Establishment of a High-Efficiency In Vitro Regeneration and Agrobacterium-Mediated Genetic Transformation System for Ajania achilleoides (Turcz.) Poljakov
This study establishes a stable and efficient regeneration and transformation system for A. achilleoides, providing a platform for functional gene analysis and molecular breeding of wild chrysanthemum species.
Abstract
Ajania achilleoides, a wild species with strong environmental adaptability, is a valuable germplasm resource for genetic improvement and functional gene studies. However, efficient in vitro regeneration and genetic transformation systems for this species remain undeveloped, limiting functional gene characterization and genetic improvement of this valuable germplasm resource. In this study, leaf explants were used to establish an efficient regeneration system by evaluating different combinations of plant growth regulators and to develop an Agrobacterium tumefaciens-mediated genetic transformation system for A. achilleoides. MS medium supplemented with 2.0 mg/L 6-benzyladenine (6-BA) and 1.0 mg/L α-naphthaleneacetic acid (NAA) achieved the highest adventitious shoot regeneration rate (99%), while 1/2 MS medium containing 0.5 mg/L indole-3-butyric acid (IBA) was optimal for rooting, resulting in a 100% rooting rate. Critical hygromycin concentrations for shoot regeneration and rooting selection were determined to be 25 mg/L and 10 mg/L, respectively. For transformation, the best results were obtained with a 3-day preculture, bacterial suspension at OD600 = 0.2, 30 min infection, and 2-day co-cultivation. PCR and sequencing confirmed the successful integration of the target genes into the genome, yielding three positive transgenic lines with a molecular confirmation rate of 30%. This study establishes a stable and efficient regeneration and transformation system for A. achilleoides, providing a platform for functional gene analysis and molecular breeding of wild chrysanthemum species.
Prunus sibirica possesses substantial ecological and economic value and is recognized as an emerging woody energy crop; however, its regeneration and genetic transformation remain challenging. The present study established an efficient regeneration and Agrobacterium tumefaciens-mediated transformation system for P. sibirica. Stem segments served as explants, and the optimal proliferation medium was Murashige and Skoog medium supplemented with 0.8 mg L-1 6-benzylaminopurine (6-BA), 0.2 mg L-1 thidiazuron (TDZ), and 0.1 mg L-1 1-naphthaleneacetic acid (NAA), resulting in a proliferation coefficient of 6.53. The genetic transformation system utilized A. tumefaciens strain GV3101 carrying reporter vectors pRI101-GFP or pCAMBIA1301-GUS, with optimal parameters including a 3-day preculture, infection at OD600 = 0.6 for 20 min, and a 2-day coculture. Transformants were selected in a medium containing 20 mg L-1 kanamycin and 300 mg L-1 cefotaxime, achieving an average transformation efficiency of 11.27%. As an initial application, PsWRKY17-overexpressing lines were generated, exhibiting transcript levels 7-18 times higher than those in the empty-vector control. Following low-temperature treatment, PsWRKY17-OE lines demonstrated increased SOD and POD activities and reduced relative electrical conductivity and MDA content compared to the control (p < 0.01). These results provide preliminary physiological evidence that PsWRKY17 overexpression is associated with altered low-temperature stress responses in P. sibirica. However, the regulatory mechanism of PsWRKY17 requires further investigation. In summary, an effective regeneration and genetic transformation system for P. sibirica was developed, providing a foundation for future research and biotechnological applications.
Shipeng Wang, Jianhua Chen, Yongqiang Sun et al.· Physiologia Plantarum : An I...· 0 citations
Efficient
Agrobacterium
-mediated transformation (AMT) is vital for the biotechnological improvement of sugarcane (
Saccharum
spp.).
Saccharum officinarum
is the main ancestor of all modern cultivars, yet little research has been conducted on its AMT system. In this work, an efficient AMT protocol for
S. officinarum
was developed, with Black Cheribon as the model genotype owing to its superior tissue culture performance and regeneration capacity. The optimized agro-infection protocol comprised the following main parameters: concentration of acetosyringone (AS) in
Agrobacterium
culture, concentration of AS for infection,
Agrobacterium
concentration at OD600 = 0.4, infection time of 30 minutes, vacuum infiltration time of 10 minutes and co-cultivation time of 3 days. To further improve transformation efficiency, 0.5 mg/L thidiazuron and 200 mg/L citric acid were added to the regeneration medium, which enhanced the regeneration of shoots. A modified stage-dependent selection strategy (FlexII) was established by using glufosinate-ammonium at concentrations of 2.0, 1.0, and 0.75 mg/L in the callus proliferation, shoot regeneration, and rooting stages, respectively. This strategy was more successful than the minimum inhibitory concentration-based strategy in
S. officinarum
transformation. The optimized protocol further boosted the transformation efficiency of Black Cheribon from 1.12% to 7.17%. The resulting transgenic lines were confirmed by PCR amplification of T-DNA regions and immunochromatographic detection of
Bar
protein expression in primary transformants, respectively. These results provide a sound technical foundation for the functional genomics and biotechnological optimization of
S. officinarum
germplasm, and may serve as a reference for future transformation studies in other sugarcane germplasm.
Chunjia Li, Xu-Juan Li, Chunyan Tian et al.· Frontiers in Plant Science· 0 citations
Solanum retroflexum
is cultivated for both its fruit and leaves in Africa, China, India, and Indonesia. However, it is still considered an underutilized crop due to limited genetic and genomic resources to support improvement. To enable gene function studies for future improvement by new breeding technologies, this study established efficient plant regeneration and
Agrobacterium tumefaciens
–mediated transformation approaches. Cotyledon and hypocotyl explants were used to test plant regeneration on a Murashige and Skoog salts-based medium supplemented with 0.5 mg L
−1
zeatin. Both explant types showed 100% regeneration; however, cotyledons produced a greater number of whole plants than hypocotyls. For transformation experiments, cotyledon explants were infected with
A. tumefaciens
AGL1 carrying the pJL33 binary vector containing the
Green Fluorescent Protein
(
GFP
) reporter and the
Neomycin Phosphotransferase-II
(
nptII
) selectable marker genes. Following cocultivation, firstly, kanamycin at various concentrations (75.0 to 400.0 mg L
−1
) was evaluated to determine the optimal concentration for maximizing transgenic line recovery. However, a significant number of escapes even at 400.0 mg L
−1
kanamycin were observed. Because the
nptII
gene also confers resistance to G418, in this study’s subsequent experiments the current authors tested the effectiveness of a range of G418 concentrations (25.0 to 150.0 mg L
−1
). Fluorescence microscopy and PCR analysis showed that 25.0 mg L
−1
G418 resulted in the highest transformation efficiency at 88% and the fewest escapes. Ploidy levels of regenerated, transgenic, and wild-type plants were assessed using flow cytometry, confirming stable ploidy. The optimized selection medium facilitated an efficient and stable transformation system that will be utilized to support crop improvement efforts of
S. retroflexum
.
Julie Thakur, Marina Martínez-López, J. Van Eck· Plant· 0 citations
Plant genetic transformation is a biotechnological tool with a high impact on plant breeding, since it offers the possibility of introducing new traits in crops to fulfill the demands of production and consumption. The most widely used transformation strategy in potatoes is based on the integration of genes via A. tumefaciens. The objective of the present work was to develop a transformation protocol for Solanum tuberosum cv. Spunta mediated by A. tumefaciens using hygromycin as a selective agent. For this, explants of potato cv. Spunta were transformed using A. tumefaciens with a binary vector containing the hygromycin resistance transgene (hpt) under the control of the NOS promoter. Molecular and phenotypic determinations were performed to validate both the efficiency of the transformation protocol and the performance of hygromycin as a selection agent. Sixty-two regenerated shoots were obtained from explants inoculated with A. tumefaciens (62% regeneration), of which 30 plants were positive for the hpt gene. A thirty percent transformation efficiency, calculated as the number of transgenic plants / the number of explants inoculated, was obtained using this protocol. The present work presents a simple and efficient protocol for the genetic transformation of potato cv. Spunta by A. tumefaciens using hygromycin as a selective agent.
Décima Oneto, Coronel, Storani et al.· Truth in Research· 0 citations
Tomato (Solanum lycopersicum L.) is one of the most economically important horticultural crops in China, and Xinjiang is the main region in China for processing tomato production. The establishment of a robust in vitro regeneration system is a fundamental prerequisite for genetic improvement and cultivar innovation in this crop. Among the factors influencing regeneration efficiency, exogenously applied plant growth regulators (PGRs) are the most important because they regulate endogenous phytohormone homeostasis and signaling pathways. In this study, true leaf explants of the processing tomato Ligeer 87-5 were used to optimize combinations of 6-benzyladenine (6-BA) and indole-3-acetic acid (IAA) and to establish an efficient in vitro regeneration system. Full strength MS medium supplemented with 2 mg/L 6-BA and 0.2 mg/L IAA was optimal for callus induction and organogenic competence, resulting in a callus induction rate of 97.00%, an embryogenic cell incidence of 63.88%, and an adventitious bud formation rate of 21.33% after 20 days; the budding rate further increased to 63.00% after 30 days. For rapid shoot induction, a medium composed of MS + 1 mg/L 6-BA + 0.2 mg/L IAA yielded the highest adventitious bud formation rate, reaching 89.00% after 10 days. Rooting was most effective on MS medium supplemented with 0.2 mg/L IAA, achieving a 100% rooting rate after 21 days, an average root length of 11.76 cm, a 76.67% acclimatization rate after 28 days, and a 100% survival rate after hardening and transplantation. Collectively, the stage-specific application of different media substantially improved true-leaf regeneration efficiency in processing tomato plants, providing a robust platform for future genetic transformation and molecular breeding.
Jiang-Yuan Zhang, Hongrun Zhou, Haoran Kan et al.· PeerJ· 0 citations
The present study addresses optimization of in-vitro regeneration via direct organogenesis and Agrobacterium-mediated genetic transformation, enabling efficient multiplex CRISPR/Cas9-based genome editing of the phytoene desaturase (PsPDS) gene in pea. Pea (Pisum sativum L.) is an important legume crop valued for food, plant-based protein, vegetable, and green manure. Although genome editing offers a precise and rapid strategy for crop improvement, its application in pea remains challenging due to inherent recalcitrance to in-vitro regeneration and genotype-dependent transformation. The regeneration and Agrobacterium-mediated transformation systems were optimized, and the dicotyledonary node (DCN) was identified as the preferred explant for multiplex CRISPR/Cas9-based genome editing in pea. Among three explant types (embryonic axis, DCN and nodal segment), DCN showed the highest regeneration efficiency, producing 100% shoot bud induction and 39.70 shoots per explant on MS medium augmented with 6-benzylaminopurine (BAP; 6.00 mg/L) and kinetin (1.00 mg/L). Shoot elongation and rooting efficiencies were improved using GA3 (1.00 mg/L), BAP (1.00 mg/L), IAA (0.10 mg/L), and NAA (0.5 mg/L), respectively. Manipulating explant type, Agrobacterium optical density, vacuum infiltration, acetosyringone concentration, infection time, and co-cultivation duration improved the transient transformation efficiency. We noted efficiency from 23.33% to 90.00% in DCN and from 6.66% to 93.33% in embryonic axis explants across 10 pea cultivars. Stable transformed lines generated from the DCN of cultivar Kashi Samridhi were confirmed by GUS staining and PCR. The optimized regeneration and transformation system facilitated targeted editing of phytoene desaturase (PsPDS) in pea, achieving ICE-estimated mutation frequencies of upto 97% in independent lines. The study provides a robust platform for functional genomics and accelerates the deployment of genome-editing technologies for pea improvement.