Overcoming roadblocks for in vitro nurseries in plants: induction of meiosis.

Efforts to increase genetic gains in breeding programs of flowering plants depend on making genetic crosses. Time to flowering, which can take months to decades depending on the species, can be a limiting factor in such breeding programs. It has been proposed that the rate of genetic gain can be increased by reducing the time between generations by circumventing flowering through the in vitro induction of meiosis. In this review, we assess technologies and approaches that may offer a path towards meiosis induction, the largest current bottleneck for in vitro plant breeding. Studies in non-plant, eukaryotic organisms indicate that the in vitro switch from mitotic cell division to meiosis is inefficient and occurs at very low rates. Yet, this has been achieved with mammalian cells by the manipulation of a limited number of genes. Therefore, to experimentally identify factors that switch mitosis to meiosis in plants, it is necessary to develop a high-throughput system to evaluate a large number of candidate genes and treatments, each using large numbers of cells, few of which may gain the ability to induce meiosis.

Studies on Pure Mlb® (Multiple Left Border) Technology and Its Impact on Vector Backbone Integration in Transgenic Cassava.

Imperfect T-DNA processing is common during Agrobacterium-mediated transformation, which integrates vector backbone sequences into the plant genome. However, regulatory restrictions prevent such transgenic plants from being developed for commercial deployment. The binary vector pCAMBIA2300 was modified by incorporating multiple left border (Mlb®) repeats and was tested in BY2 cells, tobacco, and cassava plants to address this issue. PCR analyses confirmed a twofold increase in the vector backbone free events in the presence of triple left borders in all three systems tested. Vector backbone read-through past the LB was reduced significantly; however, the inclusion of Mlbs® did not effectively address the beyond right border read-through. Also, Mlbs® increased the frequency of single-copy and vector backbone free events (clean events) twice compared to a single LB construct. Here, we briefly narrate the strength and limitations of using Mlb® technology and reporter genes in reducing the vector backbone transfer in transgenic events.

Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event.

Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (Lolium perenne L.)-associated Epichloë endophyte strains using A. tumefaciens-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.

Mesophyll Protoplasts and PEG-Mediated Transfections: Transient Assays and Generation of Stable Transgenic Canola Plants.

Plant protoplasts are derived by controlled enzymatic digestion that removes the plant cell wall without damaging the cell membrane. Protoplasts represent a true single-cell system and are useful for various biochemical and physiological studies. Protoplasts from several agriculturally important crop species can be regenerated into a fertile whole plant, extending the utility of protoplasts from transient expression assays to the generation of stable transformation events. Here we describe procedures for transient and stable transformation of leaf mesophyll protoplasts obtained from axenic shoot cultures of canola (Brassica napus). Key steps including enzymatic digestion for protoplast release, density gradient-based protoplast purification, PEG-mediated transfection, bead-type culturing (sea-plaque agarose and sodium alginate), and the recovery of putative transgenic canola plants are described. This method has been used for double-stranded DNA break-mediated genome editing and for the routine generation of stable transgenic canola events at commercial scale.

Clovers (Trifolium spp.).

Clovers (Trifolium spp.) constitute one of the major forage legumes widely grown for its rich protein content and its major role in maintaining environmental sustainability by improving the soil fertility. Gene technology can assist plant improvement efforts in clovers (Trifolium spp.), aiming to improve forage quality, yield, and adaptation to biotic and abiotic stresses. An efficient and reproducible protocol for Agrobacterium-mediated transformation of a range of Trifolium species, using cotyledonary explants and different selectable marker genes, is described. The protocol is robust and allows for genotype and Agrobacterium strain-independent transformation of clovers. Stable meiotic transmission of transgenes has been demonstrated for selected transgenic clovers carrying single T-DNA inserts recovered from Agrobacterium-mediated transformation. This methodology can also be successfully used for "isogenic transformation" in clovers: the generation of otherwise identical plants with and without the transgene from the two cotyledons of a single seed. Stable transgenes may be used in further functional genomics, develop new traits and profile gene expression using reporters, and facilitate purification of tissue or single cells.