Protoplast

About: Protoplast is a research topic. Over the lifetime, 5474 publications have been published within this topic receiving 122468 citations.

Plant cell culture : a practical approach

Abstract: 1: Isolation and maintenance of callus and cell suspension cultures. 2: Isolation, culture, and regeneration of protoplasts. 3: Applications of protoplast technology. 3.A: Fusion and selection of somatic hybrids. 3.B: Transient gene expression and stable transformation. 3.C: Studies with viruses. 4: Selection of plant cells for desirable characteristics. 4.A: Inhibitor resistance. 4.B: Cold tolerance. 4.C: In vitro selection for salt tolerance. 4.D: In vitro selection for disease/toxin resistance. 5: Plant regeneration via embryonic suspension cultures. 6: Applied aspects of plant regeneration. 6.A: Micropropagation. 6.B: Virus-free plants. 6.C: Artificial seeds. 7: Cryopreservation. 8: Secondary products from cultured cells and organs I: molecular and cellular approaches. 9: Secondary products from cultured cells and organs II: large scale culture

Self‐fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs

Abstract: A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R0 and R1 progeny plants. Segregation of the PAT activity and herbicide resistance in R1 progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R1 progeny.

High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Abstract: Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).

Transgenic rye plants obtained by injecting DNA into young floral tillers

Abstract: The most successful method currently used to produce transgenic plants is based on the ability of Argobacterium tumefaciens to infect a number of higher plant species and transfer a defined DNA fragment (T-DNA) to the genome of the infected cells1. This procedure cannot be used with the agriculturally important cereals, although preliminary data have suggested2 infection of maize seedlings by A. tumefaciens strains. Purified exogenous DNA can be taken up, integrated and expressed in cells of a variety of plant species including some cereals following direct gene transfer into isolated protoplasts3–7. However, although it is possible to grow isolated cereal protoplast into unorganized tissue (calli)5,7,8, only rice protoplasts have so far been shown to regenerate mature plants9–11. Here we report an alternative approach to transformation of cereal plants which does not involve tissue culture techniques.