-derived ipt gene allows transgenic cells to produce cytokinins autonomously [79,80], while rolC expression has auxin-like effects [81,82]. These genes allow for lower concentrations of exogenous phytohormones while creating synergistic regeneration stimuli.
However, excessive cytokinin in the presence of WUS can cause excessive meristem formation without proper shoot development. Therefore, some protocols use hormone-lean regeneration media to allow morphogenesis genes to dominate the process and avoid competing morphogenetic signals. When using growth factor regulators (GRFs), which enhance general cell proliferation, slightly elevated cytokinin levels can help steer proliferation towards organogenesis, otherwise undifferentiated cell masses can form. This fine-tuning requires culture-specific optimization [13].

7. Emerging Applications of Morphogenetic Regulators in Plant Biotechnology

The integration of MTFs in plant genetic engineering is becoming more widespread. The use of morphogens extends the range of species and cultivars that can be transformed, overcoming barriers to transformation in previously difficult crops. This is especially important for woody plants, medicinal species, and high-yielding agricultural cultivars with low in vitro regeneration potential. Novel combinations of regulators (e.g., PLT and GRF-GIF) can enable transformation of species that are difficult to transform using classical methods. Researchers expect that further developments and discoveries in this area will make the transformation of difficult crops routine and highly effective [15,45,107].
MTFs significantly reduce the time required to produce transgenic plants by accelerating the regeneration process. Protocols now exist that allow for the production of rooted transgenic shoots from certain tomato and soybean varieties in less than 5–6 weeks, bypassing lengthy callus phases [143]. This enables faster experimentation and reduces costs, while also reducing the need for complex micropropagation equipment, as some stages occur in vivo. These advances democratize transformation technology, making it accessible beyond specialized laboratories [13,103].
The search for novel genes that enhance regeneration continues, with ongoing testing of WIND1, PLT5, and variants of WUS/BBM from various plants [43,45]. This repertoire is likely to expand to include family-specific factors, such as conifer morphogens [98]. Another promising avenue is the use of synthetic regulators, which are engineered transcription factors that combine domains from multiple natural proteins, such as WUS-BBM fusions [43].
Innovative DNA/RNA delivery methods are actively being developed. Key advancements include various techniques, such as ribonucleoprotein delivery to minimize explant damage [144]. Biolistic delivery of MTFs-constructs is particularly promising for monocots, as demonstrated by Lowe et al., who achieved 10x higher regeneration in sorghum using biolistically delivered WUS (controlled by a weak nos promoter to prevent necrosis) and BBM (driven by a strong maize ubiquitin promoter). While effective for crops like soybean [112,113], biolistics can cause developmental abnormalities [145].
Viral vectors carrying morphogens [16], such as WUS, can induce meristems systemically, as demonstrated in tobacco [146], wheat [147,148], and tomato [149]. However, this approach requires the use of Cas9-expressing plants and is genotype-dependent, making it labor-intensive.
Future priorities include improving MTFs-constructs to eliminate residual transgenes and phenotypic abnormalities. Emerging strategies include: non-integrated, transient expression; self-excision systems; and precision regulation (light-inducible/tissue-specific promoters). These approaches aim to use morphogens as temporary stimulants with no genomic or epigenetic effects [13].
Because many MTF-enabled pipelines are ultimately used to generate genome-edited crops, it is important to understand that regulatory treatment depends on the final genomic state, rather than on temporary tools used during tissue culture. In many jurisdictions, current global regulations exempt certain genome edits (SDN-1/2, no foreign DNA) from GMO classification, while other countries, such as the EU, still regulate these products as GMOs under existing rules. Reforms are currently being debated (e.g., the EU’s NGT proposal) [150,151]. Broader policy analyses agree that documentation of the absence of foreign DNA and molecular characterization of edited loci are crucial for dossiers [152,153]. Accordingly, when MTFs are used transiently or excised prior to regeneration, the resulting line’s regulatory status will typically track that of a marker-free edited plant, in countries that distinguish between product-based and process-based methods (for more details, see references [150,153]).
In addition to transformation, MTFs can serve as standalone biotechnological tools. For instance, WUS/ipt co-expression can enhance shoot multiplication for biomass production, and ex vivo applications, such as treating saplings with morphogen-bearing Agrobacteria, can produce branched clones. However, these uses require caution to avoid unintended genomic alterations.

8. Conclusions

Thus, the application of MTFs to enhance the in vitro regeneration capacity of agricultural crops represents one of the key trends in plant genetic engineering biotechnology. The use of morphogens is already helping to overcome a major limitation in plant biotechnology—the low transformation and regeneration efficiency of many crops. As morphogen-based approaches advance, the list of easily and routinely transformable and regenerable crop varieties is expected to expand encompassing genotypes of soybean, rice, rapeseed, maize, cucurbits, conifers, and other plant species that are currently recalcitrant to in vitro manipulation [13,59,91,111]. Despite successes, many unresolved issues remain. It is crucial to thoroughly study the pleiotropic effects of morphogens before their widespread use. This will help identify the most effective morphogens for specific crops and methods to control their function in plants. Most studies on new morphogens focus more on their application in dicots than in monocots. It may be worthwhile for new studies to include both dicot and monocot crops (e.g., soybean and rice, tomato and maize) as research objects. This would enable comparison of morphogen effects across different crops and accelerate the selection of suitable morphogens for each crop and variety. Overcoming the barrier of genetic transformation in most woody and medicinal plants remains a significant area for research. It is possible that, along with MTFs, gene cassettes could include genes (transcription factors like MYB, bHLH, WD40 [154]) that block the synthesis of phenols, which hinder regeneration processes. The question of effective delivery methods for MTFs alongside other genes into plants is also relevant. Viral vectors and CRISPR/Cas systems could theoretically facilitate the delivery of large constructs or individual genes. Also, techniques of morphogen’s direct delivery are highly desired in plant biotechnology, which could help to eliminate in vitro routine fully or partly [155]. In the future, combining synthetic biology advances, novel vector systems, and MTFs could lead to a radical enhancement of the genetic potential of agricultural plants [16].

Author Contributions

Writing—Review & Editing, S.D.B., S.G.M., S.A.B. All authors have read and agreed to the published version of the manuscript.

Funding

S.A.B. were supported by the state assignment of VIGG RAS 125040704886-1.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Somssich, M. A short history of plant transformation. PeerJ Prepr. 2019, 28. [Google Scholar] [CrossRef]
  2. McCabe, D.E.; Swain, W.F.; Martinell, B.J.; Christou, P. Stable Transformation of Soybean (Glycine max) by Particle Bombardment. Nat. Biotechnol 1988, 6, 923–926. [Google Scholar] [CrossRef]
  3. Umbeck, P.; Johnson, G.; Barton, K.; Swain, W. Genetically Transformed Cotton (Gossypium hirsutum L.) Plants. Nat. Biotechnol 1987, 5, 263–266. [Google Scholar] [CrossRef]
  4. Toriyama, K.; Arimoto, Y.; Uchimiya, H.; Hinata, K. Transgenic Rice Plants After Direct Gene Transfer into Protoplasts. Nat. Biotechnol 1988, 6, 1072–1074. [Google Scholar] [CrossRef]
  5. Horsch, R.B.; Fry, J.E.; Hoffmann, N.L.; Wallroth, M.; Eichholtz, D.; Rogers, S.G.; Fraley, R.T. A Simple and General Method for Transferring Genes into Plants. Science 1985, 227, 1229–1231. [Google Scholar] [CrossRef]
  6. Moloney, M.M.; Walker, J.M.; Sharma, K.K. High Efficiency Transformation of Brassica napus Using Agrobacterium Vectors. Plant Cell Rep. 1989, 8, 238–242. [Google Scholar] [CrossRef]
  7. Fromm, M.E.; Morrish, F.; Armstrong, C.; Williams, R.; Thomas, J.; Klein, T.M. Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Nat. Biotechnol 1990, 8, 833–839. [Google Scholar] [CrossRef]
  8. Vasil, V.; Castillo, A.M.; Fromm, M.E.; Vasil, I.K. Herbicide Resistant Fertile Transgenic Wheat Plants Obtained by Microprojectile Bombardment of Regenerable Embryogenic Callus. Nat. Biotechnol 1992, 10, 667–674. [Google Scholar] [CrossRef]
  9. Zhao, Z.Y.; Cai, T.; Tagliani, L.; Miller, M.; Wang, N.; Pang, H.; Rudert, M.; Schroeder, S.; Hondred, D.; Seltzer, J.; et al. Agrobacterium-Mediated Sorghum Transformation. Plant Mol. Biol. 2000, 44, 789–798. [Google Scholar] [CrossRef]
  10. Bower, R.; Birch, R.G. Transgenic sugarcane plants via microprojectile bombardment. Plant J. 1992, 2, 409–416. [Google Scholar] [CrossRef]
  11. Skoog, F.; Miller, C.O. Chemical Regulation of Growth and Organ Formation in Plant Tissues Cultured In Vitro. Symp. Soc. Exp. Biol. 1957, 11, 118–131. [Google Scholar]
  12. Steward, F.C. Growth and Organized Development of Cultured Cells. Am. J. Bot. 1958, 45, 693–703. [Google Scholar] [CrossRef]
  13. Gordon-Kamm, B.; Sardesai, N.; Arling, M.; Lowe, K.; Hoerster, G.; Betts, S.; Jones, T. Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants. Plants 2019, 8, 38. [Google Scholar] [CrossRef]
  14. Zhang, Y.; Wang, X.; Zhang, J.; He, X.-Q. Plant in situ tissue regeneration: Dynamics, mechanisms and implications for forestry research. For. Res. 2023, 3, 8. [Google Scholar] [CrossRef]
  15. Lowe, K.; Wu, E.; Wang, N.; Hoerster, G.; Hastings, C.; Cho, M.J.; Scelonge, C.; Lenderts, B.; Chamberlin, M.; Cushatt, J.; et al. Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation. Plant Cell 2016, 28, 1998–2015. [Google Scholar] [CrossRef]
  16. Ramkumar, T.R.; Lenka, S.K.; Arya, S.S.; Bansal, K.C. A Short History and Perspectives on Plant Genetic Transformation. Methods Mol. Biol. 2020, 2124, 39–60. [Google Scholar] [CrossRef]
  17. Chu, U.C.; Kumar, S.; Sigmund, A.; Johnson, K.; Li, Y.; Vongdeuane, P.; Jones, T.J. Genotype-independent transformation and genome editing of Brassica napus using a novel explant material. Front. Plant Sci. 2020, 11, 579524. [Google Scholar] [CrossRef]
  18. Lowe, K.; La Rota, M.; Hoerster, G.; Hastings, C.; Wang, N.; Chamberlin, M.; Wu, E.; Jones, T.; Gordon-Kamm, W. Rapid genotype “independent” Zea mays L. (maize) transformation via direct somatic embryogenesis. Vitr. Cell. Dev. Biol. 2018, 54, 240–252. [Google Scholar] [CrossRef]
  19. Wang, K.; Liu, H.; Du, L.; Ye, X.; Wu, H. The Gene TaWOX5 Overcomes Genotype Dependency in Wheat Genetic Transformation. Nat. Plants 2022, 8, 110–117, Erratum in Nat. Plants 2022, 8, 717–720. [Google Scholar] [CrossRef]
  20. Kong, J.; Martin-Ortigosa, S.; Finer, J.; Orchard, N.; Gunadi, A.; Batts, L.A.; Thakare, D.; Rush, B.; Schmitz, O.; Stuiver, M.; et al. Overexpression of the Transcription Factor GROWTH-REGULATING FACTOR5 Improves Transformation of Dicot and Monocot Species. Front. Plant Sci. 2020, 11, 572319. [Google Scholar] [CrossRef]
  21. Bennur, P.L.; O’Brien, M.; Fernando, S.C.; Doblin, M.S. Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species. J. Exp. Bot. 2025, 76, 52–75. [Google Scholar] [CrossRef]
  22. Youngstrom, C.; Wang, K.; Lee, K. Unlocking regeneration potential: Harnessing morphogenic regulators and small peptides for enhanced plant engineering. Plant J. 2025, 121, e17193. [Google Scholar] [CrossRef]
  23. Laux, T.; Mayer, K.F.X.; Berger, J.; Jürgens, G. The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Development 1996, 122, 87–96. [Google Scholar] [CrossRef]
  24. Tvorogova, V.E.; Krasnoperova, E.Y.; Potsenkovskaia, E.A.; Kudriashov, A.A.; Dodueva, I.E.; Lutova, L.A. What Does the WOX Say? Review of Regulators, Targets, Partners. Mol. Biol. 2021, 55, 362–391. [Google Scholar] [CrossRef]
  25. Duan, H.; Maren, N.A.; Ranney, T.G.; Liu, W. New opportunities for using WUS/BBM and GRF-GIF genes to enhance genetic transformation of ornamental plants. Ornam. Plant Res. 2022, 2, 4. [Google Scholar] [CrossRef]
  26. Arroyo-Herrera, A.; Gonzalez, A.K.; Moo, R.C.; Quiroz-Figueroa, F.R.; Loyola-Vargas, V.M.; Rodriguez-Zapata, L.C.; Burgeff, D. ′Hondt, C.; Suárez-Solís, V.M., Castaño, E. Expression of WUSCHEL in Coffea canephora causes ectopic morphogenesis and increases somatic embryogenesis. Plant Cell Tissue Organ. Cult. 2008, 94, 171–180. [Google Scholar] [CrossRef]
  27. Mursyanti, E.; Purwantoro, A.; Moeljopawiro, S.; Semiarti, E. Induction of somatic embryogenesis through overexpression of ATRKD4 genes in Phalaenopsis “Sogo Vivien”. Indones. J. Biotechnol. 2016, 20, 42–53. [Google Scholar] [CrossRef]
  28. Acereto-Escoffié, P.O.M.; Chi-Manzanero, B.H.; Echeverría-Echeverría, S.; Grijalva, R.; Kay, A.J.; González-Estrada, T.; Castaño, E.; Rodríguez-Zapata, L.C. Agrobacterium-mediated transformation of Musa acuminata cv. “Grand Nain” scalps by vacuum infiltration. Sci. Hortic. 2005, 105, 350–371. [Google Scholar] [CrossRef]
  29. Bouchabké-Coussa, O.; Obellianne, M.; Linderme, D.; Montes, E.; Maia-Grondard, A.; Vilaine, F.; Pannetier, C. Wuschel overexpression promotes somatic embryogenesis and induces organogenesis in cotton (Gossypium hirsutum L.) tissues cultured in vitro. Plant Cell Rep. 2013, 32, 675–686. [Google Scholar] [CrossRef]
  30. Zuo, J.; Niu, Q.-W.; Frugis, G.; Chua, N.-H. The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis. Plant J. 2002, 30, 349–359. [Google Scholar] [CrossRef]
  31. Nelson-Vasilchik, K.; Hague, J.P.; Tilelli, M.; Kausch, A.P. Rapid transformation and plant regeneration of sorghum (Sorghum bicolor L.) mediated by altruistic Baby boom and Wuschel2. Vitr. Cell. Dev. Biol.-Plant 2022, 58, 331–342. [Google Scholar] [CrossRef]
  32. 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]
  33. van der Graaff, E.; Laux, T.; Rensing, S.A. The WUS homeobox-containing (WOX) protein family. Genome Biol. 2009, 10, 248. [Google Scholar] [CrossRef]
  34. Ogura, N.; Sasagawa, Y.; Ito, T.; Tameshige, T.; Kawai, S.; Sano, M.; Doll, Y.; Iwase, A.; Kawamura, A.; Suzuki, T.; et al. WUSCHEL-RELATED HOMEOBOX 13 suppresses de novo shoot regeneration via cell fate control of pluripotent callus. Sci. Adv. 2023, 9, eadg6983. [Google Scholar] [CrossRef]
  35. Zhou, X.; Han, H.; Chen, J.; Han, H. The emerging roles of WOX genes in development and stress responses in woody plants. Plant Sci. 2024, 349, 112259. [Google Scholar] [CrossRef]
  36. Liu, J.; Sheng, L.; Xu, Y.; Li, J.; Yang, Z.; Huang, H.; Xu, L. WOX11 and 12 Are Involved in the First-Step Cell Fate Transition during de Novo Root Organogenesis in Arabidopsis. Plant Cell 2014, 26, 1081–1093. [Google Scholar] [CrossRef]
  37. Wang, D.; Ma, X.; Hao, Z.; Long, X.; Shi, J.; Chen, J. Overexpression of Liriodenron WOX5 in Arabidopsis Leads to Ectopic Flower Formation and Altered Root Morphology. Int. J. Mol. Sci. 2023, 24, 906. [Google Scholar] [CrossRef]
  38. Wójcik, A.M. Root regeneration under miRNA control. Nat. Plants 2025, 11, 949–950. [Google Scholar] [CrossRef]
  39. Boutilier, K.; Offringa, R.; Sharma, V.K.; Kieft, H.; Ouellet, T.; Zhang, L.; Hattori, J.; Liu, C.M.; van Lammeren, A.A.; Miki, B.L.; et al. Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell 2002, 14, 1737–1749. [Google Scholar] [CrossRef]
  40. Srinivasan, C.; Liu, Z.; Heidmann, I.; Supena, E.D.; Fukuoka, H.; Joosen, R.; Lambalk, J.; Angenent, G.; Scorza, R.; Custers, J.B.; et al. Heterologous expression of the BABY BOOM AP2/ERF transcription factor enhances the regeneration capacity of tobacco (Nicotiana tabacum L.). Planta 2007, 225, 341–351. [Google Scholar] [CrossRef]
  41. 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]
  42. Florez, S.L.; Erwin, R.L.; Maximova, S.N.; Guiltinan, M.J.; Curtis, W.R. Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor. BMC Plant Biol. 2015, 15, 121. [Google Scholar] [CrossRef]
  43. Lian, Z.; Nguyen, C.D.; Liu, L.; Wang, G.; Chen, J.; Wang, S.; Yi, G.; Wilson, S.; Ozias-Akins, P.; Gong, H.; et al. Application of developmental regulators to improve in planta or in vitro transformation in plants. Plant Biotechnol. J. 2022, 20, 1622–1635. [Google Scholar] [CrossRef]
  44. Debernardi, J.M.; Tricoli, D.M.; Ercoli, M.F.; Hayta, S.; Ronald, P.; Palatnik, J.F.; Dubcovsky, J. A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants. Nature 2020, 38, 1274–1279. [Google Scholar] [CrossRef]
  45. Zhao, Y.; Cheng, P.; Liu, Y.; Liu, C.; Hu, Z.; Xin, D.; Wu, X.; Yang, M.; Chen, Q. A highly efficient soybean transformation system using GRF3-GIF1 chimeric protein. J. Integr. Plant Biol. 2025, 67, 3–6. [Google Scholar] [CrossRef]
  46. Li, X.; Cao, C.; Liu, Y.; Bolaños-Villegas, P.; Wang, J.; Zhou, R.; Hou, J.; Li, Q.; Mao, W.; Wang, P.; et al. Enhancing genetic transformation efficiency of melon (Cucumis melo L.) through an extended sucrose-removal co-culture. Plant Cell Rep. 2025, 44, 123. [Google Scholar] [CrossRef] [PubMed]
  47. Lotan, T.; Ohto, M.; Yee, K.M.; West, M.A.; Lo, R.; Kwong, R.W.; Yamagishi, K.; Fischer, R.L.; Goldberg, R.B.; Harada, J.J. Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 1998, 93, 1195–1205. [Google Scholar] [CrossRef] [PubMed]
  48. 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. Proc. Natl. Acad. Sci. USA 2001, 98, 11806–11811. [Google Scholar] [CrossRef] [PubMed]
  49. Yamaji, N.; Kyo, M. Two promoters conferring active gene expression in vegetative nuclei of tobacco immature pollen undergoing embryogenic dedifferentiation. Plant Cell Rep. 2006, 25, 749–757. [Google Scholar] [CrossRef]
  50. Valvekens, D.; Van Montagu, M.; Van Lijsebettens, M. Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 1988, 85, 5536–5540. [Google Scholar] [CrossRef]
  51. Banno, H.; Ikeda, Y.; Niu, Q.W.; Chua, N.H. Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Plant Cell 2001, 13, 2609–2618. [Google Scholar] [CrossRef]
  52. Ikeda, Y.; Králová, M.; Zalabák, D.; Kubalová, I.; Aida, M. Post-Embryonic Lateral Organ Development and Adaxial-Abaxial Polarity Are Regulated by the Combined Effect of ENHANCER OF SHOOT REGENERATION 1 and WUSCHEL in Arabidopsis Shoots. Int. J. Mol. Sci. 2021, 22, 10621. [Google Scholar] [CrossRef]
  53. 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]
  54. Wan, Q.; Zhai, N.; Xie, D.; Liu, W.; Xu, L. WOX11: The founder of plant organ regeneration. Cell Regen. 2023, 12, 1. [Google Scholar] [CrossRef]
  55. Kshetry, A.O.; Ghose, K.; Alok, A.; Devkar, V.; Raman, V.; Stupar, R.M.; Herrera-Estrella, L.; Zhang, F.; Patil, G.B. Shoot at Site: Advancing in planta transformation, regeneration and gene-editing through a cascade of wounding-mediated developmental regulators. bioRxiv 2025. [Google Scholar] [CrossRef]
  56. Waki, T.; Hiki, T.; Watanabe, R.; Hashimoto, T.; Nakajima, K. The Arabidopsis RWP-RK protein RKD4 triggers gene expression and pattern formation in early embryogenesis. Curr. Biol. 2011, 21, 1277–1281. [Google Scholar] [CrossRef] [PubMed]
  57. Chardin, C.; Girin, T.; Roudier, F.; Meyer, C.; Krapp, A. The plant RWP-RK transcription factors: Key regulators of nitrogen responses and of gametophyte development. J. Exp. Bot. 2014, 65, 5577–5587. [Google Scholar] [CrossRef]
  58. Schauser, L.; Roussis, A.; Stiller, J.; Stougaard, J. A plant regulator controlling development of symbiotic root nodules. Nature 1999, 402, 191–195. [Google Scholar] [CrossRef]
  59. Purwestri, Y.A.; Lee, Y.S.; Meehan, C.; Mose, W.; Susanto, F.A.; Wijayanti, P.; Fauzia, A.N.; Nuringtyas, T.R.; Hussain, N.; Putra, H.L.; et al. RWP-RK Domain 3 (OsRKD3) induces somatic embryogenesis in black rice. BMC Plant Biol. 2023, 23, 202. [Google Scholar] [CrossRef]
  60. Liu, X.; Bie, X.M.; Lin, X.; Li, M.; Wang, H.; Zhang, X.; Yang, Y.; Zhang, C.; Zhang, X.S.; Xiao, J. Uncovering the transcriptional regulatory network involved in boosting wheat regeneration and transformation. Nat. Plants 2023, 9, 908–925, Erratum in Nat. Plants 2024, 10, 194. [Google Scholar] [CrossRef]
  61. Ebinuma, H.; Sugita, K.; Matsunaga, E.; Yamakado, M. Selection of marker-free transgenic plants using the isopentenyl transferase gene. Proc. Natl. Acad. Sci. USA 1997, 94, 2117–2121. [Google Scholar] [CrossRef]
  62. Ijaz, S.; Bashir, A.; Malik, K.A. Expression of Agrobacterium Isopentenyl transferase (IPT) gene in wheat improves drought tolerance. Transgenic Res. 2025, 34, 7. [Google Scholar] [CrossRef]
  63. Yang, W.; Zhai, H.; Wu, F.; Lei, D.; Yu, C.; Meng, X.; Chen, Q.; Liu, C.; Bie, X.; Sun, C.; et al. Peptide REF1 is a local wound signal promoting plant regeneration. Cell 2024, 187, 3024–3038. [Google Scholar] [CrossRef]
  64. Gallois, J.L.; Woodward, C.; Reddy, G.V.; Sablowski, R. Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis. Development 2002, 129, 3207–3217. [Google Scholar] [CrossRef]
  65. Ille, K.; Melzer, S. Efficient and Versatile Rapeseed Transformation for New Breeding Technologies. bioRxiv 2024. [Google Scholar] [CrossRef]
  66. Gordon, S.P.; Chickarmane, V.S.; Ohno, C.; Meyerowitz, E.M. Multiple feedback loops through cytokinin signaling control stem cell number within the Arabidopsis shoot meristem. Proc. Natl. Acad. Sci. USA 2009, 106, 16529–16534. [Google Scholar] [CrossRef]
  67. Lenhard, M.; Bohnert, A.; Jürgens, G.; Laux, T. Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS. Cell 2001, 105, 805–814. [Google Scholar] [CrossRef]
  68. Maher, M.F.; Nasti, R.A.; Vollbrecht, M.; Starker, C.G.; Clark, M.D.; Voytas, D.F. Plant gene editing through de novo induction of meristems. Nat. Biotechnol. 2020, 38, 84–89. [Google Scholar] [CrossRef]
  69. Gallois, J.L.; Nora, F.R.; Mizukami, Y.; Sablowski, R. WUSCHEL induces shoot stem cell activity and developmental plasticity in the root meristem. Genes. Dev. 2004, 18, 375–380. [Google Scholar] [CrossRef]
  70. Mookkan, M.; Nelson-Vasilchik, K.; Hague, J.; Zhang, Z.J.; Kausch, A.P. Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2. Plant Cell Rep. 2017, 36, 1477–1491. [Google Scholar] [CrossRef]
  71. Deveaux, Y.; Toffano-Nioche, C.; Claisse, G.; Thareau, V.; Morin, H.; Laufs, P.; Moreau, H.; Kreis, M.; Lecharny, A. Genes of the most conserved WOX clade in plants affect root and flower development in Arabidopsis. BMC Ecol. Evol. 2008, 8, 291. [Google Scholar] [CrossRef]
  72. Zhang, T.; Xiang, Y.; Ye, M.; Yuan, M.; Xu, G.; Zhou, D.-X.; Zhao, Y. The uORF-HsfA1a-WOX11 module controls crown root development in rice. New Phytol. 2025, 247, 760–773. [Google Scholar] [CrossRef]
  73. Heidmann, I.; de Lange, B.; Lambalk, J.; Angenent, G.C.; Boutilier, K. Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor. Plant Cell Rep. 2011, 30, 1107–1115. [Google Scholar] [CrossRef]
  74. Kareem, A.; Durgaprasad, K.; Sugimoto, K.; Du, Y.; Pulianmackal, A.; Trivedi, Z.; Abhayadev, P.; Pinon, V.; Meyerowitz, E.; Scheres, B.; et al. PLETHORA Genes Control Regeneration by a Two-Step Mechanism . Curr. Biol. 2015, 25, 1017–1030. [Google Scholar] [CrossRef]
  75. Schmidt, E.D.L.; Guzzo, F.; Toonen, M.A.J.; 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]
  76. Ikeuchi, M.; Ogawa, Y.; Iwase, A.; Sugimoto, K. Plant regeneration: Cellular origins and molecular mechanisms. Development 2016, 143, 1442–1451. [Google Scholar] [CrossRef]
  77. Iwase, A.; Mitsuda, N.; Ikeuchi, M.; Ohnuma, M.; Koizuka, C.; Kawamoto, K.; Imamura, J.; Ezura, H.; Sugimoto, K. Arabidopsis WIND1 Induces Callus Formation in Rapeseed, Tomato, and Tobacco. Plant Signal. Behav. 2013, 8, e27432. [Google Scholar] [CrossRef]
  78. Richael, C.M.; Kalyaeva, M.; Chretien, R.C.; Yan, H.; Adimulam, S.; Stivison, A.; Weeks, J.T.; Rommens, C.M. vectors mediate marker-free and backbone-free plant transformation. Transgenic Res. 2008, 17, 905–917. [Google Scholar] [CrossRef]
  79. Ondrej, M.; Macháčková, I.; Ĉatský, J.; Eder, J.; Hrouda, M.; Pospisilová, J.; Synkova, H. Potato transformation by T-DNA Cytokinin synthesis gene. Biol. Plant. 1990, 32, 401–406. [Google Scholar] [CrossRef]
  80. Smigocki, A.C.; Owens, L.D. Cytokinin-to-Auxin Ratios and Morphology of Shoots and Tissues Transformed by a Chimeric Isopentenyl Transferase Gene. Plant Physiol. 1989, 91, 808–811. [Google Scholar] [CrossRef]
  81. Casanova, E.; Valdés, A.E.; Zuker, A.; Fernández, B.; Vainstein, A.; Trillas, M.I.; Moysset, L. rolC-transgenic carnation plants: Adventitious organogenesis and levels of endogenous auxin and cytokinins. Plant Sci. 2004, 167, 551–560. [Google Scholar] [CrossRef]
  82. Gorpenchenko, T.Y.; Kiselev, K.V.; Bulgakov, V.P.; Tchernoded, G.K.; Bragina, E.A.; Khodakovskaya, M.V.; Koren, O.G.; Batygina, T.B.; Zhuravlev, Y.N. The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses. Planta 2006, 223, 457–467. [Google Scholar] [CrossRef]
  83. Zhu, S.; Wang, J.; Ye, J.; Zhu, A.-D.; Guo, W.; Deng, X. Isolation and Characterization of LEAFY COTYLEDON 1-LIKE Gene Related to Embryogenic Competence in Citrus Sinensis. Plant Cell Tiss. Organ. Cult. 2014, 119, 1–13. [Google Scholar] [CrossRef]
  84. Hu, W.; Li, W.; Xie, S.; Fagundez, S.; McAvoy, R.; Deng, Z.; Li, Y. Kn1 Gene Overexpression Drastically Improves Genetic Transformation Efficiencies of Citrus Cultivars. Plant Cell Tiss. Organ. Cult. 2016, 125, 81–91. [Google Scholar] [CrossRef]
  85. Chen, J.; Tomes, S.; Gleave, A.P.; Hall, W.; Luo, Z.; Xu, J.; Yao, J.-L. Significant Improvement of Apple (Malus Domestica Borkh.) Transgenic Plant Production by Pre-Transformation with a Baby Boom Transcription Factor. Hortic. Res. 2022, 9, uhab014. [Google Scholar] [CrossRef]
  86. Zhao, H.; Xie, Y.; Zheng, Q.; Yu, Q.; Lv, K.; Hao, X.; Zhang, N.; Xu, W. A Simple and Efficient Non-Tissue Culture Method for Genetic Transformation of Grape Immature Zygotic Embryos via VvBBM Overexpression. Sci. Hortic. 2024, 337, 113581. [Google Scholar] [CrossRef]
  87. Sanchez, E.R.; Price, R.J.; Marangelli, F.; McLeary, K.; Harrison, R.J.; Kundu, A. Overexpression of Vitis GRF4-GIF1 Improves Regeneration Efficiency in Diploid Fragaria Vesca Hawaii 4. Plant Methods 2024, 20, 160. [Google Scholar] [CrossRef]
  88. Rekik, I.; Drira, N.; Grubb, D.; Elleuch, A. Molecular Characterization and Evolution Studies of a SERK like Gene Transcriptionally Induced during Somatic Embryogenesis in Phoenix dactylifera L. v Deglet Nour. Genetika 2015, 47, 323–337. [Google Scholar] [CrossRef]
  89. Osorio-Montalvo, P.; De-la-Peña, C.; Oropeza, C.; Nic-Can, G.; Córdova-Lara, I.; Castillo-Castro, E.; Sáenz-Carbonell, L. A Peak in Global DNA Methylation Is a Key Step to Initiate the Somatic Embryogenesis of Coconut Palm (Cocos nucifera L.). Plant Cell Rep. 2020, 39, 1345–1357. [Google Scholar] [CrossRef]
  90. Xu, N.; Kang, M.; Zobrist, J.D.; Wang, K.; Fei, S.Z. Genetic Transformation of Recalcitrant Upland Switchgrass Using Morphogenic Genes. Front. Plant Sci. 2022, 12, 781565. [Google Scholar] [CrossRef]
  91. Segatto, R.; Paggi, G.M.; Taylor, N.J. Overexpression of GRF-GIF Genes Enhances Plant Regeneration in Cassava (Manihot esculenta). Vitr. Cell. Dev. Biol.-Plant 2024, 61, 593–607. [Google Scholar] [CrossRef]
  92. Li, Y.; Wang, N.; Feng, J.; Liu, Y.; Wang, H.; Deng, S.; Dong, W.; Liu, X.; Lv, B.; Sun, J.; et al. Enhancing Genetic Transformation Efficiency in Cucurbit Crops through AtGRF5 Overexpression: Mechanistic Insights and Applications. JIPB 2025, 67, 1843–1860. [Google Scholar] [CrossRef]
  93. Feng, Q.; Xiao, L.; He, Y.; Liu, M.; Wang, J.; Tian, S.; Zhang, X.; Yuan, L. Highly Efficient, Genotype-independent Transformation and Gene Editing in Watermelon (Citrullus lanatus) Using a Chimeric ClGRF4-GIF1 Gene. JIPB 2021, 63, 2038–2042. [Google Scholar] [CrossRef]
  94. Zhang, Z.-A.; Liu, M.-Y.; Ren, S.-N.; Liu, X.; Gao, Y.-H.; Zhu, C.-Y.; Niu, H.-Q.; Chen, B.-W.; Liu, C.; Yin, W.; et al. Identification of WUSCHEL-Related Homeobox Gene and Truncated Small Peptides in Transformation Efficiency Improvement in Eucalyptus. BMC Plant Biol. 2023, 23, 604. [Google Scholar] [CrossRef]
  95. Deng, W.; Luo, K.; Li, Z.; Yang, Y. A Novel Method for Induction of Plant Regeneration via Somatic Embryogenesis. Plant Sci. 2009, 177, 43–48. [Google Scholar] [CrossRef]
  96. Pan, C.; Li, G.; Malzahn, A.A.; Cheng, Y.; Leyson, B.; Sretenovic, S.; Gurel, F.; Coleman, G.D.; Qi, Y. Boosting Plant Genome Editing with a Versatile CRISPR-Combo System. Nat. Plants 2022, 8, 513–525. [Google Scholar] [CrossRef]
  97. Wang, L.-Q.; Li, Z.; Wen, S.-S.; Wang, J.-N.; Zhao, S.-T.; Lu, M.-Z. WUSCHEL-Related Homeobox Gene PagWOX11/12a Responds to Drought Stress by Enhancing Root Elongation and Biomass Growth in Poplar. J. Exp. Bot. 2019, erz490. [Google Scholar] [CrossRef]
  98. Uddenberg, D.; Abrahamsson, M.; von Arnold, S. Overexpression of PaHAP3A stimulates differentiation of ectopic embryos from maturing somatic embryos of Norway spruce. Tree Genet. Genomes 2016, 12, 18. [Google Scholar] [CrossRef]
  99. Lin, W.; Wang, Y.; Coudert, Y.; Kierzkowski, D. Leaf Morphogenesis: Insights From the Moss Physcomitrium Patens. Front. Plant Sci. 2021, 12, 736212. [Google Scholar] [CrossRef]
  100. Klein, T.M.; Wolf, E.D.; Wu, R.; Sanford, J.C. High velocity microprojectiles for delivering nucleic acids into living cells. Nature 1987, 327, 70–73. [Google Scholar] [CrossRef]
  101. Christou, P.; Ford, T.L.; Kofron, M. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA Into Immature Zygotic embryos. Nat. Biotechnol. 1991, 9, 957–962. [Google Scholar] [CrossRef]
  102. Hiei, Y.; Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat. Protoc. 2008, 3, 824–834. [Google Scholar] [CrossRef] [PubMed]
  103. Hiei, Y.; Ohta, S.; Komari, T.; Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994, 6, 271–282. [Google Scholar] [CrossRef] [PubMed]
  104. Allahi, S.; Khodaparast, S.A.; Sohani, M.M. Agrobacterium-Mediated Transformation of Indica Rice: A Non-Tissue Culture Approach. Int. J. Agric. Innov. Res. 2014, 3, 467–472. [Google Scholar]
  105. Cardoza, V.; Stewart, C.N. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep. 2003, 21, 599–604. [Google Scholar] [CrossRef]
  106. Zhang, K.; He, J.; Liu, L.; Xie, R.; Qiu, L.; Li, X.; Yuan, W.; Chen, K.; Yin, Y.; Kyaw, M.M.M.; et al. A convenient, rapid and efficient method for establishing transgenic lines of Brassica napus. Plant Methods 2020, 16, 43. [Google Scholar] [CrossRef]
  107. Liu, F.; Wang, P.; Xiong, X.; Fu, P.; Gao, H.; Ding, X.; Wu, G. Comparison of three Agrobacterium-mediated co-transformation methods for generating marker-free transgenic Brassica napus plants. Plant Methods 2020, 16, 81. [Google Scholar] [CrossRef]
  108. Christou, P.; McCabe, D.E.; Swain, W.F. Stable Transformation of Soybean Callus by DNA-Coated Gold Particles. Plant Physiol. 1988, 87, 671–674. [Google Scholar] [CrossRef]
  109. Hinchee, M.A.W.; Connor-Ward, D.V.; Newell, C.A.; McDonnell, R.E.; Sato, S.J.; Gasser, C.S.; Fischhoff, D.A.; Re, D.B.; Fraley, R.T.; Horsch, R.B. Production of Transgenic Soybean Plants Using Agrobacterium-Mediated DNA Transfer. Nat. Biotechnol. 1988, 6, 915–922. [Google Scholar] [CrossRef]
  110. Paz, M.M.; Martinez, J.C.; Kalvig, A.B.; Fonger, T.M.; Wang, K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep. 2006, 25, 206–213. [Google Scholar] [CrossRef]
  111. Wang, G.; Xu, Y. Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference. Plant Cell Rep. 2008, 27, 1177–1184. [Google Scholar] [CrossRef]
  112. Jia, Y.; Yao, X.; Zhao, M.; Zhao, Q.; Du, Y.; Yu, C.; Xie, F. Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process. Int. J. Mol. Sci. 2015, 16, 18522–18543. [Google Scholar] [CrossRef]
  113. Rech, E.L.; Vianna, G.R.; Aragão, F.J. High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat. Protoc. 2008, 3, 410–418. [Google Scholar] [CrossRef]
  114. Nagle, M.F.; Yuan, J.; Kaur, D.; Ma, C.; Peremyslova, E.; Jiang, Y.; Goralogia, G.S.; Magnuson, A.; Li, J.Y.; Muchero, W.; et al. Genome-wide association study and network analysis of in vitro transformation in Populus trichocarpa support key roles of diverse phytohormone pathways and cross talk. New Phytol. 2024, 242, 2059–2076. [Google Scholar] [CrossRef] [PubMed]
  115. McCormick, S.; Niedermeyer, J.; Fry, J.; Barnason, A.; Horsch, R.; Fraley, R. Leaf disc transformation of cultivated tomato (L. esculentum) Using Agrobacterium tumefaciens. Plant Cell Rep. 1986, 5, 81–84. [Google Scholar] [CrossRef] [PubMed]
  116. Sun, H.-J.; Uchii, S.; Watanabe, S.; Ezura, H. A Highly Efficient Transformation Protocol for Micro-Tom, a Model Cultivar for Tomato Functional Genomics. Plant Cell Physiol. 2006, 47, 426–431. [Google Scholar] [CrossRef]
  117. Fuentes, A.D.; Ramos, P.L.; Sánchez, Y.; Callard, D.; Ferreira, A.; Tiel, K.; Cobas, K.; Rodríguez, R.; Borroto, C.; Doreste, V.; et al. A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors. Biotechnol. J. 2008, 3, 1088–1093. [Google Scholar] [CrossRef]
  118. Sun, S.; Kang, X.P.; Xing, X.J.; Xu, X.Y.; Cheng, J.; Zheng, S.W.; Xing, G.M. Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv. Hezuo 908) with improved efficiency. Biotechnol. Biotechnol. Equip. 2015, 29, 861–868. [Google Scholar] [CrossRef]
  119. Frary, A.; Earle, E.D. An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Rep. 1996, 16, 235–240. [Google Scholar] [CrossRef] [PubMed]
  120. Soniya, E.V.; Banerjee, N.S.; Das, M.R. Genetic analysis of somaclonal variation among callus-derived plants of tomato. Curr. Sci. 2001, 80, 1213–1215. [Google Scholar]
  121. Karami, O. Factors affecting Agrobacterium-mediated transformation of plants. Transgenic Plant 2008, 2, 127–137. [Google Scholar]
  122. Conti, G.; Xoconostle-Cázares, B.; Marcelino-Pérez, G.; Hopp, H.E.; Reyes, C.A. Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies. Front. Plant Sci. 2021, 12, 768197. [Google Scholar] [CrossRef] [PubMed]
  123. Khadgi, A.; Sagawa, C.H.D.; Vernon, C.; Mermaz, B.; Irish, V.F. Optimization of in Planta Methodology for Genome Editing and Transformation in Citrus. Front. Plant Sci. 2024, 15, 1438031. [Google Scholar] [CrossRef] [PubMed]
  124. Singh, S.; Tarannum, Z.; Kokane, S.; Ghosh, D.K.; Sharma, A.K.; Chauhan, H. Efficient Transformation and Regeneration of Transgenic Plants in Commercial Cultivars of Citrus Aurantifolia and Citrus Sinensis. Transgenic Res. 2023, 32, 523–536. [Google Scholar] [CrossRef]
  125. Bahariah, B.; Masani, M.Y.A.; Fizree, M.P.M.A.A.; Rasid, O.A.; Parveez, G.K.A. Multiplex CRISPR/Cas9 Gene-Editing Platform in Oil Palm Targeting Mutations in EgFAD2 and EgPAT Genes. J. Genet. Eng. Biotechnol. 2023, 21, 3. [Google Scholar] [CrossRef]
  126. Nie, H.; Batool, S.; Htwe, Y.M.; Fang, X.; Zhang, D.; Shi, P.; Li, Z.; Ma, M.; Su, H.; Yu, Q.; et al. Development of Protoplast-Based Gene Editing System for Areca Palm. Plants 2025, 14, 832. [Google Scholar] [CrossRef]
  127. Zhao, H.; Zhang, J.; Zhao, J.; Niu, S.; Zhao, H.; Zhang, J.; Zhao, J.; Niu, S. Genetic Transformation in Conifers: Current Status and Future Prospects. For. Res. 2024, 4, e010. [Google Scholar] [CrossRef]
  128. Bruegmann, T.; Fendel, A.; Zahn, V.; Fladung, M. Genome Editing in Forest Trees. In A Roadmap for Plant Genome Editing; Ricroch, A., Eriksson, D., Miladinović, D., Sweet, J., Van Laere, K., Woźniak-Gientka, E., Eds.; Springer Nature: Cham, Switzerland, 2024; pp. 347–372. ISBN 9783031461491. [Google Scholar]
  129. Kui, L.; Chen, H.; Zhang, W.; He, S.; Xiong, Z.; Zhang, Y.; Yan, L.; Zhong, C.; He, F.; Chen, J.; et al. Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium Officinale. Front. Plant Sci. 2017, 7, 2036. [Google Scholar] [CrossRef]
  130. Li, Y.; Yang, J.; Zhang, Q.; Zhang, K.; Xue, Q.; Liu, W.; Ding, X.; Niu, Z. CRISPR-Cas9 Mediated Gene Editing Platform Through Callus-to-Plant Regeneration and Functional Analysis of DoALA4─DoALA6 in Dendrobium Officinale. Plant Cell Environ. 2025, 48, 2923–2936. [Google Scholar] [CrossRef]
  131. Semiarti, E.; Nopitasari, S.; Setiawati, Y.; Lawrie, M.D.; Purwantoro, A.; Widada, J.; Yoshioka, Y.; Matsumoto, S.; Ninomiya, K.; Asano, Y. Application of CRISPR/Cas9 Genome Editing System for Molecular Breeding of Orchids. Indones. J. Biotechnol. 2020, 25, 61. [Google Scholar] [CrossRef]
  132. Ren, R.; Gao, J.; Yin, D.; Li, K.; Lu, C.; Ahmad, S.; Wei, Y.; Jin, J.; Zhu, G.; Yang, F. Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops. Front. Plant Sci. 2021, 12, 626015. [Google Scholar] [CrossRef]
  133. Guo, M.; Chen, H.; Dong, S.; Zhang, Z.; Luo, H. CRISPR-Cas Gene Editing Technology and Its Application Prospect in Medicinal Plants. Chin. Med. 2022, 17, 33. [Google Scholar] [CrossRef]
  134. Dey, A. CRISPR/Cas Genome Editing to Optimize Pharmacologically Active Plant Natural Products. Pharmacol. Res. 2021, 164, 105359. [Google Scholar] [CrossRef]
  135. Lee, K.; Wang, K. Strategies for Genotype-Flexible Plant Transformation. Curr. Opin. Biotechnol. 2023, 79, 102848. [Google Scholar] [CrossRef] [PubMed]
  136. DeMaggio, A.E. Morphogenetic Factors Influencing the Development of Fern Embryos. Linn. Soc. Lond. Proc. Bot. J 1963, 58, 361–376. [Google Scholar] [CrossRef]
  137. Johnson, K.; Cao Chu, U.; Anthony, G.; Wu, E.; Che, P.; Jones, T.J. Rapid and highly efficient morphogenic gene-mediated hexaploid wheat transformation. Front. Plant Sci. 2023, 14, 1151762. [Google Scholar] [CrossRef] [PubMed]
  138. Hoerster, G.; Wang, N.; Ryan, L.; Wu, E.; Anand, A.; McBride, K.; Lowe, K.; Jones, T.; Kamm, B.G. Use of non-integrating Zm-Wus2 vectors to enhance maize transformation. Vitr. Cell. Dev. Biol.-Plant 2020, 56, 265–279. [Google Scholar] [CrossRef]
  139. Che, P.; Wu, E.; Simon, M.K.; Anand, A.; Lowe, K.; Gao, H.; Sigmund, A.L.; Yang, M.; Albertsen, M.C.; Gordon-Kamm, W.; et al. Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum. Commun. Biol. 2022, 5, 344. [Google Scholar] [CrossRef] [PubMed] [PubMed Central]
  140. Stroud, H.; Ding, B.; Simon, S.A.; Feng, S.; Bellizzi, M.; Pellegrini, M.; Wang, G.L.; Meyers, B.C.; Jacobsen, S.E. Plants regenerated from tissue culture contain stable epigenome changes in rice. Elife 2013, 2, e00354. [Google Scholar] [CrossRef]
  141. Hsieh, J.-W.A.; Chang, P.; Kuang, L.-Y.; Hsing, Y.-I.C.; Chen, P.-Y. Rice transformation treatments leave specific epigenome changes beyond tissue culture. Plant Physiol. 2023, 193, 2–1297, Erratum in Plant Physiol. 2023, 193, 1713. [Google Scholar] [CrossRef]
  142. Ong-Abdullah, M.; Ordway, J.M.; Jiang, N.; Ooi, S.E.; Kok, S.Y.; Sarpan, N.; Azimi, N.; Hashim, A.T.; Ishak, Z.; Rosli, S.K.; et al. Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm. Nature 2015, 525, 533–537. [Google Scholar] [CrossRef]
  143. Zhong, H.; Li, C.; Yu, W.; Zhou, H.P.; Lieber, T.; Su, X.; Wang, W.; Bumann, E.; Lunny Castro, R.M.; Jiang, Y.; et al. A fast and genotype-independent in planta Agrobacterium-mediated transformation method for soybean. Plant Commun. 2024, 5, 101063. [Google Scholar] [CrossRef]
  144. Karp, H.; Zoltek, M.; Wasko, K.; Vazquez, A.L.; Brim, J.; Ngo, W.; Schepartz, A.; Doudna, J. Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing. bioRxiv 2024. [Google Scholar] [CrossRef] [PubMed]
  145. Hamada, H.; Linghu, Q.; Nagira, Y.; Miki, R.; Taoka, N.; Imai, R. An in planta biolistic method for stable wheat transformation. Sci. Rep. 2017, 7, 11443, Erratum in Sci. Rep. 2017, 7, 17573. [Google Scholar] [CrossRef] [PubMed]
  146. Ali, Z.; Abul-faraj, A.; Li, L.; Ghosh, N.; Piatek, M.; Mahjoub, A.; Aouida, M.; Piatek, A.; Baltes, N.J.; Voytas, D.F.; et al. Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System. Mol. Plant 2015, 8, 1288–1291. [Google Scholar] [CrossRef] [PubMed]
  147. Chen, H.; Su, Z.; Tian, B.; Liu, Y.; Pang, Y.; Kavetskyi, V.; Trick, H.N.; Bai, G. Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat. Plant Biotechnol. J. 2022, 20, 1018–1020. [Google Scholar] [CrossRef]
  148. Wang, W.; Yu, Z.; He, F.; Bai, G.; Trick, H.N.; Akhunova, A.; Akhunov, E. Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system. Plant Biotechnol. J. 2022, 20, 2332–2341. [Google Scholar] [CrossRef]
  149. Liu, D.; Ellison, E.E.; Myers, E.A.; Donahue, L.I.; Xuan, S.; Swanson, R.; Qi, S.; Prichard, L.E.; Starker, C.G.; Voytas, D.F. Heritable gene editing in tomato through viral delivery of isopentenyl transferase and single-guide RNAs to latent axillary meristematic cells. Proc. Natl. Acad. Sci. USA 2024, 121, e2406486121. [Google Scholar] [CrossRef]
  150. Sprink, T.; Wilhelm, R.; Hartung, F. Genome editing around the globe: An update on policies and perceptions. Plant Physiol. 2022, 190, 1579–1587. [Google Scholar] [CrossRef]
  151. Buchholzer, M.; Frommer, W.B. An increasing number of countries regulate genome editing in crops. New Phytol. 2023, 237, 12–15. [Google Scholar] [CrossRef] [PubMed]
  152. Ahmad, A.; Munawar, N.; Khan, Z.; Qusmani, A.; Khan, S.; Jamil, A.; Ashraf, S.; Ghouri, M.; Aslam, S.; Mubarik, M.; et al. An Outlook on Global Regulatory Landscape for Genome-Edited Crops. Int. J. Mol. Sci. 2021, 22, 11753. [Google Scholar] [CrossRef]
  153. Voigt, B. EU regulation of gene-edited plants—A reform proposal. Front. Genome Ed. 2023, 5, 1119442. [Google Scholar] [CrossRef]
  154. Zagoskina, N.Z.; Zubova, M.Y.; Nechaeva, T.L.; Kazantseva, V.V.; Goncharuk, E.A.; Katanskaya, V.M.; Baranova, E.N.; Aksenova, M.A. Polyphenols in Plants: Structure, Biosynthesis, Abiotic Stress Regulation, and Practical Applications (Review). Int. J. Mol. Sci. 2023, 24, 13874. [Google Scholar] [CrossRef]
  155. Steinberger, A.R.; Voytas, D.F. Virus-induced gene editing free from tissue culture. Nat. Plants 2025, 11, 1241–1251. [Google Scholar] [CrossRef]
Table 1. Functional diversity of major morphogenetic transcription factor groups and their applications in in vitro regeneration of plants.
Table 1. Functional diversity of major morphogenetic transcription factor groups and their applications in in vitro regeneration of plants.
Group Name of Morphogenetic FactorsGenesFunctions in PlantsObserved
Impact In Vitro		References
WUS (WUSCHEL)WUSMaintenance of cell populations in shoot and floral meristems; regulation of meristem differentiationOverexpression enhances shoot regeneration and meristem growth[25,26,27,28,29,30,31,64,65,66,67,68,69,70]
WOX (WUSCHEL-related homebox) WOX1, WOX2, WOX3, WOX4, WOX5, WOX11Regulation of cell differentiation
(WOX5 maintains root meristems; WOX11 controls lateral root branching)		Stimulation organo- and embryogenesis; WOX11 improves in vitro rhizogenesis efficiency[32,33,34,35,36,37,38,71,72]
BBM (BABY BOOM) BBMControl of embryogenesis; regulation of cell proliferation and embryo developmentSomatic embryogenesis induction without exogenous hormones[39,40,41,42,68,73]
GRF-GIF (GROWTH-REGULATING FACTOR–GIF) GRF1–GRF9, GIF1–GIF3Management of shoot and leaf growth; cell proliferationImprovement of regeneration efficiency by promoting prolific shoot formation[20,44,45,46]
PLT (PLETHORA) PLT1, PLT2, PLT3, PLT5Maintenance of root meristems; regulation of embryo development; control of auxin signalingRoot regeneration; overexpression enhances somatic embryogenesis[43,74]
LEC (LEAFY COTYLEDON) LEC1, LEC2, FUS3Control of embryo maturation and seed germination; regulation of nutrient storageSomatic embryogenesis induction and embryoid yield increasing[15,47,48,49]
SERK (SOMATIC EMBRYOGENESIS RECEPTOR KINASE) SERK1, SERK2Enhancement of somatic embryogenesis; co-factor in signaling pathwaysEnhancement of embryogenic competence; and regeneration efficiency[25,75,76]
ESR (Embryo Surrounding Region) ESR1Processes of endosperm formation and seed coat developmentShoot formation efficiency increasing[50,51,52]
WIND (WOUND INDUCED DEDIFFERENTIATION) WIND1Wound-induced promotion dedifferentiation; regulation of callus formation Cell division induction and proliferation without phytohormones, improving transformation efficiency[53,54,55,76,77]
RKD (RWP-RK DOMAIN-CONTAINING) RKD1, RKD2, RKD4Regulation of egg cell differentiation and maintenance of embryogenic totipotencySomatic embryogenesis induction and callusogenesis[56,57,58,59]
DOF (DNA-binding One Zinc Finger)DOF3.4, DOF5.6Development of shoot and root apical meristemsCallus induction[60]
ipt (isopentenyltransferase) iptOncogeneEnhancement of shoot formation efficiency[55,61,62,78,79,80]
rol (root loci) rolB, rolCAlteration of plant cell hormonal balanceEfficient root or shoot initiation[13,81,82]
REF1 (REGENERATION FACTOR1)-Initiation of callusogenesis via WIND1 activation in response to woundingRegeneration efficiency improving via callusogenesis[63]
Table 2. Applications of morphogenetic transcription factors in regeneration of different crops and model plants organisms.
Table 2. Applications of morphogenetic transcription factors in regeneration of different crops and model plants organisms.
CropCultivarGenesObserved Impact In VitroReference
O. sativassp. indica IRV95OsBBM + ZmWUS243% regeneration efficiency[15]
ZmBBM + ZmWUS227% regeneration efficiency[15]
KitaakeGRF4-GIF1Enhancement of regeneration efficiency 2-fold (from 20 to 42.8%)[44]
-WOX11Enabling root system modifications without affecting shoot traits[72]
Cempo IrengOsRKD3Enhancement of transformation 23.5-fold, enhancement of somatic embryogenesis by activating a gene network (AP2/ERF, MYB, COL)[59]
B. napusTopas DH 4079BnBBMDevelopment of somatic embryos in the absence of exogenous phytohormones[39]
BNS3AtGRF5Increasing shoot regeneration to 19.6% (vs. 3% in controls) without developmental defects[20]
G. maxDN50, DN252, DN254, SN4, SN14, ZJ602GmGRF3-GIF1Enhancement of regeneration up to 5.5–13.8% (vs. 2–5% in controls)[45]
CD215, JackAtGRF5Enhancement of shoot regeneration up to 50%[20]
Dongnong-50REF1Enhancement of transformation and regeneration efficiency 5- and 9-fold respectively[63]
S. lycopersicumBig BeefPLT5Enhancement of transformation efficiency up to 13.3%[43]
WUSEnhancement of transformation efficiency up to 3.3%[43]
Alisa Craig, CastlemartREF1Enhancement of regeneration efficiency 3-fold[63]
Citrus × sinensis (L.) OsbeckValenciaL1L (LEC1)Somatic embryogenesis induction in one month after Agrobacterium-mediated transformation [83]
‘Pineapple’, ‘Hamlin’, ‘Sucarri’, ‘Valencia’ZmKN1Enhancement of transformation efficiency by 3–15 times[84]
Citrus reticulata BlancoRed, BendizaoL1L (LEC1)Somatic embryogenesis induction in one month after Agrobacterium-mediated transformation [83]
Citrus limon (L.) OsbeckEurekaZmKN1Enhancement of transformation efficiency by 3–15 times[84]
Citrus medica L.CarrizoGRF-GIFEnhancement of regeneration efficiency 4.7-fold[44]
Malus domestica L.Royal GalaMdBBM1Increasing the frequency of transgenic shoot regeneration to ~31%[85]
Vitis vinifera L.Cabernet SauvignonVvBBMOverexpression enhances somatic embryogenesis to 42.85%[86]
-GRF-GIFShoot formation efficiency increasing by more than 4-fold[44]
Fragaria vesca L.Hawaii 4VvGRF4-GRF1Enhancement of transformation efficiency by more than 2-fold, reaching 40%[87]
Theobroma cacao L.6–1, ICS1TcBBMEnhancement of somatic embryogenesis induction 5.5-fold without exogenous hormones[42]
Phoenix dactylifera L.Deglet NourPdSERK1Studying SERK1 expression which is highly expressed during embryogenic competence acquisition and globular embryo formation in culture[88]
Cocos nucifera L.MI-192-17WUS, BBM, SERK, LECInvestigation of MTF expression during explant treatment with 5-Azacytidine[89]
Sorghum bicolor (L.) MoenchTX430ZmBBM + ZmWUS2Somatic embryogenesis induction and enhancement of transformation efficiency from 2 to 18%[15]
Saccharum officinarum L.CP01-1372ZmBBM + ZmWUS2Somatic embryogenesis induction and enhancement of transformation efficiency from 2 to 273%[15]
Panicum virgatum L.Summer, BlackwellZmBBM + ZmWUS2Enhancement of transformation efficiency to 6% and regeneration efficiency to over 40%[90]
Manihot esculenta Crantz60444, NASEVvGRF4-GRF1, AtGRF5Enhancement of transformation and regeneration efficiency to 50% without exogenous hormones[91]
Cucumis sativus L.Cu2AtGRF5Improvement of regeneration efficiency by promoting prolific shoot formation[92]
Citrullus lanatus (Thunb.) Matsum. & NakaiTCClGRF4-GIF1Improvement of regeneration efficiency by promoting prolific shoot formation by 47.02%[93]
Beta vulgaris L.9BS0448, 1RV6183, 7RV5706H, 8RV6921AtGRF5Enhancement of transformation efficiency 6-fold and regeneration efficiency to 20.7%[20]
E. urophylla × E. grandis‘DH32-29′WUSEnhancement of transformation efficiency 3-fold, embryogenic callus and somatic embryogenesis induction 40%[94]
Populus L.-BBM
WUS		Embryogenic callus and somatic embryogenesis induction 39%[95]
-PtWUS + PtWOX11Enhancement of callus induction, shoot regeneration and rhizogenesis efficiency, increase in leaf area to 25%[96]
84KWUSEnhancement of rhizogenesis efficiency 1.5–2-fold[97]
Picea abies (L.) H. Karst88.22, 61.21PaHAP3ASomatic embryogenesis induction in maturated embryo 5.5%[98]
Physcomitrium patens (Hedw.) Bruch & Schimp.-LSH1Studies on the maintenance of meristematic cell activity[99]
Table 3. Strategies for controlled expression of morphogens in plants.
Table 3. Strategies for controlled expression of morphogens in plants.
Type of StrategyPrinciple of FunctionReference
Transient Expression (Non-Integrated)Short-term expression without genomic integration, non-integrated cassettes due to
T-DNA border termination		[15]
Chemically Inducible PromotersThe morphogen is expressed under a chemically inducible promoter only in the presence of a specific substance in the medium (e.g., dexamethasone-, tetracycline-, or estrogen-responsive promoters)[13,39,40]
Tissue-Specific PromotersThe morphogen is expressed under a special promoter only in target cells and tissues (calli or embryoids)[15]
Gene Excision SystemsPost-regeneration removal of morphogenes via recombinase-based systems (Cre/LoxP, FLP/FRT)[76,77,78]
Combining Multiple Morphogenetic FactorsCo-expression of synergistic regulators via separate promoters or self-cleaving peptides for balanced expression[15,16,43,87,90,91,95,96]
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Bakulin, S.D.; Monakhos, S.G.; Bruskin, S.A. Morphogenetic Factors as a Tool for Enhancing Plant Regeneration Capacity During In Vitro Transformation. Int. J. Mol. Sci. 2025, 26, 8583. https://doi.org/10.3390/ijms26178583

AMA Style

Bakulin SD, Monakhos SG, Bruskin SA. Morphogenetic Factors as a Tool for Enhancing Plant Regeneration Capacity During In Vitro Transformation. International Journal of Molecular Sciences. 2025; 26(17):8583. https://doi.org/10.3390/ijms26178583

Chicago/Turabian Style

Bakulin, Semyon D., Sokrat G. Monakhos, and Sergey A. Bruskin. 2025. "Morphogenetic Factors as a Tool for Enhancing Plant Regeneration Capacity During In Vitro Transformation" International Journal of Molecular Sciences 26, no. 17: 8583. https://doi.org/10.3390/ijms26178583

APA Style

Bakulin, S. D., Monakhos, S. G., & Bruskin, S. A. (2025). Morphogenetic Factors as a Tool for Enhancing Plant Regeneration Capacity During In Vitro Transformation. International Journal of Molecular Sciences, 26(17), 8583. https://doi.org/10.3390/ijms26178583

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