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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 species Plasmids Strains Genes Promoters Ref. Pinus Pinus pinea p35SGUSint EHA105/LBA4404/
C58GV3850uidA 35S [28] Pinus strobus pGIN/pBIV/pBIVSAR/pBINm-gfp5-ER C58pMP90 GUS 35S/2 × 35S [9] pCAMBIA1301 GV3101 GUS 35S [10] Pinus taeda pAD1289/pToK47/pBISN1/pWWS006 LBA4404/GV3101/EHA105 GUS 35S [51] pPCV6NFHygGUSINT GV3101 GUS 35S [52] pGUS3/pSSLa.3 EHA101/EHA105 GUS 35S/RbcS [53] pCAMBIA1301 EHA105 GUS 35S [54] pCAMBIA1301 GV3101/EHA105/LBA4404 GUS 35S [55] pBIGM LBA4404 Mt1D/GutD 35S [22] Pinus radiata pBI121 LBA4404 GUS 35S [56] pGA643 AGL1 GUS 35S [11] pGUL/pKEA EHA105 NPTII/uidA/Bar 35S [57] pMP2482/pTGUS/ p4CL/pSLJ1111/pLN27/pLUG KYRT1 GFP 35S/CoA ligase 1 [32] Pinus pinaster pPCV6NFGUS C58pMP90 GUS 35S [58] pBINUbiGUSint EHA105/AGL1/LBA4404 GUS ubi1 [59] Pinus patula pAHC25 LBA4404 GUS ubiquitin [12] Pinus elliottii pCAMBIA1301 EHA105/GV3101/LBA4404 GUS 35S [30] Pinus massoniana pBI121 EHA105 CslA2 35S [13] Pinus tabuliformis pBI121 GV3101 GUS 35S [31] Larix Larix decidua pRi11325 Rhizogenes strains 11325 Ri plasmid / [7] pCGN1133/pWB139 strains 11325 Bt/aroA 35S [21] hybrid larch pMRKE70Km C58pMP90 NPTII 35S [60] pCAMBIA1301 GV3101 GUS 35S [61] Larix olgensis pCAMBIA1300/pBI121 GV3101 GUS 35S/PtHCA2-1 [35] VB191103 GV3101 LoHDZ2 35S [25] Larix kaempferi Super1300-GFP GV3101 LaCDKB1;2 Super [24] Picea Picea sitchensis MOG23 LBA4404/strain 1065 GUS 35S [62] Picea abies pAD1289/pToK47/pBISN1/pWWS006 LBA4404/GV3101/EHA105 GUS 35S [51] pBIV10 C58/pMP90 GUS 2 × 35S [63] pET-22b LBA4404 Cry3A 35S [23] Picea mariana pBIV10 C58/pMP90 GUS 2 × 35S [63] Picea glauca pBIV10 C58/pMP90 GUS 2 × 35S [63] pBI121 EHA105/GV3101/
LBA4404GUS 35S [29] pUC19 C58pMP90 WUS/CHAP3A G10 [64] Abies Abies spp. pTS2 AGLO GUS 2 × 35S [65] Abies koreana pBIV10/MP90 C58/pMP90/LBA4404 GUS 2 × 35S [66] Taxus Taxus brevifolia/Taxus baccata / Bo542/C58 / / [8] Chamaecyparis Chamaecyparis obtusa pBin19-sgfp C58/pMP90 GFP 35S [67] Cryptomeria Cryptomeria japonica pIG121-Hm/pUbiP-GFP-Hyg GV3101/pMP90 GFP/GUS 35S/ubiquitin [68] pIG121-Hm GV3101/pMP90 GFP 35S [69] Types of promoters
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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].
Transformed exogenous genes
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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[14−16, 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.
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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 species Acceptor materials Co-culture time OD600nm Results Ref. Pinus Pinus pinea Cotyledons 3 d 1 Cotyledons forming buds [28] Pinus strobus Embryogenic tissues 2 d 0.6 Regenerated plant [9] Mature zygotic embryos 12 h 0.8−1.0 Regenerated plant [10] Pinus taeda Embryogenic tissues 2 d 1 Transient expression [51] Mature zygotic embryos 3−5 d / Regenerated plant [52] Shoot apex 7 d / Transgenic plants [53] Mature zygotic embryos 3−5 d 0.8−1.0 Transgenic plants [54] Mature zygotic embryos 3−5 d 0.8−1.0 Transgenic plants [55] Mature zygotic embryos 3−5 d 0.5−1.0 Improve salt tolerance [22] Pinus radiata Embryogenic tissues 1 d 0.6 Stable transformation [56] Cotyledons 5−60 min OD550nm = 0.4 Transgenic plants [11] Embryogenic tissues 5 d OD550nm = 0.5−0.8 Transgenic plants [57] Micropropagated shoot 3 d OD550nm = 0.35−0.4 Transgenic plants [32] Pinus pinaster Embryogenic tissues 36 h 0.6 Transgenic plants [58] Embryogenic tissues 3 d 0.3 Transgenic plants [59] Pinus patula Embryogenic tissues 2 d 0.5−0.75 Transgenic tissues [12] Pinus elliottii Mature zygotic embryos 3 d 0.9 Transgenic plants [30] Pinus massoniana Mature zygotic embryos 3 d 0.5 Transgenic plants [13] Pinus tabuliformis Callus/hypocotyls/Needles 3 d 0.8 Transient expression [31] Larix Larix decidua Hypocotyls 2−3 d / Regenerated plant [7] Hypocotyls 4 d / Regenerated plant [21] hybrid larch Embryogenic tissues 2 d 0.3 Regenerated plant [60] Embryogenic tissues 2 d 0.5 Regenerated plant [61] Larix olgensis Embryogenic tissues 3 d 0.6 Transgenic plants [35] Embryogenic tissues 2 d 0.5 Enhance stress resistance [25] Larix kaempferi Embryogenic tissues 2 d 0.1 Promotes cell proliferation [24] Picea Picea sitchensis Embryogenic cell lines 3 d 0.8−1.1 Stable transformation [62] Picea abies Embryogenic tissues 2 d 1 Transient expression [51] Embryogenic tissues 2 d 0.6 Transgenic plants [63] Embryogenic tissues 2 d / Transgenic plants [23] Picea mariana Embryogenic tissues 2 d 0.6 Transgenic plants [63] Picea glauca Embryogenic tissues 2 d 0.6 Transgenic plants [63] Embryogenic tissues 2 d 1 Transgenic plants [29] Embryogenic tissues / / Transgenic plants [64] Abies Abies spp. Embryogenic tissues 2 d 0.6 Transgenic plants [65] Abies koreana Embryogenic tissues 3 d 0.6 Transgenic plants [66] Taxus Taxus brevifolia/Taxus baccata Shoot segments 3 d / Gall formation [8] Chamaecyparis Chamaecyparis obtusa Embryogenic tissues 2 d 0.3 Transgenic plants [67] Cryptomeria Cryptomeria japonica Embryogenic tissues 2 d 0.15 Enhance transformation [68] Embryogenic tissues 2 d 0.2−0.6 Transgenic 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.
Genetic transformation via biolistic bombardment
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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, 77−80]. 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 species Acceptor materials Plasmids Promoters Genes Results Ref. Pinus Pinus taeda Cotyledons pBI221 35S GUS Transient expression [73] Pinus radiata Suspension cells pBI221 35S GUS Transient expression [87] Embryogenic tissues pCW103/pCWI22 2 × 35S gusA Transient expression [88] Cotyledons pBI121/pCGUΔl/
pAIGusN/pActl-D35S/UbBI/Adhl/Actl gusA Transient expression [89] Embryogenic tissues pRC101/pCW122 35S/Emu uidA Transgenic plants [83] Embryogenic tissues pAHC25/pCW122 maize ubiquitin/35S GUS/Bar Transgenic plants [14] Calli pCW122/pCADsense 35S npt II/Cad Transgenic calli [90] Embryogenic tissues pMYC3425/pAW16/
pCW132/pRN2Emu/ubi Cry1Ac Transgenic plants [15] Pinus concorta/Pinus banksiana Mature pollen pBM113Kp/pRT99GUS/
pAct1-D/pGA98435S/rice actin GUS Transient expression [81] Pinus sylvestris Calli/Vegetative buds/
Suspension cellspBI221 35S GUS Transient expression [91] Pollen pBI221/pRT99/pBI410/
pBI426/pBM11335S/EmP/UbB1 GUS Transient expression [79 ] Pinus strobus Embryonal masses p35S-GFP/mGFP4 35S GFP Transient expression [72] Pinus aristata/Pinus griffithii/Pinus monticola Pollen tubes pBI221 35S GUS Transient expression [92] Pinus patula Embryogenic tissues pAHC25 35S Bar/GUS Somatic embryos [48] Pinus nigra Embryogenic tissues pCW122 2 × 35S GUS Somatic embryos [86] Pinus roxbughii Mature zygotic embryos pAHC25 maize ubiquitin Bar/GUS Transgenic plants [16] Picea Picea glauca Zygotic embryos/Seedlings/
embryogenic calluspUC19 35S GUS Transient expression [82] Somatic embryos pBI426 35S GUS Stable transformation [93] Somatic embryos pTVBT41100 35S GUS/Bt Transgenic plants [49] Embryonal masses p35S-GFP/mGFP4 35S GFP Transient expression [72] Embryogenic tissues pKUB/pBI426 maize ubiquitin/35S cry1Ab Transgenic plants [26] Picea mariana Embryogenic tissues pRT99GUS/pBM113Kp 35S GUS Transient expression [94] Embryogenic tissues pRT99GUS/pGUSInt/
pMON990935S/Em protein of wheat/Rbcs/NOS/
Actin/ArabinGUS Transient expression [76] Mature pollen pBM113Kp/pRT99GUS/
pAct1-D/pGA98435S/rice actin GUS Transient expression [81] Embryonal masses pRT99GUS/pBI426 35S GUS Transgenic plants [84] Pollen/Embryonal masses/ Somatic embryos p35S-GFP/mGFP4 35S GFP Transient expression [72] Mature somatic embryos pBI221.23 35S GUS Transgenic plants [17] Picea abies Somatic embryo pRT99gus 35S GUS Stable transformation [77] Embryogenic tissues pRT99gus/pJIT65/
Dc8gus/pBMI13Kp35S/2 × 35S/
Act1-D/Dc8GUS Transient expression [80] Pollen pBI221/pRT99/pBI410/
pBI426/pBM11335S/EmP/UbB1 GUS Transient expression [79] Embryogenic tissues pCW122 35S GUS Transgenic plants [95] Embryogenic tissues pAHC25 maize ubiquitin Bar Transgenic plants [78] Embryogenic tissues pAHC25/pCW122 maize ubiquitin/35S GUS/Bar Transgenic plants [14] Embryogenic tissues pAHC25 maize ubiquitin CCR Transgenic plants [27] Larix Larix × eurolepis Embryogenic tissues pRT99GUS/pGUSInt/
pMON990935S/Em protein of wheat/Rbcs/NOS/
Actin/ArabinGUS Transient expression [76] Larix laricina Embryonal masses pBI426/pRT99gus/
pRT66gus/pRT55gus35S/2 × 35S GUS Transient expression [75] Larix gmelinii Zygotic embryos pUC-GHG/pBI221-HPT 35S GUS/GFP Transgenic plants [34] Pseudotsuga Pseudotsuga menziesii Cotyledons pTVBTGUS 35S GUS Transient expression [74] Chamaecyparis Chamaecyparis nootkatensis Mature pollen pBM113Kp/pRT99GUS/
pAct1-D/pGA98435S/rice actin GUS Transient expression [81] Tsuga Tsuga heterophylla Mature pollen pBM113Kp/pRT99GUS/
pAct1-D/pGA98435S/rice actin GUS Transient expression [81] Abies Abies nordmanniana Embryogenic tissues pCW122 35S GUS Transgenic 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 transformation
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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[98−100]. 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).
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Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.
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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
Genetic transformation in conifers: current status and future prospects
- Received: 11 December 2023
- Revised: 30 January 2024
- Accepted: 28 February 2024
- Published online: 21 March 2024
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.
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Key words:
- Conifer /
- Genetic transformation /
- Regeneration /
- Prospects