WO2010140961A1

WO2010140961A1 - Plant protocol

Info

Publication number: WO2010140961A1
Application number: PCT/SE2010/050595
Authority: WO
Inventors: Li-hua ZHU
Original assignee: Zhu li-hua
Priority date: 2009-06-04
Filing date: 2010-05-31
Publication date: 2010-12-09

Abstract

The present invention provides methods for regeneration of whole plants from the explants obtained from the Crambe abyssinica. In addition,the present invention also provides methods for transforming Crambe abyssinica, plant cells and tissues with the use of recombinant Agrobacterium strain. An efficient method for regeneration and transformation of Crambe abyssinica is also disclosed. The invention also relates to transformed Crambe abyssinica, plant cells and tissues for improved properties,such as bio-based wax ester production.

Description

PLANT PROTOCOL

FIELD OF INVENTION

The present invention provides methods for regeneration of whole plants from the explants obtained from the Crambe abyssinica. In addition, the present invention also provides methods for transforming Crambe abyssinica, plant cells and tissues with the use of recombinant Agrobacterium strain An efficient method for regeneration and transformation of Crambe abyssinica is also disclosed. The invention also relates to transformed Crambe abyssinica, plant cells and tissues for improved properties, such as bio-based wax ester production.

BACKGROUND OF INVENTION

Genetic improvement of oilseed crops aiming at increasing oil yield or altering the fatty acid composition for nutritional, pharmaceutical and industrial uses has been a long held concept (Murphy, 1994, 1996, 1999). Since the late 1980s, many efforts have been made in this area and impressive achievements in isolating oil-related genes and producing transgenic plants with modified oil compositions have been reported (Lardizabal et al., 2000, Ujjal et al., 2009; Wiberg et al., 2000). However, commercialization of genetic modified (GM) oilseed crops with acceptable oil quality and quantity has not become the reality yet, especially for those with special or novel non-edible oil components (James, 2004). Apart from technological and social barriers surrounding gene technology, one major challenge about GM oilseed crops is how to separate them from food oilseed crops in large scale production. Currently, most oilseed crops in commercial cultivation are mainly for food purposes and strict regulatory legislations for approving these commodity varieties exist for the sake of food safety. On the other hand, GM oilseed crops with nonfood oils often have different or even novel components which may be undesirable in the food and feed chain. Although there is no evidence that such oil components are toxic, mixing of them into food oil at even trace levels may have far-reaching effects on commercial food oil production with the present regulatory legislation surrounding GM plants. Such a mistake has already been reported in maize (Macilwain, 2005). Apart from handling mixture during seed processing, outcross is the main risk causing a gene flow from GM plants into other oilseed crops. Up to now most potential GM oilseed crops with improved or novel oil quality are not acceptable by commercial production. One major reason is that these modified crops are quite ready to outcross with the major food oilseed crops.

Crambe abyssinica, belonging to the Brassicacea family, is an underexploited oil crop. As a potential dedicated oil crop for industrial oil production, Crambe has recently attracted more and more attention because it does not hybridize with any existing food oilseed crop, therefore eliminating the risk of gene flow into major food oilseed crops (Wang, 1998). The seed oil of Crambe contains 55-60 % erucic acid which makes the oil non-edible, but is useful starting material for developing other industrial oil products, such as, long chain wax esters. Crambe has already been commercially cultivated on small scales and the novel varieties can yield the same amount of oil per hectare as spring rapeseed does. Some research programmes through conventional breeding are going on to improve importantly agronomic traits, particularly yield and stress tolerance.

Genetic modification is an important complementary method to conventional breeding for improving plant properties. However, it had not been possible to transform Crambe abyssinica due to lacking of regeneration and transformation methods. Although Crambe belongs to the same family as other food oilseed crops, the regeneration and transformation protocols used in the same family do not work well on Crambe abyssinica. In the genus Crambe there are a huge amount of different species (crambe abyssinica; crambe aspera; crambe filiformis; crambe gigantea; crambe glabrata; crambe hispanica; crambe juncea; crambe laevigata; crambe maritime; crambe orientalis; crambe pinnatifida; crambe scaberrima and crambe tataria), which behave in different ways when it comes to transformation and regeneration.

A number of factors affect regeneration and transformation frequency and those factors need to be optimised prior to transformation of a new plant species. These include developing a regeneration process, developing a transformation process, finding a suitable genotype, selection system, choice of explant, types of bacteria to be used, etc. If the regeneration frequency could be increased the transformation efficiency will also be increased since more transformed cells will be regenerated into shoots, therefore there is a need of focusing on the regeneration process prior to the transformation process.

WO2009/067498 dicloses a method to transform Crambe species in which they utilises 0.5 mg/ml of TDZ as well as 0.5 mg/L NAA in the regeneration medium (see page 29). By the use of such a medium they claim that they obtain transformation frequencies of at least 1% up to at least 25%. The regeneration capacity was 10 shoots from 100 seedlings, i.e., 10 %. There is no discussion within the hole document about the importance of the NAA as well as TDZ concentrations or BAP, which is an indication that a person skilled in the art would focus on other things, such as the different explants and the different ways of transforming a plant. In the example I they are using different Agrobacterium strains, different amounts of geneticin and Timenton. In example II they show that the regeneration does not work and to increase the regeneration capacity they focus on producing a cell line having an increased regeneration capacity. Example II is focused on using a sub- population for regeneration purposes. In example V they use particle bombardment. This means that they are analysing a lot of different parameters to improve the transformation without mentioning the importance of the NAA, TDZ and BAP concentration and a person skilled in the art would focus on the parameters mention in the application, since there are so many parameters to evaluate upon setting up a new transformation and regeneration protocol for a new plant species and all the different species are unique. US4,665,031 discloses a process for vegetative propagation of Crambe

Maritima in which BAP is used in an amount of 1-2 mg/ml, preferably 1.5 mg/ml. Crambe Maritima being another species than Crambe abysinnica and a person skilled in the art being aware of how different species function would not even look into such a document SUMMARY OF THE INVENTION

Accordingly, an object of the invention is to provide regeneration as well as a transformation process for Crambe abyssinica. After evaluation of a number of parameters including the regeneration process, transformation process, suitable genotype, selection system, choice of explants, and evaluation of different types of bacteria the inventor has finally managed to develop a protocol for regeneration and transformation that efficiently works on Crambe abyssinica in such a way that the number of plants obtained in the end are enough to make any evaluation and in the end identify transgenic plants of interest that contains the new genes stably integrated in the genome over generations as well as express the integrated gene or genes. Thereby it is for the first time possible to regenerate new plants from Crambe abyssinica explants as well as introduce new heterologous genes into Crambe abyssinica in an efficient way. By focusing on the regeneration process and increasing this process by altering the amount of cytokinin it was surprisingly found that the regeneration could be increased up to more than 60 %. Thereby the number of transformants, i.e., cells which have been transformed will be increased as well since there will be an increase in the amount of the transformed cells. This is important as a large number of transgenic plants are required to be able to evaluate stable transgenic lines expressing the new introduced gene to an acceptable level which permits the production of offsprings which still express the heterologous gene. Thereby it may be possible to use Crambe abyssinica for the production of industrial wax esters in an industrial scale.

In a first aspect the invention relates to a method of regeneration of Crambe abyssinica plants, wherein said method comprising a) obtaining explants from either seeds, seedlings or whole plant, wherein the explants are selected from a group consisting of hypocotyl, cotyledon with petiole, embryo, immature embryo, anther, and root b) culturing the explants on a regeneration medium containing cytokinin and auxin to obtain multiple shoot buds or callus c) culturing the shoot buds on a rooting medium to obtain rooted plantlets; and d) transferring the rooted plantlets in soil, to obtain regenerated plants. In a second aspect the invention relates to a method for transforming Crambe abyssinica, wherein the said method comprising the steps of a) surface sterilising the seeds, and imbibing the seeds with water, b) germinating the seeds of step (a) in a suitable tissue culture medium to obtain seedlings, c) obtaining the explants from step (a) or step (b), d) co-cultivating the explants of step (c) with recombinant Agrobacterium strain, e) culturing the explants of step (d) on a suitable selection medium to select transformed plant cells and tissues, f) culturing the explants on a regeneration medium containing cytokinin and auxin to obtain multiple shoot buds or callus g) culturing the shoot buds on a rooting medium to obtain rooted plantlets; and h) transferring the rooted plantlets in soil, to obtain regenerated plants. By inventing two new methods a number of problems have been solved including, choices of regeneration protocol including cytokinins and auxins as well as the amounts required. To be able to develop an efficient regeneration protocol, a number of auxines as well as cytokinines were evaluated as well as the different amounts to be able to find the optimal ones. Accordingly, a number of explants were evaluated to identify that the best candidate was hypocotyl.

During the development of the transformation protocol a large number of parameters were needed to be evaluated which would work, including again choice of explant, transformation system such as Agrobacterium, biolistic etc, which type of Agrobacterium strain as well as how to perform the transformation to obtain stable transgenic plants expressing the introduced genes. BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1 Hypocotyl explants with regenerated buds 15 days after regeneration (A) and GUS staining of one transgenic line (B) Fig 2 Southern blotting of seven transgenic clones (Lanes 1-7) transformed with the binary vector pSCVl .6. The DNA was digested with EcoRI and hybridized with the nptll probe.

Fig 3 Southern blotting of the transgenic clones transformed with the binary vector pMS9 (Lanes 1-5) and vector pKan-Wax-FAR (Lane 6). The DNA of all transgenic clones was digested with BamRI and hybridized with the nptll probe. C = untransformed control

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The term "Explant" is used to refer to target material for transformation, comprising meristematic tissues. It may refer to plant tissues including, without limitation, one or more embryos, cotyledons, hypocotyls, leaf bases, mesocotyls, plumules, protoplasts, and embryonic axes.

The term "transgene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell or which has been manipulated by experimental manipulations by man. Preferably, said sequence is resulting in a genome which is different from a naturally occurring organism (e.g., said sequence, if endogenous to said organism, is introduced into a location different from its natural location, or its copy number is increased or decreased). A transgene may be an "endogenous DNA sequence", "an "exogenous DNA sequence" (e.g., a foreign gene), or a "heterologous DNA sequence". The term "endogenous DNA sequence" refers to a nucleotide sequence, which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally-occurring sequence. One embodiment of the present invention is to provide a method of regenerating whole plant from explants of Crambe abyssinica species, wherein the said method comprising the steps of a) obtaining explanst from either seeds, seedlings or whole plant, wherein the explants are selected from a group consisting of hypocotyl, cotyledon with petiole, embryo, immature embryo, anther, and root; b) culturing the explants on a regeneration medium containing cytokinin and auxin to obtain multiple shoot buds or callus c) culturing the shoot buds on a rooting medium to obtain rooted plantlets; and d) transferring the rooted plantlets in soil, to obtain regenerated plants. Further, the invention provides for regeneration of plants from Crambe abyssinica species. One example being Crambe abyssinica, such as the cultivar Galactica. Another embodiment of the present invention provides a method of regeneration of the plants, wherein the explants are obtained from seed, wherein the surface sterilized seeds are washed and imbibed for a period of 0 to 48 hours, and then placed on MS medium for germination [1/2 MS, 10 g/L sucrose, 7g/L agar at pH5.8]. The surface sterilisation may be performed by the use of 15% calcium hypochlorite for about 25 minutes.

Further, the invention provides for use of various explants for regeneration of the plant wherein the explants are selected from a group consisting of cotyledon with petiole, hypocotyls, embryo, immature embryo, leaf lamina, cotyledonary axil, shoot tip, anther, root, callus or other suitable explants. For example may the hypocotyls be efficiently used.

Another embodiment of the present invention is to provide a method of regeneration of the plant wherein the explants are transferred onto the medium defined above for germination. The explants are placed in boxes in a climate chamber in dark for about 3 days or in light for 3 days at 25/18⁰C in a climate chamber. The chamber has a temperature of 25/18°C (day/night) and 16 h

-2 -1 photoperiod with a light intensity of 40 μmol m s (cool white fluorescent tubes). Another embodiment of the present invention is to provide a method of regeneration of the plant wherein the hypocotyls are excised and transferred onto a regeneration medium containing either MS or Lepovire, sucrose or glucose and cytokinin such as BAP (6-benzylaminopurine in the range 1 to 5 mg/1, for example 2 mg/L, such as 1.5-2.5 mg/L or 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 mg/L or TDZ (thidiazuron) in the range 1.0 to 2.7 mg/1, such as 2.2 mg/1, such as 1.7-2.7 mg/L or 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5 or 2.6 mg/L in combination with auxin, such as NAA (a-naphtaleneacetic acid in the range of 0.01 to 1 mg/L, for example from 0.3 to 0.8 such as 0.5 or 0.4, 0.6 or 0.7 mg/L and kept in light for induction of shoot buds. The hypocotyls were cut into 2-3 mm in length prior to regeneration was initiated. The amount of the cytokinin being important since it was surprisingly found that by increasing the amount more shoots were regenerated compared to when the amount was below 1 mg/L. Other amounts of TDZ and NAA may be TDZ in about 1.7 to about 2.7 mg/L and said BAP in an amount about 1.7 to about 2.5 mg/L. Another example is wherein NAA is in an amount of about 0.01 to about 1 mg/L and another example wherein said TDZ is in an amount about 2.0-2.5 mg/L ad NAA in and amount of 0.4 to about 0.6 mg/L and a final example wherein said TDZ is in an amount of 2.2 mg/L and NAA in an amount of 0.5 mg/L.

Another embodiment of the present invention is to provide a method of regeneration of Crambe abyssinica plant wherein the said explant is transferred regularly onto fresh regeneration medium, such as every 2 or 3 weeks and allowed to grow to a length of 0.1 cm to 0.8cm, preferably 0.5 cm.

Another embodiment of the present invention is to provide a method of shoot multiplication of Crambe abyssinica plant wherein, the regenerated buds or shoots are transferred to an elongation medium with different growth regulators for further elongation of shoot and the induction and growth of roots to obtain plantlets. The medium may contain full MS or Lepovire, 20g/L sucrose, 0.05 mg/L BA (0.01- 0.5 mg/L), 0.03 mg/L GA₃, and 7 g/L agar at pH 5.8

Another embodiment of the invention is to provide a method of regeneration of Crambe abyssinica plants wherein said rooting is performed by the use of a rooting medium comprising ¹A MS or Lepovire, 10 g/L sucrose and 7 g/L agar at pH 5.8 and auxin with the range of 0.03-0.15 mg/L.

Another embodiment of the present invention is to provide a method of regeneration of Crambe abyssinica plant wherein the plantlet is transferred in soil for further growth. The regenerated plantlet grown is phenotypically normal and/or mutant and fertile and is capable of producing fertile seeds in subsequent generations. Further the invention provides for advancing the generation of the regenerated Crambe abyssinica plants.

Still another embodiment of the present invention is to provide a method for transforming Crambe abyssinica species, wherein the said method comprising the steps of a) surface sterilizing the seeds, and imbibing the seeds with water, b) germinating the seeds of step (a) in a suitable culture medium to obtain seedlings, c) obtaining the explants from step (a) or step (b), d) co-cultivating the explants of step (c) with recombinant Agrobacterium strain, e) culturing the explants of step (d) on a suitable tissue culture selection medium to select transformed plant cells and tissues, f) culturing the explants on a regeneration medium containing cytokinin and auxin to obtain multiple shoot buds or callus g) culturing the shoot buds on a rooting medium to obtain rooted plantlets; and h) transferring the rooted plantlets in soil, to obtain regenerated plants.

Example of a cultivar which may be used is Galactica. Another embodiment of the present invention provides a method of regeneration and transformation of Crambe abyssinica plant, wherein the surface sterilized seeds are washed and imbibed for a period of 0 to 48 hours, and then placed on MS medium for germination [1/2 MS, 10 g/L sucrose, 7g/L agar at pH5.8]. The surface sterilisation may be performed by the use of 15% calcium hypochlorite for about 25 minutes.

Still another embodiment of the present invention is to provide a method for transforming Crambe abyssinica species, wherein the surface sterilized seeds are used to isolate the embryos in sterile conditions by pressing with tweezers or any other means and these embryos are washed in sterile water and blotted dry on filter paper and later placed on soaked sterile filter paper.

Another embodiment of the present invention is to provide a method for transforming Crambe abyssinica species, wherein the explants are selected from a group consisting of cotyledon with petiole, hypocotyls, embryo, immature embryo, leaf lamina, cotyledonary axil, shoot tip, anther, root and callus or other suitable explants. Example of suitable explants is hypocotyl.

Another embodiment of the present invention is to provide a method for transforming Crambe abyssinica species, wherein the recombinant Agrobacterium strain carrying DNA/RNA sequence comprises of a coding or non-coding gene sequence, inclusive or not, of terminator or promoter, as an expressing or non- expressing cassette.

In designing a vector for the transformation process, one or more genetic components are selected that are introduced into the plant cell or tissue. Genetic components can include any nucleic acid that is introduced into a plant cell or tissue using the method according to the invention. In one embodiment, the genetic components are incorporated into a DNA composition such as a recombinant, double-stranded plasmid or vector molecule comprising at least one or more of following types of genetic components: (a) a promoter that functions in plant cells to cause the production of an RNA sequence, (b) a structural DNA sequence that causes the production of an RNA sequence that encodes a product of agronomic utility, and (c) a 3' non-translated DNA sequence that functions in plant cells to cause the addition of polyadenylated nucleotides to the 3' end of the RNA sequence.

The vector may contain a number of genetic components to facilitate transformation of the plant cell or tissue and regulate expression of the structural nucleic acid sequence. In one preferred embodiment, the genetic components are oriented so as to express an mRNA, that in an optional embodiment can be translated into a protein. The expression of a plant structural coding sequence (a gene, cDNA, synthetic DNA, or other DNA) that exists in double-stranded form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme and subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' non-translated region that adds polyadenylated nucleotides to the 3' ends of the mRNA. Means for preparing plasmids or vectors containing the desired genetic components are well known in the art. When a DNA construct contains more than one T-DNA, these T-DNAs and the transgenes contained within may be integrated into the plant genome at separate loci. This is referred to as "co-transformation" (U.S. Pat. No. 5,731,179, WO 00/18939). The process of co-transformation, where two T-DNAs are at different loci in the plant genome and therefore segregate independently in the progeny, can be achieved by delivery of the T-DNAs with a mixture of Agrobacteria transformed with plasmids carrying the separate T-DNA. Co-transformation can also be achieved by transforming one Agrobacterium strain with two binary DNA constructs, each containing one T-DNA (e.g. Daley et al., 1998). Two T-DNAs may also be designed on a single DNA vector, followed by transforming the vector into a plant cell and then identifying the transgenic cells or plants that have integrated the T-DNAs at different loci (U.S. Pat. No. 5,731,179, WO 00/18939, Komari et al, 1996; U.S. Pat. No. 7,288,694).

A two T-DNA system is a useful method to segregate the marker gene from the bio based wax ester genes of interest (GOI) in a transgenic plant. The marker gene generally has no further utility after it has been used to select or score for the transformed plant cell. A single DNA vector carrying the two-T-DNAs is one method to construct a two T-DNA transformation system. However because of the occurrence of both T-DNAs on a single DNA construct, both may be transferred into the plant genome at the same locus. This occurs when one of the border DNA molecule of the first T-DNA is not recognized during the integration process. This reduced efficiency adds to the cost of producing the events and selecting for the individuals that have T-DNAs integrated at an independent locus. It thus also may be desirable to have DNA constructs and a method wherein it is possible to chemically select against individuals that have incorporated the two T-DNAs at the same locus, while screening for the presence/absence and linkage status of each of the T-DNAs.

Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the "promoter". The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription into mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA. A number of promoters that are active in plant cells have been described in the literature. Such promoters would include but are not limited to the nopaline synthase (NOS) and octopine synthase (OCS) promoters that are carried on Ti plasmids of Agrobacterium tumefaciens, the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters and the Figwort mosaic virus (FMV) 35S promoter, and the enhanced CaMV35S promoter (e35S). A variety of other plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals, also can be used for expression of heterologous genes in plant cells, including, for instance, promoters regulated by (1) heat (Callis et al., 1988, (2) light (e.g., pea RbcS-3A promoter, Kuhlemeier et al., (1989); maize RbcS promoter, Schaffner et al., (1991); (3) hormones, such as abscisic acid (Marcotte et al., 1989, (4) wounding (e.g., Wuni, Siebertz et al., 1989); or other signals or chemicals. Tissue specific expression is also known. As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of the gene product of interest. Additional promoters that may find use are a nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987), the CaMV 35S promoter (Odell et al., 1985), the figwort mosaic virus 35S-promoter (Walker et al., 1987; U.S. Pat. Nos. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang et al., 1990), the R gene complex promoter (Chandler et al., 1989), and the chlorophyll a/b binding protein gene promoter, PClSV (U.S. Pat. No. 5,850,019), and AGRtu.nos (GenBank Accession V00087; Depicker et al, 1982; Bevan et al., 1983) promoters. Promoter hybrids can also be constructed to enhance transcriptional activity (U.S. Pat. No. 5,106,739), or to combine desired transcriptional activity, inducibility and tissue specificity or developmental specificity. Promoters that function in plants include but are not limited to promoters that are inducible, viral, synthetic, constitutive as described, and temporally regulated, spatially regulated, and spatio-temporally regulated. Other promoters that are tissue-enhanced, tissue- specific, or developmentally regulated are also known in the art and envisioned to have utility in the practice of this invention.

The promoters used in the DNA constructs (i.e. chimeric/recombinant plant genes) of the present invention may be modified, if desired, to affect their control characteristics. Promoters can be derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters may be altered to contain multiple "enhancer sequences" to assist in elevating gene expression.

The mRNA produced by a DNA construct of the present invention may also contain a 5' non-translated leader sequence. This sequence can be derived from the promoter selected to express the gene and can be specifically modified so as to increase or decrease translation of the mRNA. The 5' non-translated regions can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. Such "enhancer" sequences may be desirable to increase or alter the translational efficiency of the resultant mRNA. The present invention is not limited to constructs wherein the non-translated region is derived from both the 5' non-translated sequence that accompanies the promoter sequence. Rather, the non-translated leader sequence can be derived from unrelated promoters or genes (see, for example U.S. Pat. No. 5,362,865). Examples of non- translation leader sequences include maize and petunia heat shock protein leaders (U.S. Pat. No.

5,362,865), plant virus coat protein leaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAntl, TEV (Carrington and Freed, 1990), and AGRtu.nos (GenBank Accession V00087; Bevan et al, 1983). Other genetic components that serve to enhance expression or affect transcription or translational of a gene are also envisioned as genetic components. The 3' non- translated region of the chimeric constructs may contain a transcriptional terminator, or an element having equivalent function, and a polyadenylation signal that functions in plants to cause the addition of polyadenylated nucleotides to the 3' end of the RNA. The DNA sequences are referred to herein as transcription- termination regions. The regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA). RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Examples of suitable 3' regions are (1) the 3' transcribed, non-translated regions containing the polyadenylation signal of Agrobacterium tumor- inducing (Ti) plasmid genes, such as the nopaline synthase (NOS; Fraley et al., 1983) gene, and (2) plant genes such as the soybean storage protein genes and the small subunit of the ribulose-l,5-bisphosphate carboxylase (ssRUBISCO) gene. An example of a preferred 3' region is that from the ssRUBISCO E9 gene from pea (European Patent Application 0385 962).

In one embodiment, the vector contains a selectable, screenable, or scoreable marker gene. These genetic components are also referred to herein as functional genetic components, as they produce a product that serves a function in the identification of a transformed plant, or a product of agronomic utility. The DNA that serves as a selection or screening device may function in a regenerable plant tissue to produce a compound that would confer upon the plant tissue resistance to an otherwise toxic compound. A number of screenable or selectable marker genes are known in the art and can be used in the present invention. Genes of interest for use as a marker would include but are not limited to GUS, green fluorescent protein (GFP), luciferase (LUX), among others. In certain embodiments, the vector comprises an aadA gene with associated regulatory elements encoding resistance to spectinomycin in plant cells. In a particular embodiment, the aadA gene comprises a chloroplast transit peptide (CTP) sequence that directs the transport of the AadA gene product to the chloroplast of a transformed plant cell. In other embodiments, the vector comprises a spectinomycin resistance gene with appropriate regulatory elements designed for expression in a bacterial cell, such as an Agrobacterium cell, so that the selection reagent may be added to a co- cultivation medium, and allowing obtention of transgenic plants for instance without further use of the selective agent after the co-culture period.

The present invention can be used with any suitable plant transformation plasmid or vector containing a selectable or screenable marker and associated regulatory elements as described, along with one or more nucleic acids expressed in a manner sufficient to confer a particular desirable trait. Examples of suitable structural genes of interest envisioned by the present invention would include but are not limited to genes for modified oils production such as wax esters which are esters of long chain fatty alcohols and fatty acids. Wax esters have lubrication properties that are superior to ordinary vegetable oil, i.e. triacylglycerols (TAGs), due to their high oxidation stabilities and resistance to hydrolysis.

High performance lubricating oils are often based on synthetic esters (such as wax esters) sometimes with the fatty acid part from plant sources. Any of these or other genetic elements, methods, and transgenes may be used with the invention as will be appreciated by those of skill in the art in view of the instant disclosure. Alternatively, the DNA sequences of interest can affect these phenotypes by encoding a an RNA molecule that causes the targeted inhibition of expression of an endogenous gene via gene silencing technologies such as antisense-, co- suppression-mediated mechanisms, RNAi technologies including miRNA (e.g., U.S. Patent Application Publication 2006/0200878). Exemplary nucleic acids that may be introduced by the methods encompassed by the present invention include, for example, DNA sequences or genes from another species, or even genes or sequences that originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. However, the term "exogenous" is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes that are normally present yet that one desires, e.g., to have over-expressed. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene. The DNA constructs used for transformation in the methods of present invention may also contain the plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp that encodes for aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin (e.g. U.S. Pat. No. 5,217,902; or Sandvang, 1999). For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, LBA4404, EHAlOl, or EHA105 carrying a plasmid having a transfer function for the expression unit. Other strains known to those skilled in the art of plant transformation can function in the present invention.

Bacterially-mediated gene delivery (e.g. Agrobacterium-mediated; U.S. Pat. Nos. 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840) can be made into cells in the hypocotyl and may be cultured in the presence of a selection agent such as kanamycin. 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 with the simultaneous formation of shoots, which arise from a single transformed cell, or small cluster of cells including transformed cells.

In light of this disclosure, numerous other possible regulatory elements, and other sequences of interest will be apparent to those of skill in the art. Therefore, the foregoing discussion is intended to be exemplary rather than exhaustive. Screenable or scorable markers can be employed to identify transgenic sectors/and or plants. Exemplary markers are known and include .beta.- glucuronidase (GUS) that encodes an enzyme for various chromogenic substrates (Jefferson et al., 1987a; Jefferson et al., 1987b); an R-locus gene, that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., 1988); a .beta. -lactamase gene (Sutcliffe et al., 1978); a gene that encodes an enzyme for that various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al., 1986); a xylE gene (Zukowsky et al., 1983) that encodes a catechol dioxygenase that can convert chromogenic catechols; an .alpha. -amylase gene (Ikatu et al., 1990); a tyrosinase gene (Katz et al., 1983) that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone that in turn condenses to melanin; green fluorescence protein (Elliot et al., 1999) and an .alpha.-galactosidase. As is well known in the art, other methods for plant transformation may be utilized, for instance as described by Miki et al., (1993), including use of microprojectile bombardment (e.g. U.S. Pat. No. 5,914,451 ; McCabe et al., 1991 ; U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880).

In another embodiment the explants are pre-cultured for a number of days on a regeneration medium containing MS or Lepovire, 30 g/L sucrose, TDZ 2.2 mg/L, NAA 0.5 mg/L, 0.5 g/L MES and Gelrite 2.5 g/L at pH5.8 prior to

Agrobacterium infection. The explants may be cultured on a filter paper on the pre- culture medium. For transformation, the bacteria may be cultured in liquid LB medium for about 18 hours. After centrifuge at 4000 rpm for 15 min, the pellet may be suspended in liquid MS-20 medium (full MS, 20 g/L sucrose, pH 5.2, 200 μM acetosyringone) to a concentration around 0.4 at OD .

^{J b J} 600

The pre-cultured explants may be washed in the bacterial suspension for 1 - 2 min and transferred to the co-culture medium with a filter paper for co-cultivation for a number of days in light. The co-culture medium may be the same as pre- culture medium, but should contain acetosyringone, such as 200 μM acetosyringone. After co-culture, the explants may be washed with 200mg/L cefotaxime to remove Agrobacterium and placed on the selection medium, which was the same as pre-culture medium, but without acetosyringone and supplemented with 0.5 mg/L AgNO , 100 mg/L cefotaxime, 100 mg/L ticarcillin and 25 mg/L kanamycin. The

3 explants may be transferred to fresh selection medium every 3 weeks. The explants were cultured in the climate chamber as stated above. Once the regenerants are about 0.5 cm in height, such as from 0.3-0.7 they may be excised from the explants and cultured on an elongation medium containing full MS or Lepovire, 20 g/L sucrose, 0.05 mg/L BA, 0.03 mg/L GA 3, 100 mg/L cefotaxime, 100 mg/L ticarcillin, 25 mg/L kanamycin and 7 g/L agar at pH 5.8. The shoots may be cultured as mentioned above. Once the shoots are big enough they will be transferred to a rooting medium which may contain Vi MS or Lepovire, 10 g/L sucrose, 100 mg/L cefotaxime and 7 g/L agar at pH 5.8 supplemented with auxin to stimulate root formation. The auxin may be NAA or IBA (indole-3-butyruc acid). NAA or IBA may be in an amount of from 0.03-0.15 mg/L, such as about 0.05 mg/L.

To confirm the presence of the exogenous DNA or "transgene(s)" in the transgenic plants a variety of assays may be performed. Such assays include, for example, "molecular biological" assays, such as Southern and Northern blotting and PCR. "biochemical" assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

Once a transgene has been introduced into a plant, that gene can be introduced into any plant sexually compatible with the first plant by crossing, without the need for ever directly transforming the second plant. Therefore, as used herein the term "progeny" denotes the offspring of any generation of a parent plant prepared in accordance with the instant invention, wherein the progeny comprises a selected DNA construct. A "transgenic plant" may thus be of any generation. "Crossing" a plant to provide a plant line having one or more added transgenes or alleles relative to a starting plant line is defined as the techniques that result in a particular sequence being introduced into a plant line by crossing a starting line with a donor plant line that comprises a transgene or allele. To achieve this one could, for example, perform the following steps: (a) plant seeds of the first (starting line) and second (donor plant line that comprises a desired transgene or allele) parent plants; (b) grow the seeds of the first and second parent plants into plants that bear flowers; (c) pollinate a flower from the first parent plant with pollen from the second parent plant; and (d) harvest seeds produced on the parent plant bearing the fertilized flower.

The present invention also provides for plant parts or a plant produced by the methods of the present invention. Plant parts, without limitation, include fruit, seed, endosperm, ovule, pollen, leaf, stem, and roots. In a preferred embodiment of the present invention, the plant part is a seed.

The following examples are for understanding the invention and should not be construed as to limit the scope of the invention.

EXAMPLES

EXAMPLE 1: Plant material

Crambe abyssinica cv. Galactica.

Methods

Seed germination

Seeds without silique were surface-sterilized with 15% calcium hypochlorite (CaCl O ) for 25 minutes and thoroughly rinsed with sterile water. The surface-sterilized seeds were planted on a germination medium in Petri dishes which were placed in sterile Magenta boxes. The germination medium contained ¹A MS, 10 g/L sucrose, 7 g/L agar at pH 5.8. The boxes were placed in a climate chamber in dark or light for 3 days. The climate chamber has a temperature of

-2 -1 25/18°C (day/night) and 16 h photoperiod with a light intensity of 40 μmol m s (cool white fluorescent tubes). Regeneration 1

Regeneration tests were performed to determine the suitable plant growth regulators and their combinations as well as concentration of AgNO before transformation was conducted. Hypocotyls from 3 days old seedlings grown in dark were used as explants. Hypocotyls of about 1 cm from the biological top were cut into 2-3 mm in length and placed on the regeneration media. The exact plant growth regulator combinations and concentrations of AgNO tested are presented in tables

1, 2, 3 and 4 in the result section. The basal medium containing full MS, 30 g/L sucrose, 0.5 mg/L AgNO 3 , 0.5 g/L MES and 2.5 g/L Gelrite at pH5.8 was used for all regeneration tests except for the AgNO test, in which TDZ was replaced by 2 mg/L BAP supplemented with different concentrations of AgNO . The explants were cultured in light in the same climate chamber as stated above. This protocol gave 30-60% regeneration (see the result section).

Regeneration 2

Hypocotyls from 3 days old seedlings grown in light were used as explants.

Hypocotyls of about 1 cm from the biological top were cut into 2-3 mm in length and placed on the regeneration media. The basal medium containing full Lepovire, 16 g/L glucose or 30 g/L sucrose, 2.2 mg/L TDZ, 0.5 mg/L NAA, 0.5 mg/L AgNO₃,

0.5 g/L MES and 2.5 g/L Gelrite at pH5.8. The Petri dishes were sealed with parafilm and the explants were cultured in light in the same climate chamber as stated above. This protocol gave more than 95% regeneration. Several hormone combinations were tested (see table 8 for details).

Transformation

Transformation vectors The binary vector pSCVl .6 (Vaughan et al., 2004) containing nptll and gus genes was used for evaluating effects of Agrobacterium stains on transformation efficiency. Both nptll and gus genes are under 35S promoter, while the gus contains a gus intron. This experiment was repeated once. Meanwhile, the binary vector pMS9 containing jojoba WAX and FAR genes and the binary vector pKAN-Wax-FAR containing Arabidopsis WAX and FAR genes were also tested in this study, i.e., synthetic genes based on the jojoba fatty acid reductase gene (Genebank ace AF149917) and jojoba wax synthase gene (Genebank ace. AF 149919) with codon optimised for expression in Crambe.

Asrobacterium strains The strains EHAlOl, EHA 105, AGL-I, GV3101 (pMP90), pGV2260 and pGV3850 carrying the vector pSCVl .6 were used to determine a suitable strain for Crambe transformation. Besides, the strain GV3101 (pMP90) also harbours the vector pMS9 and the strain EHA 105 harbours the vector pKAN-Wax- FAR.

Transformation method Hvpocotyl explants as stated for the regeneration tests were used in all transformation tests. The explants were first pre-cultured for 3 days on the regeneration medium containing 30 g/L sucrose, TDZ 2.2 mg/L, NAA 0.5 mg/L, 0.5 g/L MES and Gelrite 2.5 g/L at pH5.8 prior to Agrobacterium infection.

The explants were cultured on a filter paper on the pre-culture medium. For transformation, the bacteria were cultured in liquid LB medium for about 18 hours.

After centrifuge at 4000 rpm for 15 min, the pellet was suspended in liquid MS-20 medium (full MS, 20 g/L sucrose, pH 5.2, 200 μM acetosyringone) to a concentration around 0.4 at OD . The pre-cultured explants were thoroughly washed in the bacterial suspension for 1 -2 min and transferred to the co-culture medium with a filter paper for co-cultivation for 3 days in light. The co-culture medium was the same as pre-culture medium, but contains 200 μM acetosyringone. After co-culture, the explants were washed with 200mg/L cefotaxime and placed on the selection medium, which was the same as pre-culture medium, but without acetosyringone and supplemented with 0.5 mg/L AgNO , 100 mg/L cefotaxime,

100 mg/L ticarcillin and 25 mg/L kanamycin. The explants were transferred to fresh selection medium every 3 weeks. The explants were cultured in the climate chamber as stated above. Multiplication of putative transgenic plants

Once the regenerants were about 0.5 cm in height, they were excised from the explants and cultured on the elongation medium containing full MS, 20 g/L sucrose, 0.05 mg/L BA, 0.03 mg/L GA₃ 100 mg/L cefotaxime, 100 mg/L ticarcilin, 25 mg/L kanamycin and 7 g/L agar at pH 5.8. The shoots were cultured in the climate chamber as stated above.

Rooting of transgenic plants

About 20 shoots from 10 transformed clones were used in each treatment. The rooting medium contains ¹A MS, 10 g/L sucrose, 100 mg/L cefotaxime and 7 g/L agar at pH 5.8. Different types of auxin at various levels were tested as indicted in Table 6 in the result section. The shoots were cultured in the climate chamber as stated above.

Verification of transgenic plants

GUS staining To evaluate the infection efficiency of different Agrobacterium strains, 20-40 explants from each treatment were collected for GUS staining at day 3, 7, 12 and 20 after the Agrobacterium infection. The GUS staining o was carried out at 37 C overnight according to Jefferson (Jefferson et al, 1987) and the results were recorded with the help of microscope.

Southern blot analysis Total genomic DNA was extracted from in vitro growing shoots using CTAB method described by Aldrich and Cullis (1993). Southern blot hybridisation was performed to confirm the integration of the nptll gene. Ten micrograms of the genomic DNA were digested with the restriction enzyme EcoRl for the vector pSCVl .6 and BamHl for the vectors pMS9 and pKAN-Wax-FAR. Southern blot hybridisation was based on the non-radioactive DIG system from Roche (the former Boehringer Mannheim) (van Miltenburg et al. 1995). The nptll probe was synthesized according to Zhu et al. (2008). Results

Regeneration

Effects of plant growth regulators on Crambe regeneration

The results of effects of different growth regulators and their combinations on Crambe regeneration are presented in Table 1. The choice of using these growth regulators and their concentrations was based on our previous studies (data not shown). The results in Table 1 clearly showed that the hormone combination of 2.2 mg/L TDZ and 0.5 mg/L NAA gave the highest regeneration frequency than all the other combinations. BAP or zeatin in combination with NAA resulted in lower regeneration frequency than TDZ. Combination of different types of cytokinin did not help to increase the regeneration frequency for Crambe abyssinica cv. Galactica. In most cases, the regenerated explants gave one shoot or one shoot cluster. This is true for all regeneration tests.

Effects of different concentrations of TDZ and NAA on Crambe regeneration

As showed in Table 2, the combination of 2.2 mg/L TDZ with 0.5 mg/L NAA gave the best regeneration result. The regeneration frequency of combination of 2.2 mg/L TDZ and 0.2 mg/L NAA was much lower than that of 2.2 mg/L TDZ and mg/L NAA 0.5, suggesting that a certain high level of NAA is required for Crambe regeneration. However, our other studies showed that a further increase in concentration of TDZ and NAA would decrease the regeneration frequency of Crambe abyssinica (data not shown).

Different tests on the combination of 2.2 mg/L TDZ and 0.5 mg/L NAA Since variation in the regeneration frequency was found for the best combination 2.2 mg/1 TDZ and 0.5 mg/1 NAA from one test to another, as shown in Tables 1 and 2, several further tests were conducted to check the range of differences. The results showed a variation in the regeneration frequency from 30 to 61% (Table 3). Some regenerated explants are showed in Figure IA. Effect of AgNO on regeneration

The effect of AgNO on regeneration was carried out to select the best

3 concentration for transformation. The results showed that the regeneration frequency was decreased with increasing the concentration of AgNO . Among the tested concentrations, 1 mg/L AgNO gave the highest regeneration frequency (Table 4).

Transformatiom GUS staining GUS staining can be used as an indicator for both transient and stable transformation. The results of GUS staining of six Agrobacterium strains are presented in Table 5. The results showed no obvious differences in GUS staining among the six strains regarding sampling date, but the strains pGV2260 and pGV3850 had a lower GUS staining frequency compared with other strains. Figure IB shows one transgenic shoot with clear GUS staining.

Recovery of transgenic plants

The transformation results from six Agrobacterium strains containing the binary vector pSCVl .6 are shown in Table 6. The strains EHAlOl and AGL-I had higher transformation frequency at 1.3% and 2.1%, respectively, while strains pGV3101 and pGV3850 had low transformation frequency at 0.2% and 0.1%, respectively. The results suggest that Agrobacterium strain plays an important roll in a successful transformation of Crambe abyssinica.

Southern blot results

Southern blot analysis was conducted to confirm the stable integration of transgenes into the plant genome. Forty putative clones transformed with the vector pSCVl .6, which recovered from different strains and grew well on the selection medium for more than 8 weeks, were chosen for Southern blot analysis on the nptll gene and 35 clones were Southern positive. Partial Southern results are shown in Figure 2. Six putative clones with Wax and FAR genes from the two vectors with the genes of interest were analysed by Southern blot and 4 clones were positive (Figure 3).

Rooting test The transgenic plants confirmed by Southern blot analysis were used for rooting tests. The results showed that, in the presence of cefotaxime, 0.1 mg/L NAA and 0.05 mg/L NAA could give 59% and 67% of rooting, respectively, while IBA 0.1 mg/L and IBA 0.05 mg/L had low rooting frequency of 21% and 18%, respectively (Table 7).

Table 1 Effects of plant growth regulators on shoot regeneration of Crambe abyssinica, cv. Galactica

Growth regulator No. of explants No. of regenerated Regeneration concentration explants %

(mg/L)

BAP 2.0 94 2 2X)

TDZ 2.2

Zeatin 0.5

NAA 0.5

BAP 2.0 90 0 0

TDZ 2.2

NAA 0.5

Zeatin 2.0 105 1.0

NAA 0.1

BAP 2.0 86 8 9.3

NAA0.5

TDZ 2.2 100 28 28.0

NAA 0.5

Table 2 Effects of different concentrations of TDZ and NAA on shoot regeneration of Crambe abyssinica, cv. Galactica

Concentration of No. of explants No. of regenerated Regeneration %

TDZ and NAA explants

(mg/L)

TDZ 0.22 80 7.5

NAA 0.5

TDZ : L l 74 10 13.5

NAA 0.5

TDZ 2.2 87 10 11.5

NAA 0.2

TDZ 2.2 98 46 46.9

NAA 0.5

Table 3 Regeneration results from different tests on Crambe abyssinica, cv. Galactica using the same growth regulator combination (2.2 mg/L TDZ and 0.5 mg/L NAA)

Different No. of No. of regenerated Regeneration % tests explants explants

A 222 89 40

B 222 67 30

C 240 146 61 Table 4 Effects Of AgNO₃ on shoot regeneration of Crambe abyssinica, cv. Galactica

Concentration of No. of explants No. of Regeneration

AgNO₃ (mg/L) regenerants %

0 68 0 0

1 66 8 12.5

2 66 5 7.6

3 73 5 6.8

4 67 3 4.5

5 70 5 7.1

Table 5 Results of GUS staining of six Agrobacterium strains sampled at day 3, 7, 12 and 20 after Agrobacterium infection (No. of GUS-positive explants/No. of total sampled explants)

Strain 3 days 7 days 12 days 20 days

EHAlOl 40/40 39/40 38/40 19/19

EHA 105 39/40 38/40 19/20 24/24

AGLl 38/40 38/40 19/20 18/18 pGV3101 32/38 16/20 36/39 17/17 pGV2260 18/39 11/20 31/40 13/37 pGV3850 6/19 15/30 24/35 21/30

Table 6 Effects of different Agrobacterium strains on transformation frequency of Crambe abyssinica cv. Galactica.

¹ The figure contains both escapes and transgenic clones. b ' TTrraannssffoorrmmaattiioonn ffrreeqquueennccyy wwaass ccaallccuullaatteedd uussiinngg tthhee nnuunmber of Southern positive clones divided by the total explants used for transformation.

Table 7 Rooting test on the transgenic lines of Crambe abyssinica, cv. Galactica

Table 8. Regeneration frequency of Crambe cv. Galactica on the Lepovire medium containing different concentrations of TDZ and NAA and 16g/L glucuose or 30g/L sucrose

Treatment (mg/L) Regeneration %

T1.1N0.2GD 35A

T2.2N0.5SD 50.7

T2.2N0.25GD 50.9

T3.3N0.5GD 59.3

T2.2N0.5GD 0.6

T2.2N0.5SL 77.8

T1.1N0.2GL 82.9

T2.2N0.25GL 92.3

T3.3N0.5GL 93.6

T2.2N0.5GL 97.6 Note: G= glucose, D=germination in dark, S= sucrose, L=germination in light, T=TDZ, N=NAA

EXAMPLE 2

Six different vector constructs were used to transform C. abyssinica cv. Galactica using the same transformation protocol under example 1. The results are shown below.

Table 9. Transgenic lines obtained from different vectors using the same transformation protocol

FAE = fatty acid elongase PLAAT = lyso-phosphatidic acid acyltransferase FAD2 = Δ12 fatty acid desaturase WS = wax synthase

Claims

1. A method of regeneration of crambe abyssinica plants, wherein said method comprising a) obtaining explants from either seeds, seedlings or whole plant, wherein b) the explants are selected from a group consisting of hypocotyl, cotyledon with petiole, embryo, immature embryo, anther, and root; c) culturing the explants on a regeneration medium containing TDZ in an amount from about 1 to about 5 mg/L or BAP in an amount of from about 1.5 to about 5 mg/L and auxin to obtain multiple shoot buds or callus d) culturing the shoot buds on a rooting medium to obtain rooted plantlets; and e) transferring the rooted plantlets in soil, to obtain regenerated plants.

2. The method according to claim 1, wherein said explants are hypocotyls.

3. The method according to claim 2, wherein said TDZ is in an amount about 1.7 to about 2.7 mg/L and said BAP in an amount about 1.7 to about 2.5 mg/L.

4. The method according to any of claims 2-3, wherein NAA is in an amount of about 0.01 to about 1 mg/L.

5. The method according to any of claims 1-4, wherein said TDZ is in an amount about 2.0-2.5 mg/L ad NAA in and amount of 0.4 to about 0.6 mg/L.

6. The method according to claim 5, wherein said TDZ is in an amount of 2.2 mg/L and NAA in an amount of 0.5 mg/L.

7. The method according to any of preceding claims, wherein a multiplication step is used between step c) and d) using an elongation medium comprising MS, 20 g/L sucrose, 0.05 mg/L BA, 0.03 mg/L GA₃ and 7g/L agar at a pH of 5.8.

8. The method according to any of preceding claims, wherein said rooting medium comprises Vi MS or Lepovire, 10 g/L sucrose and 7 g/L agar at pH 5.8 and auxin with the range of 0.03-0.15 mg/L.

9. A method for transforming crambe abyssinica, wherein the said method comprising the steps of a) surface sterilizing the seeds, and imbibing the seeds with water, b) germinating the seeds of step (a) in a suitable culture medium to obtain seedlings, c) obtaining explants from step (a) or step (b), d) co-cultivating the explants of step (c) with recombinant Agrobacterium strain, e) culturing the explants of step (d) on a suitable tissue culture selection medium to select transformed plant cells and tissues, f) culturing the explants on a regeneration medium comprising TDZ in an amount from about 1 to about 5 mg/L or BAP in an amount of from about 1.5 to about 5 mg/L and auxin to obtain multiple shoot buds or callus g) culturing the shoot buds on a rooting medium to obtain rooted plantlets; and h) transferring the rooted plantlets in soil, to obtain regenerated plants.

10. The method according to claim 9, wherein said explants are hypocotyls.

11. The method according to claim 9-10, wherein said TDZ is in an amount about 1.7 to about 2.7 mg/L and said BAP in an amount about 1.7 to about 2.5 mg/L.

12. The method according to any of claims 9-11, wherein NAA is in an amount of about 0.01 to about 1 mg/L.

13. The method according to any of claims 9-12, wherein said TDZ is in an amount about 2.0-2.5 mg/L ad NAA in and amount of 0.4 to about 0.6 mg/L.

14. The method according to claim 13, wherein said TDZ is in an amount of 2.2 mg/L and NAA in an amount of 0.5 mg/L.

15. The method according to any of preceding claims, wherein said explants were exposed to a pre-cultivating step prior to said co-cultivating step.