Aug 2026· Plant Cell Tissue and Organ Culture· Vol 166· 0 citations· 84 references
TL;DR
An efficient protocol for protoplast isolation from embryogenic calli and protoplast-to-plant regeneration in the allotetraploid species Coffea arabica is presented and polyethylene glycol-mediated transfection of coffee protoplasts with a CRISPR–Cas9 plasmid targeting the coffee xanthosine methyltransferase gene involved in caffeine biosynthesis is demonstrated.
Abstract
Conventional coffee breeding is a long and complex process that limits genetic improvement, highlighting the need for advanced biotechnological tools. Protoplast-based CRISPR genome editing is a promising transgene-free approach, which enables precise genetic modifications and whole-plant regeneration while avoiding the stable integration of foreign DNA. However, reliable plant regeneration from protoplasts remains a significant challenge in most crops, especially in tropical tree species like coffee. Here, we present an efficient protocol for protoplast isolation from embryogenic calli and protoplast-to-plant regeneration in the allotetraploid species Coffea arabica. Culture conditions were optimised by comparing protoplast-derived cell growth in liquid medium versus alginate layers and by assessing medium supplementation with the peptide growth factor phytosulfokine-α (PSK) or polyamines (spermine and putrescine). The alginate-embedding system, combined with 0.1 µM or 1 µM of PSK, significantly enhanced early cell division frequencies (up to 50%) and facilitated the formation of up to 270 microcalli.cm−2, enabling subsequent whole-plant regeneration using somatic embryogenesis. Although spermine also favoured cell division, its effect was weaker than that of PSK, and no synergistic interaction was observed when combined with PSK. Furthermore, we demonstrated successful polyethylene glycol-mediated transfection of coffee protoplasts with a CRISPR–Cas9 plasmid targeting the coffee xanthosine methyltransferase (XMT) gene involved in caffeine biosynthesis. Subgenome-specific PCR and deep sequencing revealed insertion/deletion mutations at target sites in ca. 4% of protoplasts. Collectively, these findings pave the way for transgene-free genome editing and functional genomics studies based on cutting-edge technologies in coffee.
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.
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
Transgene-free genome edited plants were regenerated from protoplasts, representing the first report of RNP mediated genome editing in eggplant protoplasts.
M. Ferrero, M. N. González, I. Perrone et al.· Frontiers in Plant Science· 0 citations
An effective system for isolating and regenerating protoplasts is crucial for research in genome engineering. This study focused on refining a protocol for the isolation and regeneration of mesophyll protoplasts from the leaves of Solanum tuberosum cv. Kufri Jyoti. Key factors influencing protoplast yield and viability, such as dark pretreatment, pre-plasmolysis, enzyme concentrations, and osmoticum levels, were thoroughly assessed and optimized. The highest protoplast yield and viability were achieved with an enzyme mixture of 1.0% cellulase R-10 and 0.5% macerozyme R-10 after 16 h of incubation. Furthermore, culturing on a Murashige and Skoog-based medium (MSPI) without ammonium nitrate, enriched with an osmoticum concentration of 0.4 M and a carefully adjusted auxin-to-cytokinin ratio, successfully facilitated protoplast division, microcalli proliferation, and minicalli formation. Callus proliferation and shoot induction were accomplished on MS13K medium supplemented with naphthaleneacetic acid (NAA) and zeatin riboside. Root initiation and elongation were promoted on MS basal medium supplemented with indole-3-butyric acid (IBA) at 1 mg/L. The regenerated plantlets were subsequently acclimatized and hardened under controlled greenhouse conditions. This robust protoplast-to-plant protocol serves as a crucial resource for the introduction of ribonucleoprotein complexes into plant cells, facilitating accurate, transgene-free genome editing.
Nehanjali Parmar, Shruti Pathania, Vanita Patial et al.· Journal of Genetic Engineeri...· 0 citations
A new protoplast-based platform enables transient transformation and proof-of-concept CRISPR/Cas12a-mediated genome editing in European beech, a key but recalcitrant forest tree species.
Virginia Zahn, Alice-Jeannine Sievers, B. Kersten et al.· Communications Biology· 0 citations
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.
Xiao-Yue Zheng, Hao Li, Shu Wang et al.· BioTech· 0 citations
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