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Genetic transformation in conifers: current status and future prospects

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  • Received: 11 December 2023
    Revised: 30 January 2024
    Accepted: 28 February 2024
    Published online: 21 March 2024
    Forestry Research  4 Article number: e010 (2024)  |  Cite this article
  • Genetic transformation has been a cornerstone in plant molecular biology research and molecular design breeding, facilitating innovative approaches for the genetic improvement of trees with long breeding cycles. Despite the profound ecological and economic significance of conifers in global forestry, the application of genetic transformation in this group has been fraught with challenges. Nevertheless, genetic transformation has achieved notable advances in certain conifer species, while these advances are confined to specific genotypes, they offer valuable insights for technological breakthroughs in other species. This review offers an in-depth examination of the progress achieved in the genetic transformation of conifers. This discussion encompasses various factors, including expression vector construction, gene-delivery methods, and regeneration systems. Additionally, the hurdles encountered in the pursuit of a universal model for conifer transformation are discussed, along with the proposal of potential strategies for future developments. This comprehensive overview seeks to stimulate further research and innovation in this crucial field of forest biotechnology.
  • The Lonicera Linn. genus is a constituent member of the Caprifoliaceae family[1]. It is the largest genus in this family and comprises at least 200 species with a notable presence in North Africa, North America, Asia, and Europe[1]. Members of the Lonicera genus possess a wide range of economic benefits from their use as ornamental plants to food and as plants credited with numerous health benefits. Conspicuous among the numerous members of this genus with known medicinal uses are L. japonica, L. macranthoides, L. hypoglauca, L. confusa, and L. fulvotomentosa[2]. Though these species feature prominently in the Chinese Pharmacopoeia, other species such as L. acuminata, L. buchananii, and L. similis are recognized as medicinal resources in certain parts of China[1]. Among the aforementioned species, L. japonica takes precedence over the rest due to its high medicinal and nutritional value[3,4]. For instance, the microRNA MIR2911, an isolate from L. japonica, has been reported to inhibit the replication of viruses[57]. Also, the water extract of L. japonica has been used to produce various beverages and health products[8]. The Lonicera genus therefore possesses huge prospects in the pharmaceutical, food, and cosmetic industries as an invaluable raw material[9].

    The main active constituents of the Lonicera genus include organic acids, flavonoids, iridoids, and triterpene saponins. Chlorogenic acids, iridoids, and flavones are mainly credited with the anti-inflammatory, antiviral, anticancer, and antioxidant effects of the various Lonicera species[1013]. Their hepatoprotective, immune modulatory, anti-tumor and anti-Alzheimer’s effects are for the most part ascribed to the triterpene saponins[1416]. As stated in the Chinese Pharmacopoeia and backed by the findings of diverse research groups, the plants of the Lonicera genus are known to possess high amounts of organic acids (specifically chlorogenic acid) and pentacyclic triterpenoid saponins[2,1719]. The flower and flower bud have traditionally served as the main medicinal parts of the Lonicera genus even though there is ample evidence that the leaves possess the same chemical composition[20]. A perusal of the current scientific literature reveals the fact that little attention has been devoted to exploring the biosynthesis of the chemical constituents of the Lonicera genus with the view to finding alternative means of obtaining higher yields. It is therefore imperative that priority is given to the exploration of the biosynthesis of these bioactive compounds as a possible means of resource protection. There is also the need for further research on ways to fully tap the medicinal benefits of other plant parts in the Lonicera genus.

    Here, we provide a comprehensive review of relevant scientific literature covering the structure, pharmacology, multi-omics analyses, phylogenetic analysis, biosynthesis, and metabolic engineering of the main bioactive constituents of the Lonicera genus. Finally, we proffer suggestions on the prospects of fully exploiting and utilizing plants of the Lonicera genus as useful medicinal plant resources.

    A total of at least 400 secondary metabolites have been reported for the Lonicera genus. These metabolites are categorized into four main groups (Fig. 1a), including not less than 50 organic acids, 80 flavonoids, 80 iridoids, and 80 triterpene saponins[2123]. Organic acids are mainly derivatives of p-hydroxycinnamic acid and quinic acid. Among the organic acids, chlorogenic acids are reported to be the main bioactive compounds in L. japonica[2426]. The organic acids are most abundant in the leaves, while the least amounts are found in the stem of L. japonica. The flowers of the plant are known to contain moderately high amounts of organic acids[27]. The basic core structure of the flavonoids is 2-phenylchromogen. Luteolin and its glycoside which are characteristic flavonoids of the Lonicera genus are most abundant in L. japonica[28]. On the whole, the flavonoid contents in L. japonica are also highest in the leaves, available in moderate amounts in the flowers, and in lowest amounts in the stem[21]. The core structures of the iridoids are iridoid alcohols, the chemical properties of which are similar to hemiacetal. The iridoids often exist in the form of iridoid glycosides in plants. Secoiridoids glycosides are predominant in the Lonicera genus[25]. In L. japonica, the contents of the iridoids are most abundant in the flowers, moderate in leaves, and lowest in the stem[21]. The characteristic saponins of the Lonicera genus are mainly pentacyclic triterpenoids, including the hederin-, oleanane-, lupane-, ursulane- and fernane-types, etc[22]. The hederin-type saponins are reported in the highest amounts in L. macranthoides[17] (Fig. 1b).

    Figure 1.  Core structures of main secondary metabolites and their distribution in five species of Lonicera. (a) 1 and 2, the main core structures of organic acids; 3, the main core structures of flavonoids; 4, the main core structures of iridoids; 5, the main core structures of triterpene saponins. (b) Comparison of dry weight of four kinds of substances in five species of Lonicera[17,28].

    The similarities between chlorogenic acid (CGA) and flavonoids can be traced back to their biosynthesis since p-coumaroyl CoA serves as the common precursor for these compounds[29]. p-coumaroyl CoA is obtained through sequential catalysis of phenylalanine and its biosynthetic intermediates by phenylalanine-ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA ligase (4CL)[3033].

    CGA is a phenolic acid composed of caffeic acid and quinic acid and is the most important bioactive compound among the organic acids. Its biosynthesis has been relatively well-established; three main biosynthetic routes have been propounded (Fig. 2a). One route relates to the catalysis of caffeoyl-CoA and quinic acid by hydroxycinnamoyl-CoA quinate transferase (HQT)/hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) to produce CGA[3437]. The HQT-mediated pathway has been deemed the major route for CGA synthesis in in different plant species[38,39]. The second biosynthetic route stems from the biosynthesis of p-coumaroyl quinate through the catalytic effect of HCT/HQT and subsequent hydroxylation of p-coumaroyl quinate under the catalysis of p-coumarate 3'-hydroxylase (C3’H)[34,36,37]. For the third route, caffeoyl glucoside serves as the intermediate to form CGA, a process that is catalyzed by hydroxycinnamyl D-glucose: quinic acid hydroxycinnamyl transferase (HCGQT)[40,41].

    Figure 2.  Biosynthetic pathways of main bioactive constituents of Lonicera. (a) Biosynthetic pathways of chlorogenic acid. (b) Biosynthetic pathways of luteoloside. (c) Biosynthetic pathways of secologanin. (d) Biosynthetic pathways of hederin-type triterpene saponins. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; C3’H, p-coumaroyl 3-hydroxylase; HQT, hydroxycinnamoyl-CoA quinate transferase; UGCT, UDP glucose: cinnamate glucosyl transferase; CGH, p-coumaroyl-D-glucose hydroxylase; HCGQT, hydroxycinnamoyl D-glucose: quinate hydroxycinnamoyl transferase; CHS, Chalcone synthase; CHI, Chalcone isomerase; FNS, Flavone synthase; F3H, flavonoid 30-monooxygenase/flavonoid 30-hydroxylase; UF7GT, flavone 7-O-β-glucosyltransferase; GPS, Geranyl pyrophosphatase; GES, geraniol synthase; G8O, geraniol 10-hydroxylase/8-oxidase; 8HO, 8-hydroxygeraniol oxidoreductase; IS, iridoid synthase; IO, iridoid oxidase; 7DLGT, 7-deoxyloganetic acid glucosyltransferase; 7DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferase; SLS, secologanin synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; β-AS, β-amyrin synthase; OAS, oleanolic acid synthetase.

    The key enzymes in the biosynthesis of p-coumaroyl CoA, and invariably CGA, thus, PAL, C4H, and 4CL have been established in diverse studies such as enzyme gene overexpression/knockdown[42], enzyme activity analysis[33] and transcriptomics[18]. However, the centrality of HQT in the biosynthesis of CGA remains disputable. While some studies have reported a strong correlation between HQT expression level with CGA content and distribution[18,35,39,43,44], others found no such link[45], bringing into question the role of HQT as a key enzyme in CGA biosynthesis.

    Few studies have been conducted on the regulation of CGA biosynthesis in the Lonicera genus. It was found that overexpression of the transcription factor, LmMYB15 in Nicotiana benthamiana can promote CGA accumulation by directly activating 4CL or indirectly binding to MYB3 and MYB4 promoters[46]. LjbZIP8 can specifically bind to PAL2 and act as a transcriptional repressor to reduce PAL2 expression levels and CGA content[47]. Under NaCl stress, increased PAL expression promoted the accumulation of phenolic substances in leaves without oxidative damage, a condition that was conducive to the accumulation of the bioactive compounds in leaves[48].

    Luteolin and its derivative luteolin 7-O- glucoside (luteoloside) are representative flavonoids of the Lonicera genus. Similar to CGA, luteolin is biosynthesized from p-coumaroyl CoA but via a different route. The transition from p-coumaroyl CoA to luteolin is underpinned by sequential catalysis by chalcone synthetase (CHS), chalcone isomerase (CHI), flavonoid synthetase (FNS), and flavonoid 3'-monooxygenase/flavonoid 3'-hydroxylase (F3'H)[45,49,50] (Fig. 2b). Luteoloside is synthesized from luteolin by UDP glucose-flavonoid 7-O-β-glucosyltransferase (UF7GT)[51]. Similar to CGA biosynthesis, the key enzymes of luteolin synthesis include PAL, C4H, and 4CL in addition to FNS[33,45,52]. The content of luteoloside was found to be highly abundant in senescing leaves relative to other tissues such as stem, flowers, and even young leaves[52]. Through transcriptomic analysis, luteoloside biosynthesis-related differentially expressed unigenes (DEGs) such as PAL2, C4H2, flavone 7-O-β-glucosyltransferase (UFGT), 4CL, C4H, chalcone synthase 2 and flavonoid 3'-monooxygenase (F3'H) genes were found to be upregulated in the senescing leaves. Biosynthesis-related transcription factors such as MYB, bHLH, and WD40 were also differentially expressed during leaf senescence[52], while bHLH, ERF, MYB, bZIP, and NAC were differentially expressed during flower growth[53]. Further analysis of the transcription factors revealed that MYB12, MYB44, MYB75, MYB114, MYC12, bHLH113, and TTG1 are crucial in luteoloside biosynthesis[52,53].

    The biosynthesis of terpenoids mainly involves three stages; formation of intermediates, formation of basic structural skeleton, and modification of basic skeleton[54]. The intermediates of terpenoids are mainly formed through the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, and eventually converted to the universal isoprenoid precursors, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) through a series of enzyme-catalyzed reactions. Under the catalysis of geranyl pyrophosphatase (GPS), IPP is then converted to geranyl pyrophosphate (GPP). Different terpenoids are subsequently derived from GPP as the intermediate product. For instance, in the formation of secoiridoid, GPP first removes the phosphoric acid group to obtain geraniol, second through a series of reactions such as oxidation and cyclization, the skeleton of iridoid, namely iridodial, can be obtained. Finally, through a series of reactions, the basic carbon skeleton of the secoiridoid, namely secologanin, is obtained[5561] (Fig. 2c). In the formation of triterpene saponins, the key step lies in the formation of the precursor, 2,3-oxidosqualene, a reaction that is catalyzed by squalene epoxidase (SE). There are many pentacyclic triterpenes in the Lonicera genus, the most important type being the hederin-type saponins with hederagenin as aglycones. Hederin-type saponins are produced after the synthesis of oleanolic acid from β-amyrin and catalyzed by β-starch synthetase (β-AS) and Oleanolic acid synthase (OAS)[62,63]. The skeletal modification of the triterpenoid saponins is mainly achieved via the activities of the CYP450 enzymes and UDP-glycosyltransferase (UGT). Hence, the corresponding aglycones are first obtained via oxidation by the CYP450 enzymes (e.g., CYP72A), and further subjected to glycosylation by the appropriate UGT enzyme[6365] (Fig. 2d). Skeletal formations of the iridoids and triterpene saponins in general have been well researched, but the same cannot be said about the enzymes involved in biosynthesis of these groups of compounds in the Lonicera genus. To fully utilize the iridoids and triterpene saponins in the Lonicera genus, it is necessary to further explore their biosyntheses with the view to enhancing and optimizing the process.

    Given the importance of the bioactive compounds in the Lonicera genus, continual isolation of these compounds using the traditional methods are not only tedious and time-consuming, but also unsustainable. With the development and application of microbial metabolic engineering, different strategies have been introduced to produce these bioactive compounds by heterologous synthesis (Table 1).

    Table 1.  Biosynthesis of Lonicera-specialized metabolites using metabolic engineering.
    Engineering bacteriaOperational methodsProductsYieldRefs
    S. cerevisiaeEliminate the tyrosine-induced feedback inhibition, delete genes involved in competing pathways and overexpress rate-limiting enzymesCaffeic acid569.0 mg/L[69]
    S. cerevisiaeEmploye a heterologous tyrosine ammonia lyase and a 4HPA3H complex composed of HpaB and HpaC derived from different speciesCaffeic acid289.4 mg/L[73]
    S. cerevisiaeSupply and recycle of three cofactors: FADH2, S-adenosyl-L-methion, NADPHCaffeic acid
    Ferulic acid
    Caffeic acid: 5.5 g/L;
    Ferulic acid: 3.8 g/L
    [117]
    E. coliKnocking out competing pathwaysCaffeic acid7,922 mg/L[118]
    E. coliArtificial microbial community, a polyculture of three recombinant Escherichia coli strainsChlorogenic acid250 μM[68]
    Cell-free biosynthesisExtract and purify spy-cyclized enzymes (CFBS-mixture)Chlorogenic acid711.26 mg/L[70]
    S. cerevisiaeThree metabolic engineering modules were systematically optimized: shikimate pathway and carbon distribution, branch pathways, CGA pathway genesChlorogenic acidFlask fermentation: 234.8 mg/L;
    Fed-batch fermentation:
    806.8 mg/L
    [119]
    E. coliUsing modular coculture engineering: construction of the defective strain improves the production and utilization of precursor substancesChlorogenic acid131.31 mg/L[122]
    E. coliIntroduce heterologous UDP-glucose biosynthetic genesLuteolin34 mg/L[120]
    Y. lipolyticaOverexpression of the key genes involved in the mevalonate pathway, the gene encoding cytochrome P450 (CYP716A12) to that encoding NADPH-P450 reductaseOleanolic acid129.9 mg/L[85]
    S. cerevisiaeImprove the pairing efficiency between Cytochrome P450 monooxygenase and reductase and the expression level of key genesOleanolic acid606.9 mg/L[121]
    S. cerevisiaeHeterologous expression and optimization of CrAS, CrAO, and AtCPR1, and regulation of ERG1 and NADPH regeneration systemOleanolic acid433.9 mg/L[123]
     | Show Table
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    Due to the demand for CGA in the food, pharmaceutical, chemical, and cosmetic industries, the traditional means of obtaining the same requires a relatively longer period for plant maturation to obtain low yields of the desired product. This therefore brings into question the sustainability and efficiency of this approach. The alternative and sustainable approach has been to produce CGA using synthetic biology and metabolic engineering.

    Current research has sought to utilize Escherichia coli (and its mutant strain) and Saccharomyces cerevisiae to synthetically generate CGA and other flavonoids[6673]. For instance, Cha et al. employed two strains of E. coli to produce a relatively good yield of CGA (78 mg/L). Their approach was based on the ability of one strain to generate caffeic acid from glucose and the other strain to use the caffeic acid produced and quinic acid as starting materials to synthesize CGA[66]. Using a bioengineered mutant of E. coli (aroD mutant), Kim et al. increased the yield of CGA to as high as 450 mg/L[67]. Others have sought to increase the yield of CGA by employing a polyculture of three E. coli strains that act as specific modules for the de novo biosynthesis of caffeic acid, quinic acid and CGA. This strategy eliminates the competition posed by the precursor of CGA (i.e., caffeic acid and quinic acid) and generally results in improved production of CGA[68]. Saccharomyces cerevisiae is a chassis widely used for the production of natural substances from plants with an intimal structure that can be used for the expression of cytochrome P450 enzymes that cannot be expressed in E. coli. Researchers have used yeast to increase the production of organic acids[69]. A de novo biosynthetic pathway for the construction of CGA in yeast has been reported new cell-free biosynthetic system based on a mixture of chassis cell extracts and purified Spy cyclized enzymes were adopted by Niu et al. to a produce the highest yield of CGA reported so far up to 711.26 ± 15.63 mg/L[70].

    There are many studies on the metabolic engineering for the synthesis of flavonoids, but few on luteolin and its glycosides. Strains of E. coli have been engineered with specific uridine diphosphate (UDP)-dependent glycosyltransferase (UGT) to synthesize three novel flavonoid glycosides. These glycosides were quercetin 3-O-(N-acetyl) quinovosamine (158.3 mg/L), luteolin 7-O-(N-acetyl) glucosaminuronic acid (172.5 mg/L) and quercetin 3-O-(N-acetyl)-xylosamine (160.8 mg/L)[71]. Since most of the flavonoid glycosides synthesized in E. coli are glucosylated, Kim et al. in their bid to synthesize luteolin-7-O-glucuronide, deleted the araA gene that encodes UDP-4-deoxy-4-formamido-L-arabinose formyltransferase/UDP-glucuronic acid C-4'' decarboxylase in E.coli and were able to obtain a yield of 300 mg/L of the desired product[72].

    Terpenoidal saponins are mostly derived from slow-growing plants and usually possess multiple chiral centers[74]. Traditional isolation and even chemical synthesis of the terpenoidal saponins are both tedious and uneconomical for large-scale production. Therefore, it is necessary to find other ways to synthesize these compounds known to have diverse pharmacological functions.

    Heterologous synthesis has become an important way to improve the target products. With the development of synthetic biology, heterologous synthesis of triterpene saponins involves chassis of both plant and microbial origin. In this regard, Nicotiana benthamiana is a model plant species for the reconstruction of the biosynthetic pathways of different bioactive compounds including monoterpenes, hemiterpenes, and diterpenes[59,7577]. Aside from Nicotiana benthamiana, other plants have also been used as heterologous hosts[78]. Heterologous synthesis using microbial hosts mainly involves Saccharomyces cerevisiae and Escherichia coli[7981], and other microorganisms[82,83]. Comparatively, plants as biosynthetic hosts have the advantages of an established photosynthetic system, abundant supply of relevant enzymes, and presence of cell compartments, etc. They are however not as fast growing as the microorganisms, and it is also difficult to extract and separate the desired synthesized compounds from them as hosts.

    Although heterologous synthesis has many advantages, the premise of successful construction of synthetic pathway in host is to elucidate the unique structure of the compound and the key enzyme reaction mechanism in the biosynthetic pathway. There is little research on metabolic engineering of the hederin-type pentacyclic triterpene saponins in Lonicera, but there are studies on the heterologous synthesis of its aglycone precursor, oleanolic acid[84,85]. There is a dearth of scientific literature on key enzymes in the biosynthesis of pentacyclic triterpenoid saponins in the Lonicera genus.

    Scientific evidence by diverse research groups has linked members of the Lonicera genus to a wide range of pharmacological effects (Fig. 3). These pharmacological effects are elicited by different chemical constituents, much of the underlying mechanisms of which have been elucidated by the omics techniques. Here, we summarize the pharmacological effects and pharmacodynamics of the Lonicera genus in the last 6 years.

    Figure 3.  Schematic summary of four main pharmacological effects (anti-inflammatory, antimicrobial, anti-oxidative and hepatoprotective effects) of the Lonicera genus and the underlying mechanisms of actions.

    Bioactive compounds of plants in the Lonicera genus have demonstrated varying degrees of anti-inflammatory actions. In a recent study, Lv et al. showed that lonicerin inhibits the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) through regulating EZH2/AtG5-mediated autophagy in bone marrow-derived macrophages of C57BL/6 mice[86]. The polysaccharide extract of L. japonica reduces atopic dermatitis in mice by promoting Nrf2 activation and NLRP3 degradation through p62[87]. Several products of Lonicera have been reported to have ameliorative effects on DSS-induced colitis. Among them, flavonoids of L. rupicola can improve the ulcerative colitis of C57BL/6 mice by inhibiting PI3K/AKT, and pomace of L. japonica can improve the ulcerative colitis of C57BL/6 mice by improving the intestinal barrier and intestinal flora[88,89]. The flavonoids can also ameliorate ulcerative colitis induced by local enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in Wistar rats by inhibiting NF-κB pathway[90]. Ethanol extract from L. Japonica has demonstrated the potential to inhibit the expressions of inflammatory cytokines in serum and macrophages of LPS-induced ICR mice[91]. The water extract of L. japonica and luteolin were found to exhibit their anti-inflammatory effects via the inhibition of the JAK/STAT1/3-dependent NF-κB pathway and induction of HO-1 expression in RAW263.7 cells induced by pseudorabies virus (PRV)[92].

    Existing scientific evidence indicates that the extracts of plants in the Lonicera genus exhibit strong inhibition against different pathogenic microorganisms. Phenolic compounds from L. japonica demonstrated a particularly significant inhibitory effect against Staphylococcus aureus and Escherichia coli, in vitro, making these compounds potential food preservatives[93]. Influenza A virus is a serious threat to human health. Recent research has found the ethanol extract of L. japonica to possess a strong inhibitory effect against H1N1 influenza virus-infected MDCK cells and ICR mice[94]. The incidence of the COVID-19 pandemic called to action various scientists in a bid to find safe and efficacious treatment[95]. Traditional Chinese medicines became an attractive alternative in this search. The water extract of the flower bud of L. japonica which has traditionally served as a good antipyretic and antitussive agent attracted the attention of researchers. Scientific evidences have confirmed that the water extract of L. japonica can induce let-7a expression in human rhabdomyosarcoma cells or neuronal cells and blood of lactating mice, inhibiting the entry and replication of the virus in vitro and in vivo[96]. In addition, the water extract of L. japonica also inhibits the fusion of human lung cancer cells Calu-3 expressing ACE2 receptor and BGK-21 cells transfected with SARS-CoV-2 spike protein, and up-regulates the expression of miR-148b and miR-146a[97].

    Oxidative stress has been implicated in the pathophysiology of many diseases, hence, amelioration of the same could be a good therapeutic approach[98,99]. In keeping with this therapeutic strategy, various compounds from the Lonicera genus have demonstrated the ability to relieve oxidative stress due to their pronounced antioxidant effects. For instance, the polyphenolic extract of L. caerulea berry was found to activate the expression of AMPK-PGC1α-NRF1-TFAM proteins in the skeletal muscle mitochondria, improve the activity of SOD, CAT and GSH-Px enzymes in blood and skeletal muscle, relieve exercise fatigue in mice by reducing oxidative stress in skeletal muscle, and enhance mitochondrial biosynthesis and cell proliferation[100]. The diverse health benefits of the anthocyanins from L. japonica have been mainly credited to their antioxidant and anti-inflammatory effects. The anthocyanin and cyanidin-3-o-glucoside have been reported to possess the potential to prolong life and delay senescence of Drosophila through the activation of the KEAP1/NRF2 signaling pathway[101].

    The liver is an essential organ that contributes to food digestion and detoxification of the body. These functions expose the liver to diverse toxins and metabolites. The Lonicera genus is rich in phytochemicals that confer protection on the liver against various toxins. The phenolic compound, 4, 5-di-O-Caffeoylquinic acid methyl ester was shown to be able to improve H2O2-induced liver oxidative damage in HepG2 cells by targeting the Keap1/Nrf2 pathway[102]. Hepatic fibrosis is a complex dynamic process, with the propensity to progress to liver cancer in severe cases. The L. japonicae flos water extract solution increased the cell viability of FL83B cells treated with thioacetamide (TAA), decreased the levels of serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP), inhibited the transformation growth factor β1 (TGF-β1) and liver collagen deposition[103]. Sweroside, a secoiridoid glucoside isolate of L. japonica is known to protect the C57BL/6 mice liver from hepatic fibrosis by up-regulating miR-29a and inhibiting COL1 and TIMP1[104].

    Aside from the aforementioned, other pharmacological effects have been ascribed to the Lonicera genus. The ethanolic extract of L. caerulea has been reported to inhibit the proliferation of SMMC-7721 and H22 hepatoma cells, while its anthocyanins induced the apoptosis of tumor cells via the release of cytochrome C and activation of caspase[105]. AMPK/PPARα axes play an important role in lipid metabolism. A chlorogenic acid-rich extract of L. Japonica was found to significantly decrease the early onset of high-fat diet-induced diabetes in Sprague-Dawley rats via the CTRPs-AdipoRs-AMPK/PPARα axes[106]. In a high-fat diet-induced non-alcoholic fatty liver disease in C57BL/6 mice, treatment with L. caerulea polyphenol extract decreased serum inflammatory factors and endotoxin levels and the Firmicutes/Bacteroidetes ratio, an indication of its modulatory effect on the gut microbiota[107]. The iridoid-anthocyanin extract from L. caerulea berry contributed to alleviating the symptoms of intestinal infection with spirochaeta in mice[108].

    The traditional classification of the Lonicera genus based on the morphology of member plants is further categorized into two subgenera, Chamaecerasus and Periclymenum. The Chamaecerasus includes four categories, Coeloxylosteum, Isika, Isoxylosteum and Nintooa. The Periclymenum includes two categories, Subsect. Lonicera and Subsect. Phenianthi (Supplemental Table S1).

    High-throughput chloroplast genome sequencing of L. japonica found its length to be 155078 bp, which is similar to the structure of the typical angiosperm chloroplast genome. It contains a pair of inverted repeat regions (IRa and IRb, 23774 bp), a large single copy region (LSC, 88858 bp) and a small single copy area (SSC, 18672 bp)[109,110]. However, compared with chloroplast genomes of other plants, the chloroplast genome of L. japonica has a unique rearrangement between trnI-CAU and trnN-GUU[110]. Based on the phylogenetic analysis of the plastid genomes of seven plants in the Lonicera genus, 16 diverging hot spots were identified as potential molecular markers for the development of the Lonicera plants[111]. The phylogeny of Lonicera is rarely researched at the molecular level and the pattern of repetitive variation and adaptive evolution of the genome sequence is still unknown. Chloroplast genome sequences are highly conserved, but insertions and deletions, inversions, substitutions, genome rearrangements, and translocations also occur and have become powerful tools for studying plant phylogeny[112,113].

    We present here the phylogenetic tree of the Lonicera genus based on the published complete chloroplast genome sequences downloaded from the National Center for Biotechnology Information (NCBI) database using the Maximum likelihood method (Fig. 4). Based on our chloroplast phylogenies, we propose to merge L. harae into Sect. Isika and L. insularis into Chamaecerasus, but whether L. insularis belongs to Sect. Isika or Sect. Coeloxylosteum is uncertain. Based on protein-coding regions (CDS) of the chloroplast genome or complete chloroplast genomes, Liu et al. and Chen et al. supported the classification of the two subgenera in Lonicera[111,114]. Sun et al. and Srivastav et al. demonstrated a classification between the two subgenera with more species by using sequences of nuclear loci generated, chloroplast genome, and restriction site-associated DNA sequencing (RADSeq)[115,116]. However, our phylogenetic analysis and that of Sun et al. show relations within the subgenus Chamaecerasus are tanglesome in some respects[116]. Plant traits are affected by the environment to varying degrees. Since evidence of plant speciation is implicit in its genome sequence, comparative analysis at the molecular level provides a relatively accurate depiction of inherent changes that might have occurred over time. These findings suggest the need for more species of the Lonicera genus to be sequenced to provide a more accurate theoretical basis for the evolution of the Lonicera plants and a more effective revision in the classification of the Lonicera genus.

    Figure 4.  Phylogenetic tree of 42 species of the Lonicera genus based on complete chloroplast genome sequence data. The phylogenetic tree was constructed by the maximum likelihood method. Coeloxylosteum, Isika, Isoxylosteum, and Nintooa belong to Chamaecerasus and Subsect. Lonicera belongs to the Periclymenum. Chamaecerasus and Periclymenum are the two subgenera of Lonicera. 'Not retrieved' indicates that the species failed to retrieve a subordinate taxon in the Lonicera.

    The Lonicera genus is rich in diverse bioactive compounds with immeasurable prospects in many fields. Members of this genus have been used for thousands of years in traditional Chinese medicine for heat-clearing and detoxification. These plants generally have a good taste and form part of the ingredients of various fruit juices. In cosmetics, they are known to possess anti-aging and moisturizing functions. Plants of the Lonicera genus are also known for their good ecological adaptability and can be used to improve soil and ecological environment. Based on the value of the Lonicera genus, besides researching their use through molecular biological means, their efficient utilization can also be promoted in the following ways: (1) The stems and leaves of the plants could be developed for consumption and use since the chemical profiles of these parts do not differ significantly from the flowers. This way, the wastage of this scarce resource could be minimized or avoided. (2) Most of the Lonicera plants are vines or shrubs and their natural regeneration speed is slow, so the introduction and domestication of species could be strengthened to avoid overexploitation of wild resources.

    At present, only the research on the biosynthesis and efficacy of chlorogenic acid is quite comprehensive and has been used widely in various fields. There is limited research on various aspects of other bioactive compounds and should therefore be given priority in future research goals. Currently, the multi-omics analytical approach has gradually evolved as a reliable and helpful analytical platform. Hence, multi-omics research on the Lonicera genus could lead to discoveries in drug discovery and human health.

    The authors confirm contribution to the paper as follows: study conception and design, draft manuscript preparation: Yin X, Chen X, Li W, Tran LSP, Lu X; manuscript revision: Yin X, Chen X, Li W, Tran LSP, Lu X, Chen X, Yin X, Alolga RN; data/literature collection: Chen X, Yin X; figure preparation: Chen X, Yin X; figure revision: Alolga RN, Yin X, Chen X, Li W, Tran LSP, Lu X. All authors reviewed the results and approved the final version of the manuscript.

    All data generated or analyzed during this study are included in this published article and its supplementary information file.

    This work was partially supported by the National Natural Science Foundation of China (NSFC, Nos 82173918 and 82373983).

  • The authors declare that they have no conflict of interest. Xiaojian Yin is the Editorial Board member of Medicinal Plant Biology who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

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  • Cite this article

    Zhao H, Zhang J, Zhao J, Niu S. 2024. Genetic transformation in conifers: current status and future prospects. Forestry Research 4: e010 doi: 10.48130/forres-0024-0007
    Zhao H, Zhang J, Zhao J, Niu S. 2024. Genetic transformation in conifers: current status and future prospects. Forestry Research 4: e010 doi: 10.48130/forres-0024-0007

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Genetic transformation in conifers: current status and future prospects

Forestry Research  4 Article number: e010  (2024)  |  Cite this article

Abstract: Genetic transformation has been a cornerstone in plant molecular biology research and molecular design breeding, facilitating innovative approaches for the genetic improvement of trees with long breeding cycles. Despite the profound ecological and economic significance of conifers in global forestry, the application of genetic transformation in this group has been fraught with challenges. Nevertheless, genetic transformation has achieved notable advances in certain conifer species, while these advances are confined to specific genotypes, they offer valuable insights for technological breakthroughs in other species. This review offers an in-depth examination of the progress achieved in the genetic transformation of conifers. This discussion encompasses various factors, including expression vector construction, gene-delivery methods, and regeneration systems. Additionally, the hurdles encountered in the pursuit of a universal model for conifer transformation are discussed, along with the proposal of potential strategies for future developments. This comprehensive overview seeks to stimulate further research and innovation in this crucial field of forest biotechnology.

    • Globally, conifers are pivotal sources of timber and pulpwood, thus holding immense economic and environmental value. The huge genome, high heterozygosity, prolonged vegetative growth period, and restricted genetic transformation system of conifers[15] limit the availability of genetic tools for investigating their developmental regulation, resulting in sluggish research progress. Studies identifying gene function in conifers have relied on heterologous expression in angiosperm model species. Since the initial report of transgenic Populus in 1987[6], significant strides have been made in achieving stable genetic transformation in various forest tree species. Subsequent to this, various genetic transformation systems for conifers have been reported. In 1991, Agrobacterium rhizogenes was employed to infect aseptic seedlings of European larch (Larix decidua Mill.), yielding transgenic plants with stable foreign gene expression[7]. Numerous Agrobacterium strains, leading to tumor development in a variety of coniferous species, have been identified[7, 8]. However, reports of successful regeneration in conifers stably transformed using Agrobacterium[913], as well as stable transformation via particle bombardment[1417], are scarce, primarily due to inadequate regeneration procedures[18]. Recent developments and explorations in transgenic methods have made the mere transfer of DNA into plant cells no longer a limiting factor. Yet, the ability to regenerate complex tissues or organs after DNA transfer remains a major challenge[19]. Additionally, the establishment of genetic transformation systems is ongoing for most coniferous species, with successful transformation limited to a few species, often hindered by issues like low efficiency[20]. Currently, the focus of conifer genetic transformation is on enhancing growth rates, wood properties, pest resistance, stress tolerance, and herbicide resistance[2127].

      This review offers a comprehensive overview of recent advancements in genetic transformation technologies and their applications in conifers. Influencing factors in genetic transformation encompass vector construction (Agrobacterium strain type, promoter types, and target genes), DNA delivery methods (Agrobacterium-mediated, biobombardment, and protoplast transformation), and plant regeneration pathways. We also propose various strategies to advance genetic transformation in conifers, including optimizing transformation protocols, elucidating molecular mechanisms, enhancing tissue culture techniques, overcoming cell wall barriers, exploring genetic variation, employing nanoparticle and non-tissue culture-mediated transformation, utilizing genome editing tools, and encouraging international collaboration.

    • The strains of Agrobacterium utilized in plant genetic transformation are categorized into three types: octopine, nopaline, and agropine (succinamopine), represented by strains LBA4404, GV3101, and EHA101/EHA105, respectively. Agrobacterium strains exhibit differential abilities to transform recipient material (Table 1). Humara et al. documented the transfer and expression of foreign chimeric genes in the cotyledons of Pinus pinea[28]. It was observed that EHA105, containing the plasmid p35SGUSint, demonstrated greater infectivity compared to LBA4404 or C58GV3850, with 49.7% of cotyledons exhibiting diffuse blue staining 7 d post-infection. Similarly, Le et al. employed three strains, EHA105, LBA4404, and GV3101, to facilitate the transformation of white spruce, yet only EHA105 proved effective[29]. In another study testing various A. tumefaciens strains (EHA105, GV3101, and LBA4404), the highest frequency (60%) of transient β-glucuronidase expression in Slash pine embryos was observed with Agrobacterium strain GV3101, yielding over 330 blue spots per embryo[30]. Liu successfully developed a high-efficiency Agrobacterium-mediated transient gene expression system for P. tabuliformis callus using strain GV3101, achieving a peak transient transformation efficiency of 70.1%[31]. Even within the same Agrobacterium strain, the effects vary significantly owing to differences in the structures of the constructed vectors. Grant et al. introduced six distinct plasmids – pMP2482, pTGUS, p4CL, pSLJ1111, pLN27, and pLUG – into A. tumefaciens strain KYRT1 and demonstrated that the pSLJ1111 and p4CL plasmids were markedly more effective than the others[32]. Consequently, trials targeting specific conifer species are essential to ascertain suitable strains for transformation.

      Table 1.  Plant expression vector construction.

      Tree speciesPlasmidsStrainsGenesPromotersRef.
      Pinus
      Pinus pineap35SGUSintEHA105/LBA4404/
      C58GV3850
      uidA35S[28]
      Pinus strobuspGIN/pBIV/pBIVSAR/pBINm-gfp5-ERC58pMP90GUS35S/2 × 35S[9]
      pCAMBIA1301GV3101GUS35S[10]
      Pinus taedapAD1289/pToK47/pBISN1/pWWS006LBA4404/GV3101/EHA105GUS35S[51]
      pPCV6NFHygGUSINTGV3101GUS35S[52]
      pGUS3/pSSLa.3EHA101/EHA105GUS35S/RbcS[53]
      pCAMBIA1301EHA105GUS35S[54]
      pCAMBIA1301GV3101/EHA105/LBA4404GUS35S[55]
      pBIGMLBA4404Mt1D/GutD35S[22]
      Pinus radiatapBI121LBA4404GUS35S[56]
      pGA643AGL1GUS35S[11]
      pGUL/pKEAEHA105NPTII/uidA/Bar35S[57]
      pMP2482/pTGUS/ p4CL/pSLJ1111/pLN27/pLUGKYRT1GFP35S/CoA ligase 1[32]
      Pinus pinasterpPCV6NFGUSC58pMP90GUS35S[58]
      pBINUbiGUSintEHA105/AGL1/LBA4404GUSubi1[59]
      Pinus patulapAHC25LBA4404GUSubiquitin[12]
      Pinus elliottiipCAMBIA1301EHA105/GV3101/LBA4404GUS35S[30]
      Pinus massonianapBI121EHA105CslA235S[13]
      Pinus tabuliformispBI121GV3101GUS35S[31]
      Larix
      Larix deciduapRi11325Rhizogenes strains 11325Ri plasmid/[7]
      pCGN1133/pWB139strains 11325Bt/aroA35S[21]
      hybrid larchpMRKE70KmC58pMP90NPTII35S[60]
      pCAMBIA1301GV3101GUS35S[61]
      Larix olgensispCAMBIA1300/pBI121GV3101GUS35S/PtHCA2-1[35]
      VB191103GV3101LoHDZ235S[25]
      Larix kaempferiSuper1300-GFPGV3101LaCDKB1;2Super[24]
      Picea
      Picea sitchensisMOG23LBA4404/strain 1065GUS35S[62]
      Picea abiespAD1289/pToK47/pBISN1/pWWS006LBA4404/GV3101/EHA105GUS35S[51]
      pBIV10C58/pMP90GUS2 × 35S[63]
      pET-22bLBA4404Cry3A35S[23]
      Picea marianapBIV10C58/pMP90GUS2 × 35S[63]
      Picea glaucapBIV10C58/pMP90GUS2 × 35S[63]
      pBI121EHA105/GV3101/
      LBA4404
      GUS35S[29]
      pUC19C58pMP90WUS/CHAP3AG10[64]
      Abies
      Abies spp.pTS2AGLOGUS2 × 35S[65]
      Abies koreanapBIV10/MP90C58/pMP90/LBA4404GUS2 × 35S[66]
      Taxus
      Taxus brevifolia/Taxus baccata/Bo542/C58//[8]
      Chamaecyparis
      Chamaecyparis obtusapBin19-sgfpC58/pMP90GFP35S[67]
      Cryptomeria
      Cryptomeria japonicapIG121-Hm/pUbiP-GFP-HygGV3101/pMP90GFP/GUS35S/ubiquitin[68]
      pIG121-HmGV3101/pMP90GFP35S[69]
    • Although a variety of promoters are utilized in angiosperms for the genetic engineering of both monocots and dicots, their use in gymnosperms remains limited (Table 1). The cauliflower mosaic virus (CaMV) 35S promoter, a prominent constitutive driver of transgene expression, is predominantly utilized in dicots[33]. However, despite their frequent use for gene overexpression, the activity of constitutive CaM35S promoters is notably lower in conifers[34, 35]. Constructs containing the uidA gene, which encodes β-glucuronidase (GUS), or the green fluorescent protein (GFP) gene, were introduced into embryogenic tissues to monitor the activities of these protein products over time. Expression levels of the uidA gene were minimal with a 35S-gus intron construct, yet increased twentyfold when using a 35S-35S-AMVgus::nptII construct[9].

      Furthermore, although the CaM35S promoter is functional in certain conifers, there remains a lack of efficient promoters capable of high-level, constitutive gene expression that can accommodate multiple transgenes within a single vector. Consequently, there is a need for diverse and robust promoters specifically tailored for gymnosperms, potentially in synergy with CRISPR/Cas-mediated gene editing technology[36]. CmYLCV[37], isolated from Cestrum yellow leaf curling virus—a double-stranded DNA plant pararetrovirus of the Caulimoviridae family—demonstrates heritable, strong, and constitutive activity in both monocot and dicot species. ZmUbi[38], a ubiquitin promoter derived from maize, exhibits high efficiency exclusively in monocot species, including maize[38], wheat[39], sugarcane[40], rice[41, 42], sorghum[43], and others[44]. Utilizing transient expression technology in Chinese fir protoplasts, an in vivo molecular biological investigation compared the activities of Cula11 and Cula08—constitutive expression promoters from Chinese fir—with CaM35S[45, 46], CmYLCV, and ZmUbi, commonly used in plant genetic engineering, revealing that Cula11 and Cula08 exhibited higher activity[36]. Seven constitutive promoters underwent screening via a dual luciferase (LUC) transient expression assay, revealing that PcUbi exhibited the highest activity in Cryptomeria japonica embryogenic tissue and was thus deemed the most suitable promoter for driving SpCas9 expression[47]. The pCAMBIA1300-PtHCA2-1 promoter-GUS binary expression vector, harboring the open reading frame (ORF) of the GUS gene under the control of the poplar high cambial PtHCA2–1 promoter, was subjected to testing, resulting in the observation of tissue-specific expression of the GUS gene in somatic embryos of transgenic larch[35].

    • Despite significant progress in transgenic methodologies for conifers, the preponderance of exogenous genes employed thus far are screening marker genes (e.g., uidA, npt II, hpt, GFP, and GUS). Reports of transformations involving target genes that hold genuine potential for practical applications in production are scarce (Table 1). The initial report on the regeneration of transgenic conifer plants, specifically larch, expressing value-added genes involved herbicide and insect resistance genes via Agrobacterium-mediated gene transfer[21]. Some research groups have successfully transferred insect and herbicide resistance genes into various conifer species[1416, 23, 26, 48, 49]. Overexpression of the LoHDZ2 gene in the embryonic tissues of L. olgensis has been suggested to confer enhanced stress resistance[25]. Simultaneously express two genes: mannitol-1-phosphate dehydrogenase (Mt1D) and glucitol-6-phosphate dehydrogenase (GutD) enhanced tolerance to salt stress in transgenic loblolly pine[22]. The overexpression of the LaCDKB1;2 gene in the embryonic tissues of L. kaempferi has been shown to promote cell proliferation and high-quality cotyledon embryo formation during somatic embryogenesis. This provides a foundation for examining the regulatory mechanisms of somatic embryogenesis in larch and for developing new breeding materials[24]. Overexpression of WUSCHEL-related HOMEOBOX 2 (WOX2) during proliferation and maturation of somatic embryos of P. pinaster led to alterations in the quantity and quality of cotyledonary embryos[50]. However, reports of transformation involving target genes that possess genuine potential for practical applications remain limited.

    • Agrobacterium-mediated transformation represents the most prevalent method for achieving stable genetic transformation. Cell lines generated through this method demonstrate enhanced stability in transgene expression among progeny and reduced instances of transcriptional and posttranscriptional gene silencing[19]. However, this method encompasses several drawbacks, such as bacterial overgrowth and tissue necrosis, arising from adverse co-cultivation conditions, potentially affecting the transformation frequency[19]. Nevertheless, from the standpoint of conversion efficiency, it remains a valuable technology[68]. Since the inaugural report of conifer transformation[7], there have been significant advancements in Agrobacterium-mediated genetic transformation. In recent years, there has made encouraging progress in the field of genetic transformation of conifers (Fig. 1a & Table 2), resulting in transgenic plants derived from European larch[21], hybrid larch[60, 61], white spruce[29, 63, 64], Norway spruce[23, 51], loblolly pine[20, 52, 53, 55], and radiata pine[11, 32, 56, 57].

      Figure 1. 

      Techniques and prospects for genetic transformation of conifers. (a) Agrobacterium-mediated genetic transformation. (b) Genetic transformation via biolistic bombardment. (c) Protoplast transformation. (d) Potential strategies for transformation improvement in conifers.

      Table 2.  Agrobacterium-mediated transformation in conifers.

      Tree speciesAcceptor materialsCo-culture timeOD600nm ResultsRef.
      Pinus
      Pinus pineaCotyledons3 d1Cotyledons forming buds[28]
      Pinus strobusEmbryogenic tissues2 d0.6Regenerated plant[9]
      Mature zygotic embryos12 h0.8−1.0Regenerated plant[10]
      Pinus taedaEmbryogenic tissues2 d1Transient expression[51]
      Mature zygotic embryos3−5 d/Regenerated plant[52]
      Shoot apex7 d/Transgenic plants[53]
      Mature zygotic embryos3−5 d0.8−1.0Transgenic plants[54]
      Mature zygotic embryos3−5 d0.8−1.0Transgenic plants[55]
      Mature zygotic embryos3−5 d0.5−1.0Improve salt tolerance[22]
      Pinus radiataEmbryogenic tissues1 d0.6Stable transformation[56]
      Cotyledons5−60 minOD550nm = 0.4Transgenic plants[11]
      Embryogenic tissues5 dOD550nm = 0.5−0.8Transgenic plants[57]
      Micropropagated shoot3 dOD550nm = 0.35−0.4Transgenic plants[32]
      Pinus pinasterEmbryogenic tissues36 h0.6Transgenic plants[58]
      Embryogenic tissues3 d0.3Transgenic plants[59]
      Pinus patulaEmbryogenic tissues2 d0.5−0.75Transgenic tissues[12]
      Pinus elliottiiMature zygotic embryos3 d0.9Transgenic plants[30]
      Pinus massonianaMature zygotic embryos3 d0.5Transgenic plants[13]
      Pinus tabuliformisCallus/hypocotyls/Needles3 d0.8Transient expression[31]
      Larix
      Larix deciduaHypocotyls2−3 d/Regenerated plant[7]
      Hypocotyls4 d/Regenerated plant[21]
      hybrid larchEmbryogenic tissues2 d0.3Regenerated plant[60]
      Embryogenic tissues2 d0.5Regenerated plant[61]
      Larix olgensisEmbryogenic tissues3 d0.6Transgenic plants[35]
      Embryogenic tissues2 d0.5Enhance stress resistance[25]
      Larix kaempferiEmbryogenic tissues2 d0.1Promotes cell proliferation[24]
      Picea
      Picea sitchensisEmbryogenic cell lines3 d0.8−1.1Stable transformation[62]
      Picea abiesEmbryogenic tissues2 d1Transient expression[51]
      Embryogenic tissues2 d0.6Transgenic plants[63]
      Embryogenic tissues2 d/Transgenic plants[23]
      Picea marianaEmbryogenic tissues2 d0.6Transgenic plants[63]
      Picea glaucaEmbryogenic tissues2 d0.6Transgenic plants[63]
      Embryogenic tissues2 d1Transgenic plants[29]
      Embryogenic tissues//Transgenic plants[64]
      Abies
      Abies spp.Embryogenic tissues2 d0.6Transgenic plants[65]
      Abies koreanaEmbryogenic tissues3 d0.6Transgenic plants[66]
      Taxus
      Taxus brevifolia/Taxus baccataShoot segments3 d/Gall formation[8]
      Chamaecyparis
      Chamaecyparis obtusaEmbryogenic tissues2 d0.3Transgenic plants[67]
      Cryptomeria
      Cryptomeria japonicaEmbryogenic tissues2 d0.15Enhance transformation[68]
      Embryogenic tissues2 d0.2−0.6Transgenic plants[69]

      Although Agrobacterium-mediated gene transfer is extensively employed in numerous biotechnology laboratories, its large-scale application in conifer transformation is hindered by challenges in propagating explant material, selection inefficiencies, and low transformation rates[51]. Wenck et al. explored co-cultivation conditions and various disarmed Agrobacterium strains to enhance transformation efficiency. They discovered that incorporating additional virulence genes, such as a constitutively active virG or extra copies of virG and virB from pTiBo542, amplified the transformation efficiency of Norway spruce by 1000-fold relative to initial experiments, which exhibited minimal or nonexistent transient expression[51]. Tang examined the influence of additional virulence (vir) genes in A. tumefaciens and the impact of sonication on the transformation efficiency of loblolly pine[54]. Utilizing plasmids with supplementary vir genes and sonication significantly enhanced the transfer efficiency, affecting not only transient expression but also the recovery of hygromycin-resistant lines. In their studies on Agrobacterium-mediated hybrid larch transformation, Levee et al. observed one to two transformation events per 100 cocultured masses[60]. Introducing 100 µM of coniferyl alcohol led to an increase in yield. Other studies demonstrated that sonication[10, 30] and the addition of chemicals, including okadaic acid, trifluoperazine, acetosyringone, thidiazuron, and others[10, 30, 35, 66, 70], significantly enhanced the transformation efficiency of conifers and further advanced the transformation system. Additionally, several groups have illustrated that cold treatment of Agrobacterium can augment transformation efficiency[13].

      Transformation frequencies depend on species, genotype, and post-cultivation protocol. In a study involving three species, Picea mariana was transformed at the highest frequency, followed by P. glauca and P. abies[63]. Furthermore, for all the species, transgenic plants were regenerated using modified protocols for somatic embryo maturation and germination. Le et al. devised an efficient method for the reproducible transformation of embryogenic white spruce tissue using A. tumefaciens-mediated gene transfer[29]. A shoot-based, genotype-independent transformation method employing A. tumefaciens facilitated plant recovery and enabled the transformation of elite germplasm[53]. Shoots from 4- to 6-week-old seedlings and adventitious shoots from cultures were inoculated with A. tumefaciens, underwent selection, and were subsequently regenerated. Micropropagated shoot explants from P. radiate have successfully been employed to produce stable transgenic plants via A. tumefaciens-mediated transformation[32]. It is crucial during the transformation process to inhibit and prevent contamination caused by excessive Agrobacterium growth. In the A. tumefaciens-mediated transformation of P. pinea cotyledons, a high cotyledon mortality rate occurs, possibly related to the plant's hypersensitive response to bacterial infection[28]. For conifers, non-toxic antibiotics to plant cells, like cefotaxime sodium (Cef) or timentin, are frequently incorporated into the medium. Also, in the post-transformation selection medium, selecting transformants is crucial for obtaining transgenic plants. If tissues are initially cultivated for 10 d on a medium with timentin (400 mg·L–1) to avert bacterial overgrowth, the recovery of kanamycin-resistant tissues is enhanced before applying selection pressure[29]. An evaluation of three antibiotics was conducted to assess their effectiveness in eliminating A. tumefaciens during the genetic transformation of loblolly pine using mature zygotic embryos[55]. Exposing the cultures to 350 mg·L–1 of carbenicillin, Cef, and timentin for a duration of up to 6 weeks failed to eliminate Agrobacterium; however, increasing the concentration to 500 mg·L–1 successfully eradicated the bacterium from co-cultured zygotic embryos[55].

      Identifying the optimal combination of infection time and concentration is crucial for successful conifer transgenesis during genetic transformation experiments. Generally, the bacterial solution concentration for infecting conifers is maintained at an OD600 of 0.3–0.8. Elevating the Agrobacterium concentration and extending the infection duration can result in excessive bacterial proliferation and hypersensitive necrosis of explants, thereby diminishing transformation efficiency[28]. Conversely, employing a low-density Agrobacterium suspension and a brief infection period often results in weak infectivity, which similarly reduces transformation efficiency[13]. Moreover, the infection duration influences T-DNA transfer and, consequently, the efficiency of genetic transformation. The infection duration, typically less than 30 min, varies depending on the explant type and the physiological status of the conifer species. However, both the concentration and infection duration of the bacterial solution must be tailored to the condition, type, and environmental factors of the explants, necessitating further research.

    • Particle bombardment, also known as biolistics, serves as an alternative method for plant genetic transformation, circumventing the limitations associated with Agrobacterium-mediated genetic transformation[71]. This method is not limited by biological constraints and is applicable to a broad spectrum of plant species. However, in the context of conifer transformation frequency, biolistic techniques are generally regarded as less effective than Agrobacterium-mediated genetic transformation[68]. Foreign genes have successfully been expressed in all tested conifer tissues via particle bombardment, encompassing embryos, seedlings, xylem, pollen, needles, buds, cell suspension cultures, embryogenic callus, cell aggregate cultures, and roots (Fig. 1b & Table 3). While most of these attempts yielded only transient expression, they have offered insightful information about the factors influencing gene expression in various tissues capable of regeneration[20]. GFP introduction into conifer tissues has been achieved through microprojectile bombardment, with transient expression subsequently observed[72]. The CaMV35S promoter facilitated GUS gene expression in loblolly pine tissues[73]. Microprojectile bombardment proves to be an effective technique for assaying transient gene expression in pine, and it harbors potential for generating transgenic pine plants. Using high-velocity microprojectiles, plasmid DNA with the GUS gene, under the control of the CaMV35S promoter, has been introduced into cultured Douglas fir cotyledons[74]. Additionally, the particle gun technique has been employed to transform a variety of receptor materials in different tree species, including callus and pollen of larch[75, 76], Chir pine[16], and Norway spruce[14, 7780]. Particle bombardment has been applied to Lodgepole pine, yellow cypress, western hemlock, jack pine, and black spruce pollen to achieve transient GUS gene expression, demonstrating the method's viability for pollen transformation[81]. Furthermore, particle bombardment has facilitated the testing of transient expression of heterologous promoters in organized tissues and angiosperm promoters in gymnosperms[82]. Comparative analyses have been conducted on the initiation strengths of transient expression for eight distinct promoter sequences, based on the relative levels of GUS expression[76].

      Table 3.  Biolistic bombardment genetic transformation in conifers.

      Tree speciesAcceptor materialsPlasmidsPromotersGenesResultsRef.
      Pinus
      Pinus taedaCotyledonspBI22135SGUSTransient expression[73]
      Pinus radiataSuspension cellspBI22135SGUSTransient expression[87]
      Embryogenic tissuespCW103/pCWI222 × 35SgusATransient expression[88]
      CotyledonspBI121/pCGUΔl/
      pAIGusN/pActl-D
      35S/UbBI/Adhl/ActlgusATransient expression[89]
      Embryogenic tissuespRC101/pCW12235S/EmuuidATransgenic plants[83]
      Embryogenic tissuespAHC25/pCW122maize ubiquitin/35SGUS/BarTransgenic plants[14]
      CallipCW122/pCADsense35Snpt II/CadTransgenic calli[90]
      Embryogenic tissuespMYC3425/pAW16/
      pCW132/pRN2
      Emu/ubiCry1AcTransgenic plants[15]
      Pinus concorta/Pinus banksianaMature pollenpBM113Kp/pRT99GUS/
      pAct1-D/pGA984
      35S/rice actinGUSTransient expression[81]
      Pinus sylvestrisCalli/Vegetative buds/
      Suspension cells
      pBI22135SGUSTransient expression[91]
      PollenpBI221/pRT99/pBI410/
      pBI426/pBM113
      35S/EmP/UbB1GUSTransient expression[79 ]
      Pinus strobusEmbryonal massesp35S-GFP/mGFP435SGFPTransient expression[72]
      Pinus aristata/Pinus griffithii/Pinus monticolaPollen tubespBI22135SGUSTransient expression[92]
      Pinus patulaEmbryogenic tissuespAHC2535SBar/GUSSomatic embryos[48]
      Pinus nigraEmbryogenic tissuespCW1222 × 35SGUSSomatic embryos[86]
      Pinus roxbughiiMature zygotic embryospAHC25maize ubiquitinBar/GUSTransgenic plants[16]
      Picea
      Picea glaucaZygotic embryos/Seedlings/
      embryogenic callus
      pUC1935SGUSTransient expression[82]
      Somatic embryospBI42635SGUSStable transformation[93]
      Somatic embryospTVBT4110035SGUS/BtTransgenic plants[49]
      Embryonal massesp35S-GFP/mGFP435SGFPTransient expression[72]
      Embryogenic tissuespKUB/pBI426maize ubiquitin/35Scry1AbTransgenic plants[26]
      Picea marianaEmbryogenic tissuespRT99GUS/pBM113Kp35SGUSTransient expression[94]
      Embryogenic tissuespRT99GUS/pGUSInt/
      pMON9909
      35S/Em protein of wheat/Rbcs/NOS/
      Actin/Arabin
      GUSTransient expression[76]
      Mature pollenpBM113Kp/pRT99GUS/
      pAct1-D/pGA984
      35S/rice actinGUSTransient expression[81]
      Embryonal massespRT99GUS/pBI42635SGUSTransgenic plants[84]
      Pollen/Embryonal masses/ Somatic embryosp35S-GFP/mGFP435SGFPTransient expression[72]
      Mature somatic embryospBI221.2335SGUSTransgenic plants[17]
      Picea abiesSomatic embryopRT99gus35SGUSStable transformation[77]
      Embryogenic tissuespRT99gus/pJIT65/
      Dc8gus/pBMI13Kp
      35S/2 × 35S/
      Act1-D/Dc8
      GUSTransient expression[80]
      PollenpBI221/pRT99/pBI410/
      pBI426/pBM113
      35S/EmP/UbB1GUSTransient expression[79]
      Embryogenic tissuespCW12235SGUSTransgenic plants[95]
      Embryogenic tissuespAHC25maize ubiquitinBarTransgenic plants[78]
      Embryogenic tissuespAHC25/pCW122maize ubiquitin/35SGUS/BarTransgenic plants[14]
      Embryogenic tissuespAHC25maize ubiquitinCCRTransgenic plants[27]
      Larix
      Larix × eurolepisEmbryogenic tissuespRT99GUS/pGUSInt/
      pMON9909
      35S/Em protein of wheat/Rbcs/NOS/
      Actin/Arabin
      GUSTransient expression[76]
      Larix laricinaEmbryonal massespBI426/pRT99gus/
      pRT66gus/pRT55gus
      35S/2 × 35SGUSTransient expression[75]
      Larix gmeliniiZygotic embryospUC-GHG/pBI221-HPT35SGUS/GFPTransgenic plants[34]
      Pseudotsuga
      Pseudotsuga menziesiiCotyledonspTVBTGUS35SGUSTransient expression[74]
      Chamaecyparis
      Chamaecyparis nootkatensisMature pollenpBM113Kp/pRT99GUS/
      pAct1-D/pGA984
      35S/rice actinGUSTransient expression[81]
      Tsuga
      Tsuga heterophyllaMature pollenpBM113Kp/pRT99GUS/
      pAct1-D/pGA984
      35S/rice actinGUSTransient expression[81]
      Abies
      Abies nordmannianaEmbryogenic tissuespCW12235SGUSTransgenic plants[85]

      Particle bombardment-mediated transformation is capable of regenerating whole plants. In P. glauca plants, the stable expression of an exogenous gene marked the first successful creation of transgenic plants using the particle gun method[49]. Walter et al. used a particle gun to bombard four embryonic cell lines of P. radiate, resulting in over 150 transgenic plants from 20 transformation experiments[83]. Analyses using Southern and Northern blotting confirmed the integration of the target gene into the genome. Particle bombardment facilitated the stable genetic transformation of P. mariana in two target tissues: mature cotyledonary somatic embryos and suspensions from embryonal masses, employing the Biolistic PDS-1000/He device[84]. The expression of the GUS gene in needles of regenerated seedlings demonstrates the potential for sustained transgene expression in spruce[17]. Using biolistic transformation, stable genetic transformation has been accomplished in embryogenic cultures of Abies nordmanniana, leading to the regeneration of transgenic plants[85]. A biolistic approach has successfully achieved stable transformation in embryogenic tissues of P. nigra Arn., specifically cell line E104[86]. Given its versatility and broad applicability, particle bombardment is anticipated to continue as a primary method in genetic transformation.

      Particle bombardment possesses significant potential for producing transgenic conifer plants. A key objective in tree breeding involves reducing lignin content or modifying its composition, which would aid in delignification during pulping processes. When the antisense construct of the cinnamoyl CoA reductase (CCR) gene was introduced into Norway spruce, a significant reduction in the total lignin content of dry wood was observed compared to controls[27]. Lachance et al. conducted a study on the accumulation of crylAb protein in embryogenic tissues, somatic seedling needles, and 5-year-old field-grown needles of white spruce[26]. Insect feeding trials, both in the laboratory and the field, indicated that multiple transgenic spruce lines proved lethal to spruce budworm larvae. Through biolistic transformation of embryogenic tissue, transgenic radiata pine plants harboring the Bacillus thuringiensis (Bt) toxin gene, cry1Ac, were successfully produced[15]. Ongoing research is being conducted on functional genes utilizing this technology[14, 16, 78].

    • Protoplast technology enables various unique approaches to the genetic improvement of plants[96]. Protoplast transient expression assays serve as versatile tools in genomics, transcriptomics, metabolic, and epigenetic studies[97]. Coupling protoplast transient expression experiments with high-resolution imaging enables simple, rapid, and efficient analysis and characterisation of gene functions and regulatory networks. This includes protein subcellular localisation, protein-protein interactions, transcriptional regulatory networks, and gene responses to external cues[98100]. Reporter genes commonly used, like LUC and GUS, are employed to assess gene activity in conifer protoplasts[87]. P. glauca protoplasts were transformed with the chloramphenicol acetyltransferase (CAT) reporter gene through electroporation[101]. Fir and pine protoplasts were successfully transformed with the LUC gene through electroporation, with gene expression enhanced by the addition of polyethylene glycol (PEG) to the mixture[102]. Developments in methods for transient gene expression have been made for protoplasts of black spruce and jack pine[103]. In electroporated protoplasts of P. glauca, P. mariana, and P. banksiana, the activity levels of exogenous genes depend on the promoter, electroporation conditions, and the targeted cell line[104]. A new transient transformation system for Chinese fir protoplasts has been established, achieving cell wall regeneration and protoplast division. This method serves as a reference for conducting functional studies on Chinese fir-related genes[105]. However, the challenges in establishing protoplast regeneration systems in conifers mean that protoplast-based genetic transformation studies primarily focus on transient gene expression and the investigation of gene function and expression regulation (Fig. 1c).

    • Establishing an effective and stable regeneration system is crucial for rapidly expanding conifer populations for seedling production and successful heritage transformation. A range of plant materials, each with unique advantages, serves as transformation receptors for conifers. These include zygotic embryos, hypocotyls, embryonic tissues, somatic embryos, protoplasts, stem tips, and pollen[7, 10, 13, 31, 51, 53, 81, 101]. Embryonic tissues have been the focus of extensive research as receptors in numerous studies[9, 27, 35, 51, 57, 58, 85]. Additionally, Agrobacterium-mediated genetic transformation using mature zygotic embryos as explants has been successfully implemented in P. taeda[22, 52, 54, 55], P. elliottii[30], and P. massoniana[13]. Cotyledons and hypocotyls are identified as suitable explants for genetic transformation[7, 11, 21, 28]. Currently, embryonic tissue of conifers is predominantly used as recipient material through the somatic embryogenesis pathway to obtain stably-transformed regenerated plants (Tables 2 & 3).

      A primary challenge in the genetic transformation of coniferous trees involves plant regeneration[106]. This challenge arises primarily from the unique biological properties and regeneration mechanisms of conifers. Tissue culture in conifers proves more challenging than in other plants. This is attributed to the cells of conifers, especially those from mature trees, which have a lower capacity for differentiation and regeneration[107]. The tissue culture process entails inducing cells or tissues from the parent plant to develop into new plants under controlled conditions, a process notably less efficient in conifers. Furthermore, during tissue culture, particularly over extended periods, the genetic stability of conifers may be affected. Cell division and differentiation, occurring during tissue culture, may introduce genetic mutations; additionally, genome doubling, leading to the formation of polyploids, can also occur. Consequently, even if plant regeneration is successful, the resultant plants may exhibit genetic variations, potentially posing challenges in subsequent applications and research[19, 106]. The regeneration of conifer tissue is notably sensitive to the balance of plant hormones and other culture conditions. Different species of conifers often require specific combinations of hormones and culture environments, thereby complicating the identification of a universal method applicable to all types[108]. Conifers generally exhibit a long regeneration process, which implies that the entire process from tissue culture to mature plant consumes a considerable amount of time, acting as a limiting factor in research and application. Variations in regeneration capabilities among different species of conifers are notable.

      In summary, although the genetic transformation and regeneration of coniferous trees are theoretically feasible, their practical implementation is fraught with several challenges, most notably in tissue culture efficiency, genetic stability maintenance, and adaptation to different species' characteristics[109]. Addressing these challenges necessitates in-depth research and substantial technological innovation.

    • Despite the numerous promising success cases mentioned, it must be acknowledged that genetic transformation continues to pose a significant challenge for most conifer researchers. To date, none of these methods have proven universally applicable across multiple species or varied genotypes. Consequently, while a method may appear promising, it often remains confined to successful implementation under specific laboratory conditions, lacking widespread applicability. Significant progress is still required to develop a universal model for conifers that is as straightforward, efficient, and reproducible as those established for angiosperm model species.

    • Conifers possess distinct and complex biological characteristics, setting them apart from commonly utilized genetic engineering plants like Arabidopsis or tobacco. Their prolonged generation times, expansive genomes, and elaborate reproductive processes contribute to the challenges in working with them[1, 2, 4].

    • Despite the establishment of transformation protocols, the efficiency of integrating foreign genes into the conifer genome frequently remains low[54, 63]. Consequently, only a minor fraction of transformed cells effectively express the introduced gene, posing significant challenges in producing stable and predictable genetically modified organisms.

    • Various conifer species exhibit unique biological traits and varying responses to transformation techniques. A technique effective in one conifer species might not yield similar results in another, necessitating tailored optimization for each species.

    • The size and complexity of conifer genomes pose challenges in the introduction and expression of foreign genes. A thorough understanding of the regulatory elements and mechanisms within conifer genomes is crucial for genetic engineering success[35]. However, such knowledge is typically less comprehensive than that available for model plant species.

    • Conifers often require specialized tissue culture techniques for regeneration and propagation. Developing suitable tissue culture methods for conifers, particularly those compatible with genetic transformation, is a significant hurdle. Studies have indicated that the induction rate of embryogenic tissues from immature seeds in conifers is influenced by both the genotype and the embryonic developmental stage[110, 111].

    • Conifers, like many plants, contain high levels of phenolic compounds, such as lignins and polyphenols[112, 113]. These compounds may exert inhibitory effects on the enzymes used in the genetic transformation process. Phenolic compounds are known to contribute to oxidative stress, DNA degradation, and may interfere with the integration of foreign genes into the plant genome.

    • Conifers produce a diverse array of secondary metabolites, including terpenoids and flavonoids, which can potentially affect the success of genetic transformation. These compounds can exhibit toxic effects on the transformed cells or may interfere with the activity of introduced genes.

    • The cell walls of conifers are notably complex and rigid, serving to provide structural support to the plant. However, this complexity may impede the delivery of foreign DNA into plant cells. Efficient transformation frequently necessitates overcoming these barriers to ensure that the introduced genetic material successfully reaches the nucleus of the target cells[114, 115].

    • The presence of genetic variation within conifer populations may influence the success of genetic transformation. Individuals within a species often exhibit varying responses to transformation protocols, and optimizing these protocols for broader applicability presents a significant challenge.

      Addressing these biochemical factors typically necessitates the development of specialized techniques and treatments within the genetic transformation process. For instance, researchers might utilize tissue culture conditions designed to mitigate the effects of phenolic compounds, or employ specialized methods to enhance the delivery of foreign DNA through the cell wall.

      Comprehending the biochemical makeup of conifers and customizing transformation methods to suit their unique characteristics is an active area of research. Advances in biotechnology, encompassing the development of more robust transformation protocols and the elucidation of genes involved in stress responses, may play a pivotal role in surmounting these biochemical barriers in the future.

    • Addressing the challenges associated with the genetic transformation of conifers necessitates a comprehensive approach that integrates advancements across multiple key domains (Fig. 1d). The following delineates potential strategies and focal areas.

    • It is imperative for researchers to persist in refining and optimizing transformation protocols tailored to various conifer species. This encompasses enhancing the efficiency of introducing foreign genes into conifer cells and developing uniform methods applicable across diverse species. The utilization of developmental genes may prove beneficial in promoting transformation. These genes, capable of acting through diverse developmental mechanisms to enhance the regeneration of transgenic cells, have seen extensive use in model plants to stimulate embryogenesis and, in some instances, organogenesis[116118]. In summary, the overexpression of regeneration-regulating transcription factors, including BBM, WUS2, WOX5, GRF4, and GIF1, could enhance genetic transformation in conifers characterized by low regeneration efficiency, substantial transformation difficulty, and genotype limitation.

    • Gaining a deeper understanding of the molecular and biochemical processes in conifers is essential. This necessitates research into the regulation of gene expression, understanding the role of secondary metabolites, and comprehending the response of conifers to stress conditions. This knowledge is crucial in informing the development of transformation methods that are synergistic with the unique biology of conifers.

    • The improvement of tissue culture techniques, crucial for supporting the regeneration and propagation of conifer plants, is vital. The development of protocols for efficient plant regeneration from transformed cells can significantly bolster the success of genetic transformation. Conversely, most prevailing methods for plant genome modification entail regenerating plants from genetically modified cells in tissue culture, a process that is technically challenging, costly, time-consuming, and limited to a narrow range of plant species or genotypes[119]. Cao et al. outlined a notably straightforward cut–dip–budding (CDB) delivery system, which includes inoculating explants with A. rhizogenes, subsequently generating transformed roots that yield transformed buds through root suckering[120]. The advancement of methods that circumvent laborious procedures, such as tissue culture, and facilitate obtaining transgenic and gene-edited plants, marks a significant breakthrough in conifer research.

    • Exploring strategies to overcome the challenges presented by the complex cell walls of conifers is imperative. This could involve employing enzymes or other agents to facilitate the penetration of foreign DNA into plant cells. In the realm of conifer biotechnology, the initial protoplast extraction in P. contorta laid the foundation for the establishment of a transient transformation system in conifers[121].

    • Recognizing and addressing genetic variation within conifer species is critical. Customizing transformation protocols to accommodate the diverse genetic backgrounds of individuals within a species can lead to broader success in genetic transformation[122].

    • The utilization of cutting-edge biotechnological tools, notably CRISPR/Cas9 gene editing, can offer more precise control over the modification of conifer genomes. These advanced technologies have the potential to overcome several challenges associated with traditional genetic transformation methods. Genome editing represents a powerful technology for functional genomic research and trait improvement. Cui et al. successfully achieved knockout of the DXS1 gene in white spruce (P. glauca) employing the conifer-specific CRISPR/Cas9 toolbox[123]. Recently, CRISPR/Cas9-mediated targeted mutagenesis has been demonstrated in radiata pine[124], Japanese cedar[47], and Chinese fir[36], underscoring its feasibility in conifers. This represents a potent genome editing system of significant importance for gene function studies and the genetic improvement of plant traits, likely to make substantial contributions to the development of molecular breeding in conifers.

    • In future research endeavors, the use of nanomaterials for genetic modification promises to expand the scope of plant molecular research, particularly for conifers, which currently lack efficient systems for regeneration and stable genetic transformation. Nanocarriers are characterized by their large surface area, facilitating efficient gene loading, alongside high biocompatibility to safeguard the loaded genes, coupled with low toxicity and enhanced safety[125, 126]. Consequently, nanoparticles hold the potential to be utilized in developing transgenic technologies for conifer regeneration without dependency on tissue culture, potentially overcoming the technical challenges in genetic transformation of recalcitrant plant genotypes. Conversely, the exploration of stable and targeted nanocarrier-mediated gene editing technologies offers the prospect of achieving genetic improvements in conifers.

    • Considering the ecological significance of conifers, comprehensive risk assessments and detailed ecological studies should accompany all attempts at genetic modification. Comprehending the potential environmental impact and addressing public concerns are imperative for the responsible and sustainable deployment of genetically modified conifers.

    • Given the global distribution of conifers, international collaboration among researchers, institutions, and regulatory bodies is essential to foster the sharing of knowledge, resources, and expertise. Such collaborative efforts can significantly accelerate progress and enhance the effectiveness in addressing challenges.

      Sustained research and ongoing technological advancements, in conjunction with a holistic and interdisciplinary approach, are crucial to unlocking the full potential of genetic transformation in conifers, while simultaneously ensuring the responsible and ethical application of these technologies.

    • Many reports have documented the successful expression of exogenous genes in conifers using Agrobacterium-mediated, particle bombardment-mediated, and protoplast-based genetic transformation methods. However, the genetic transformation of conifers faces several challenges, including low transformation efficiency, high dependence on recipient genotypes, difficulties in plant regeneration. Overall, the genetic transformation of conifers remains heavily reliant on extensive experience and sophisticated technical skills, rendering its widespread application challenging for most conifer researchers. Overcoming these challenges will usher in a new era of productivity and quality in forestry. Several potential strategies have been proposed to improve conifer transformation, including the optimization of transformation protocols, understanding molecular mechanisms, improving tissue culture techniques, overcoming cell wall barriers, understanding genetic variation, employing nanoparticle- and non-tissue culture-mediated genetic transformation, utilizing genome editing tools, fostering international collaboration, and more. In conclusion, with the ongoing development of molecular biotechnology and enhancement of various regeneration and transformation systems, research on the genetic transformation of conifer species is poised for continued progress and broader applicability.

    • The authors confirm contribution to the paper as follows: study conception and design: Zhao J, Niu S, Zhang J; draft manuscript preparation: Zhao H; Figure creation: Zhao H. All authors reviewed the results and approved the final version of the manuscript.

    • Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

      • This work was supported by the National Key R&D Program of China (Grant No. 2023YFD2200102), the National Natural Science Foundation of China (Grant No. 32371834), the National Natural Science Foundation of China (Grant No. 32271836), the National Key R&D Program of China (Grant No. 2023YFD2200104).

      • The authors declare that they have no conflict of interest. Shihui Niu is the Editorial Board member of Forestry Research who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (1)  Table (3) References (126)
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    Zhao H, Zhang J, Zhao J, Niu S. 2024. Genetic transformation in conifers: current status and future prospects. Forestry Research 4: e010 doi: 10.48130/forres-0024-0007
    Zhao H, Zhang J, Zhao J, Niu S. 2024. Genetic transformation in conifers: current status and future prospects. Forestry Research 4: e010 doi: 10.48130/forres-0024-0007

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