Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review examines the criteria to verify plant transformation; the biological and practical requirements for transformation systems; the integration of tissue culture, gene transfer, selection, and transgene expression strategies to achieve transformation in recalcitrant species; and other constraints to plant transformation including regulatory environment, public perceptions, intellectual property, and economics. Because the costs of screening populations showing diverse genetic changes can far exceed the costs of transformation, it is important to distinguish absolute and useful transformation efficiencies. The major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. This will require answers to a series of biological and technical questions, some of which are defined.

PLANT TRANSFORMATION: Problems and Strategies for Practical Application

Plant transformation via Agrobacterium can be limited by both host specificity and the inability of Agrobacterium to reach the proper cells in the target tissue. Described here is a new and efficient Agrobacterium-based transformation technology that overcomes these barriers and enhances DNA transfer in such diverse plant groups as dicots, monocots, and gymnosperms. This new technology, called sonication-assisted Agrobacterium-mediated transformation (SAAT), involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. Scanning electron and light microscopy reveal that SAAT treatment produces small and uniform fissures and channels throughout the tissue allowing the Agrobacterium easy access to internal plant tissues. Unlike other transformation methods, this system has the potential to transform meristematic tissue buried under several cell layers. SAAT increases transient transformation efficiency in several different plant tissues including leaf tissue, immature cotyledons, somatic and zygotic embryos, roots, stems, shoot apices, embryogenic suspension cells and whole seedlings. A 100- to 1400-fold increase in transient β- glucuronid ase expression has been demonstrated in various tissues of soybean, Ohio buckeye, cowpea, white spruce, wheat and maize. Stable transformation of both soybean and Ohio buckeye has been obtained using SAAT of embryogenic suspension culture tissues. For soybean, SAAT treatment was necessary to obtain stable transformation with this tissue

SAAT: sonication-assisted Agrobacterium-mediated transformation

An Agrobacterium-mediated plant transformation system was developed for the generation of transgenic banana (Musa spp. van Grand Nain). This system allowed for the recovery of putative transformants within four weeks after co-cultivation of tissue samples with Agrobacterium. Two or more cycles of meristem rooting and micropropagation allowed for the selection of plants from this putative transformant population which demonstrated chromosomal integration of foreign DNA by Southern analysis with no indication of chimeric tissues. Since plant breeding strategies aimed at banana crop improvement are extremely complex and long-term, virtually all commercial production is from clonal derivatives of naturally occurring variants. The genetic transformation technology reported herein will provide an additional tool for crop breeders who wish to introduce value-added traits into the banana and plantain cultivars that serve as vital food sources and a means of generating export income for producing nations.

Generation of transgenic banana (musa acuminata) plants via agrobacterium-mediated transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

Agrobacterium-mediated sorghum transformation.

A major limitation in producing transgenic soybeans [Glycine max (L.) Merrill] using the Agrobacterium-mediated cotyledonary-node method is low-frequency T-DNA transfer from Agrobacterium tumefaciens into cotyledonary-node cells. We increased Agrobacterium infection from 37% to 91% of explants in the cotyledonary-node region by amending the solid co-cultivation medium with L-cysteine, which resulted in a fivefold increase in stable T-DNA transfer in newly developed shoot primordia. Southern analysis detected greater than a twofold increase in transformation efficiency, as determined by the number of independent fertile, transgene plants per explants inoculated. Enzymatic browning on explant tissue was also reduced, which suggests cysteine may interact with wound- and pathogen-defense responses in the soybean explant, resulting in an increased T-DNA delivery into the cotyledonary-node cells.

L -Cysteine increases Agrobacterium -mediated T-DNA delivery into soybean cotyledonary-node cells

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.

Lynn Sims

Papers

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens.