Isolation of Protoplasts from Nepenthes - A Plant Carnivore

Sweat, Jaylen; Bodri, Michael S.

doi:10.3329/ptcb.v24i1.19217

Cited by 4 publications

(3 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The type and concentration of enzyme used to isolate protoplasts from suspension-cultured cells is selected based on the species used. Cellulase degrades cell walls by modifying celluloses, lichenin, and cereal β-D-glucans (Sweat and Bodri, 2014), whereas pectolase breaks down the pectins in cell walls. Cellulase R-10 and Pectolase Y-23 are commonly used to isolate protoplasts from suspension cells in wheat (Triticum aestivum) (Kenzhebaeva et al, 2001), tobacco (Cynodon dactylon L.) (Li et al, 2006), populus (Populus alba L.) (Jing et al, 2007), and apple (Wei et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.)

Yao

Liao

Gan

et al. 2016

Scientia Horticulturae

View full text Add to dashboard Cite

Transient gene expression in plant protoplasts is a powerful tool for analyzing gene function and for performing biotechnical manipulations. Here we report the isolation of viable protoplasts from the fruit flesh of sweet cherry (Prunus avium L.) cv. Hong Deng and their polyethylene glycol (PEG)-mediated transient transfection using green fluorescent protein (GFP) as a marker gene. We investigated the main factors affecting the efficacy of protoplast isolation and transfection, including the composition of the enzymolysis solution, enzymolysis time, pH of the enzymolysis solution, PEG concentration, and transfection time. Protoplast isolation was optimal when the tissue was incubated in enzymolysis solution composed of 1.0% Cellulase R-10, 0.5% Pectolase Y-23, and 0.6 M mannitol (pH 5.8) for 18 h, resulting in a protoplast yield of 4.3×10 6 protoplasts/g fresh weight [FW] and viability of 84.1%. Protoplast transformation efficiency was measured by transient expression of the GFP reporter gene, and transformation efficiency was highest when protoplasts were incubated in transfection medium containing 40% PEG for 15 min. Collectively,

show abstract

Section: Discussionmentioning

confidence: 99%

Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.)

Yao

Liao

Gan

et al. 2016

Scientia Horticulturae

View full text Add to dashboard Cite

show abstract

“…The macerozyme R-10, as a pectin-degrading enzyme, is commonly used to break down the pectin in plant tissues, causing the cells bound together to dissociate into living individual cells. A suitable concentration of the macerozyme R-10 may significantly enhance the efficiency to degrade the pectin and isolate single cells in Populus simonii × P. nigra [46][47][48][49].…”

Section: Discussionmentioning

confidence: 99%

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

Yang,

Sun,

Sun

et al. 2024

Plant Methods

View full text Add to dashboard Cite

Background Populus simonii × P. nigra is an ideal material for studying the molecular mechanisms of woody plants. In recent years, research on Populus simonii × P. nigra has increasingly focused on the application of transgenic technology to improve salt tolerance. However, the rapid characterization of gene functions has been hampered by the long growth cycle and exceedingly poor transformation efficiency. Protoplasts are an important tool for plant gene engineering, which can assist with challenging genetic transformation and the protracted growth cycle of Populus simonii × P. nigra. This study established an optimized system for the preparation and transformation of protoplasts from Populus simonii × P. nigra leaves, making genetic research on Populus simonii × P. nigra faster and more convenient. Major Latex Protein (MLP) family genes play a crucial role in plant salt stress response. In the previous study, we discovered that PsnMLP328 can be induced by salt treatment, which suggested that this gene may be involved in response to salt stress. Protein localization is a suggestion for its function. Therefore, we conducted subcellular localization analysis using protoplasts of Populus simonii × P. nigra to study the function of the PsnMLP328 gene preliminarily. Results This study established an optimized system for the preparation and transformation of Populus simonii × P. nigra protoplasts. The research results indicate that the optimal separation scheme for the protoplasts of Populus simonii × P. nigra leaves included 2.5% cellulase R-10, 0.6% macerozyme R-10, 0.3% pectolyase Y-23, and 0.8 M mannitol. After enzymatic digestion for 5 h, the yield of obtained protoplasts could reach up to 2 × 107 protoplasts/gFW, with a high viability of 98%. We carried out the subcellular localization analysis based on the optimized transient transformation system, and the results indicated that the MLP328 protein is localized in the nucleus and cytoplasm; thereby proving the effectiveness of the transformation system. Conclusion In summary, this study successfully established an efficient system for preparing and transforming leaf protoplasts of Populus simonii × P. nigra, laying the foundation for future research on gene function and expression of Populus simonii × P. nigra.

show abstract

“…N. adrianii is dioecious or possessing male and female flowers separated in different individuals. In their natural habitat, male N. adrianii individuals are much more easily found than females, as is the case in most other pitcher plant species (Adam et al, 2011;Sweat & Bodri, 2014;Handayani, 2017). Meanwhile, sex determination in dioecious plants is crucial, mainly when each sex will be developed as individuals of economic significance, and distinguishing sex at an early stage of development is considerably challenging to do (Zarek, 2018).…”

Section: Introductionmentioning

confidence: 91%

Sex identification in Nepenthes adrianii from Baturraden Botanical Garden: Genetic analysis using RAPD Markers

Dwiati

Aziiza

Susanto

2021

bio

View full text Add to dashboard Cite

Nepenthes adrianii is one of pitcher plant species that grows endemically in Mount Slamet, Central Java. At present, it is one of pitcher plant collections of Baturraden Botanical Garden. Since N. adrianii is dioecious and both sexes are difficult to distinguish morphologically, early sex determination supporting its conservation at Baturraden Botanical Garden is needed. One approach can be performed with the use of RAPD molecular markers. Hence, this study aims to know whether differences in RAPD pattern between male and female N. adrianii exist or not and also to find out what the differences are. Genomic DNAs were extracted from leaves of 4 males, 2 females, and 2 sexually unidentified individuals. The extracted DNAs were then used to analyze DNA variation between male and female N. adrianii employing RAPD technique. As many as five oligonucleotide primers (OPA-15, OPK-16, OPP-15, OPP-08, and OPO-08) were used to amplify N. adrianii DNA. The results showed that one primer, i.e. OPK-16 (5’-GAGCGTCGAA-3’), produces a specific band of approximately 290 bp which is only found in female plants. It is assumed that this band is related to gene(s) controlling sex determination in N. adrianii. The RAPD marker can be used for the sex determination of young N. adrianii seedlings.

show abstract

Isolation of Protoplasts from Nepenthes - A Plant Carnivore

Cited by 4 publications

References 9 publications

Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.)

Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.)

Optimization of preparation and transformation of protoplasts from Populus simonii × P. nigra leaves and subcellular localization of the major latex protein 328 (MLP328)

Sex identification in Nepenthes adrianii from Baturraden Botanical Garden: Genetic analysis using RAPD Markers

Contact Info

Product

Resources

About