The present invention relates to plant cell transformation and regeneration into a differentiated transformed plant. More particularly, the invention relates to a method for transforming soybean (Glycine max) using Agrobacterium-mediated transformation of a plant tissue explant and subsequent regeneration of the transformed cells into a whole plant.
Cultivated soybean (Glycine max) has a substantial commercial value throughout the world. Over 50 million hectares worldwide are used to produce an annual crop of soybeans in excess of 100 metric tons with an estimated value exceeding 20 billion dollars. The development of scientific methods useful in improving the quantity and quality of this crop is, therefore, of significant commercial interest.
Modern biotechnological research and development has provided useful techniques for the improvement of agricultural products by plant genetic engineering. Plant genetic engineering involves the transfer of a desired gene or genes into the inheritable germline of crop plants such that those genes can be bred into or among the elite varieties used in modern agriculture. Gene transfer techniques allow the development of new classes of elite crop varieties with improved disease resistance, herbicide tolerance, and increased nutritional value. Various methods have been developed for transferring genes into plant tissues including high velocity microprojection, microinjection, electroporation, direct DNA uptake, and Agrobacterium-mediated gene transformation.
Agrobacterium-mediated gene transformation is the most widely used gene transfer technique in plants. This technique takes advantage of the pathogenicity of the soil dwelling bacteria, Agrobacterium tumefaciens or Agrobacterium rhizogenes. Agrobacterium tumefaciens natively has the ability to transfer a portion of its DNA, called T-DNA, into the genome of the cells of a plant to induce those cells to produce metabolites useful for the bacterium""s nutrition. Agrobacterium-mediated transformation takes advantage of this concept by replacing the T-DNA of an Agrobacterium with a foreign set of genes, thus, making the bacterium a vector capable of transferring the foreign genes into the genome of the plant cell. Typically, the foreign gene construct that is transferred into the plant cell involves a specific gene of interest, which is desired to be introduced into the germline of the plant, coupled with a selectable marker that confers upon the plant cell a resistance to a chemical selection agent. Typically, the Agrobacterium-mediated gene transfer is into an undifferentiated cell cultivated in tissue culture, known as a callus cell, or the transfer is made into a differentiated plant cell from a leaf or stem, which is then induced to become an undifferentiated callus culture.
Although significant advances have been made in the field of Agrobacterium-mediated transformation methods, a need continues to exist for improved methods to facilitate the ease, speed and efficiency of such methods for transformation of soybean plants.
The present invention provides a novel and efficient method of performing germline transformation of soybean using Agrobacterium-mediated transformation directly on meristematic cells of soybean embryos. Direct shoot induction from transformed meristematic cells results in germline transgenic plants. The overall process is rapid and efficient.
One significant aspect of this invention is that the reduction of the pretreatment period of soybean seeds has improved the shoot production in surviving explants as well as reduced the time taken to produce plants that are transferable to a greenhouse. Also, the reduction of time and materials provides a system that is economically beneficial to those who implement it.
It is an object of the invention to provide a rapid and efficient method to perform soybean genetic transformation using Agrobacterium-mediated gene transfer.
It is another object of the present invention to provide a soybean transformation method not requiring a step of callus culture so that the method can be used on any soybean variety.
Another aspect of the present invention is to provide novel methods of wounding to increase transformation efficiency. One method of wounding involved exposing soybean embryos to ultrasonic sound waves (i.e., sonication). Another method involves wounding via a plasma blast of an electric gene gun.
Other objects, advantages, and features of the present invention will become apparent from the following specification.
The present invention is a method for the direct germline genetic transformation of varieties of soybean, Glycine max. This method is based on Agrobacterium-mediated gene delivery into individual soybean cells in the meristem of a soybean embryo. The transformed cells are then induced to form shoots that are, at a high frequency, germline soybean transformants that can be cultivated into whole sexually mature and fertile transgenic soybean plants. The method does not involve a phase of callus culture, and hence the time period of the entire process from seed to transgenic seed is remarkably concise.
The method described here is based on Agrobacterium-mediated gene delivery into growing cells in an embryonic meristem. Agrobacterium-mediated techniques typically only result in gene delivery into one, or only a few, cells in the targeted tissue. Typically, a selective agent is applied post-transformation to kill all of the cells in the targeted tissues that are not transformed or to identify transformed cells through a selective advantage. Then a callus or other proliferative growth of transformed cells can be grown from which plants can be ultimately regenerated.
The method described here does not utilize a callus or proliferative phase. Instead, the Agrobacterium-mediated gene delivery is made into cells in the living meristem of a soybean embryo excised from a soybean seed. Then the meristematic region is cultured in the presence of a selection agent and a hormone to induce direct shoot formation. Preferably, the meristem is cultivated in the presence of the herbicide glyphosate, which acts both as a selection agent as well as a shoot-inducing hormone. The result of this step is the termination or at least growth retardation of most of the cells into which the foreign genetic construction has not been delivered and the simultaneous induction of the formation of soybean shoots, which arise from a small cluster of cells including a transformed meristematic cell. The meristem can also be cultivated in the presence of a selection agent, including, but not limited to, kanamycin.
This method is cultivar independent. The soybean tissue manipulations in this process are analogous to those in prior particle-mediated transformation methods, which have proven to be adaptable to all tested elite soybean varieties. This method is equally adapted for direct genetic transformation into elite soybean cultivars, thus potentially avoiding the need for extensive cross-breeding between varieties.
The time period required for this method is greatly reduced compared to other Agrobacterium-mediated transformation protocols. The soybean embryos are exposed to Agrobacterium transformation as soon as 6-14 hours after seed imbibition, are co-cultured for one to four days, and are then subjected to post-transformation selection. Viable phenotypically positive soybean shoots can be collected 3 to 6 weeks from the initiation of the procedure. The entire R0 (primary transformant) plant life cycle is not greatly longer than the minimum required for a soybean plant to grow to maturity in a greenhouse.
As with other Agrobacterium-mediated methods, the foreign genetic construction, or transgene, to be inserted into the soybean genome is created in vitro by normal techniques of recombinant DNA manipulations. The genetic construct is then transformed into the Agrobacterium strain for delivery into the soybean cells. The Agrobacterium is non-oncogenic, and several such strains are now widely available. The foreign genetic construction includes a selectable marker gene. Several such selectable marker genes are known, such as the gene for neomycin phosphotransferase II (NPT II), which expresses an enzyme conferring resistance to the antibiotic kanamycin and the related antibiotics neomycin, paromomycin, gentamicin, and G418. However, a preferred type of selectable marker gene is one of the genes conferring resistance to the herbicide glyphosate, such as the EPSP synthase gene described in U.S. Pat. No. 5,633,435 or the glyphosate oxidoreductase gene described in U.S. Pat. No. 5,463,175.
The starting material for the transformation process is a soybean seed. The seed is first soaked for softening and then induced to initiate germination. The seeds are imbibed in water for approximately 3 minutes and then allowed to soften for up to 2 hours. The softening step is not required for every seed lot. High quality seeds need less softening than low quality seeds. The softening step is to help keep the meristem intact. The seeds are then put on germination media and permitted to begin germination for a time period of about 6-24 hours, preferably for about 6-14 hours, and most preferably for about 8-12 hours.
Then the embryo is excised from the seed, and any primary leaf tissues are removed to expose the meristem of the soybean embryo.
For Agrobacterium-mediated gene transfer, wounding of the plant tissue is known to facilitate gene transfer. Therefore it is preferred, but not necessary, that the embryonic meristem is wounded at this step of the process. Many methods of wounding can be used, including, for example, cutting, abrading, piercing, poking, penetration with fine particles or pressurized fluids, plasma wounding, application of hyperbaric pressure, or sonication. Wounding can be performed using objects such as, but not limited to, scalpels, scissors, needles, abrasive objects, airbrush, particles, electric gene guns, or sound waves. Another alternative is vacuum infiltration. The procedure to this point will have typically taken 12-14 hours.
Preferably the wounding is done by sonication or plasma blast wounding. Sonication can be done in a bath sonicator or with a probe sonicator. A wetting agent, such as, but not limited to, Triton X-100 may or may not be used. Sonication can be done for between about 5 sec and about 10 min, preferably between about 5 seconds and about 40 seconds. Sonication is preferably done in the presence of the Agrobacterium.
Plasma blast wounding is done using an electric gene gun. Embryos are placed from 3-6 cm, preferably about 4 cm from the exposed electrodes, with 10 xcexcL of water between them. A Plexiglass dome is then placed over the chamber and a partial vacuum is applied concurrent with an influx of helium gas. A minimum of about 16,000 volts is discharged through the water, vaporizing the water, and creating a plasma and shock wave engulfing the embryos. The embryos are then placed into the Agrobacterium culture.
The explants are then inoculated with the Agrobacterium culture for a few minutes to a few hours, typically about 0.5-3 hours. The excess media is drained and the Agrobacterium are permitted to co-cultivate with the meristem tissue for several days, typically three days in the dark. During this step, the Agrobacterium transfers the foreign genetic construct into some cells in the soybean meristem.
Next the explants are transferred to a medium containing the selection agent and appropriate antibiotics. This step is intended to terminate or at least retard the growth of the non-transformed cells and kill the remaining Agrobacterium cells. The length of culture depends, in part, on the toxicity of the selection agent to untransformed cells. For glyphosate selection, a two-day culture is effective, but the length of this culture step is variable, extending from one to seven days. For kanamycin selection, the explants are cultured from one to seven days.
Following this step, the meristems are placed in a medium conducive to shoot development for 3-7 days. The MSR medium used in the examples below contains benzylaminopurine (BAP), a shoot-inducing hormone. Glyphosate itself has also been found to induce shoot formation in soybean. The term hormone also includes cell growth regulating compounds that induce shoot formation, including, but not limited to, IAA, NAA, IBA, cytokinins, auxins, kinetins, glyphosate, and thiadiazorun. Whichever hormonal treatment is used, the individual transformed cells in a meristem give rise to transgenic sectors of tissue that are incorporated to a varying extent into the shoot arising directly from the explant. After culture on the MSR medium, the explants are transferred to WPM-BAP (a medium suitable for shoot development) for 4-5 weeks.
The elongated shoots are ready for harvest 3-6 weeks after the start of the entire transformation process. The shoots are evaluated for phenotypic regularity and health, and only shoots with elongated stems (approximately 1 inch) and full trifoliate leaf formation are harvested. The collected shoots are placed on a rooting medium to induce root formation. Root formation takes approximately 1-4 weeks, following which the plants can be transferred to soil and grown to full maturity. Ideally, the rooting medium also contains the selection agent, to help to terminate any non-transformants.
The R0 plants created by this technique are transgenic plants and are regularly recovered with quite reasonable yields. The number of independent germline plant lines recovered is usually in the single digit percentage number range. Thus, a repeat of this procedure on 100 planted soybean meristems would typically yield 1-10 independent lines of transgenic soybean.