FIELD OF INVENTION
The present invention relates to the field of transgenic plants in particular it relates to transgenic sorghum plant, tissues and cells.
BACKGROUND OF THE INVENTION
Sorghum is a genus of numerous species of grasses, some of which are raised for grain and many of which are used as fodder plants, either cultivated or as part of pasture. The plants are cultivated in warmer climates worldwide. Species are native to tropical and subtropical regions of all continents, in addition to Oceania and Australasia. Sorghum is an important crop of semi-arid tropics. Ninety percent of the world's area cultivated to sorghum is in developing countries, mainly in the arid or semi-arid regions of Africa and Asia. Being highly adaptable to harsh conditions, it plays a major role by supplying staple food and fodder.
The genetic improvement of sorghum through classical plant breeding has resulted in the successful development and deployment of highly adapted high-yielding cultivars that are stable across years. However, to further enhance productivity, quality and resistance to constraints such as drought, diseases e. g. striga, grain mold, and insect pests that is so common in the tropics, much more needs to be done. The resistance level available in cultivated sorghum types is not adequate to build durable resistance to some of the constraints, especially those caused by insect pests. Therefore, the emerging technologies of genetic engineering, such as genetic transformation, have attracted much attention for sorghum improvement, as they provide novel means to supplement the traditional breeding methods (Seetharama N, Godwin I (2004) Sorghum tissue culture and transformation, Enfield, NH : Science Publishers).
Plant cells can be grown in isolation from intact plants in tissue culture systems. The cells have the characteristics of callus cells, rather than other plant cell types. Since the early demonstration of this ability, viz. totipotency and differentiation in vitro, plant tissue culture techniques have been widely used in the clonal
multiplication of plants (Herberlandt, 1902 Haberlandt, G (1902), Kulturversuche mit isolierten Pflanzenzellen. Sber. Akad Wiss.Wien 111: 69-92).
Although the process of tissue culture is known in the art, the exact conditions required to initiate and sustain plant cells in culture, or to regenerate intact plants from cultured cells, are different for each plant species. Each variety of a species will often have a particular set of cultural requirements. Despite all the knowledge that has been obtained about plant tissue culture during the twentieth century, these conditions have to be identified for each variety through experimentation.
The first report of successful transformation of sorghum appeared as early as the 1990s. Yet, sorghum is considered to be the most recalcitrant crop for tissue culture and plant regeneration, thereby for genetic transformation. Recalcitrance in sorghum tissue culture is reportedly due to the release of phenolic compounds, lack of regeneration in long-term in vitro cultures, and a high degree of genotype dependence. The release of phenolics into the culture medium can be overcome by frequent subculture and by the addition of polyvinyl pyrrolidone phosphate (PVPP) in the medium. However, transformation followed by regeneration remains extremely complicated in sorghum transgenic technology (Visarada KBRS and Sai Kishore N (2007) Improvement of Sorghum through transgenic technology, ISB News, March 2007, 1-3). The most effective method till date is Agrobacterium-mediated transformation using super binary vector. However, transformation of sorghum with binary vector has not been reported successful/efficient in the sorghum transformation.
A super binary vector carries additional vir genes, which are responsible for the super virulence phenotype of an A. tumefaciens strain. Since the total size of vector components is relatively large in the super binary system, it is not a realistic choice to introduce additional genes of interest into a super binary vector by ordinary sub cloning methods. Therefore, co-integration of an intermediate vector and an acceptor vector via homologous recombination between the shared DNA segments in A. tumefaciens, is employed in the final step of a super binary vector construction. Moreover, super binary vector systems are not readily and commonly
available for use in molecular biology laboratories. An additional burden of IP and allied costs is involved in the use of super binary vectors, whereas, binary vectors are easily available for molecular cloning in most laboratories, at no additional cost. Binary vectors also have an advantage of delivering a comparatively larger number or size of genes of interest as against super binary vector systems which have a limitation on number or size due to the presence of additional vir genes, necessary for their increased efficiency. In summary, binary vector systems have advantages over super binary vector system in the following ways: 1) Construction of a binary vector is simple, 2) commonly available in laboratories, 3) more cost effective and 4) easier to deliver more genes of interest with binary vectors.
Zhao et al (US 6369298, WO 98/48332) describes sorghum transformation using super binary vector that carries additional virulence genes from a Ti plasmid. The efficiency of transformation is increased because of the additional virulence region inserted. But construction of super binary vector is tedious and laborious even to person skilled in art and the same is not commonly available.
Nguyen et. al, (Nguyen Tuong-Van, Thu TT, Claeys M and Angenon G (2007) Agrobacterium-mediated transformation of sorghum (Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system. Plant cell, Tissue and Organ culture, vol.91 (2): 155-166) describes improved regeneration method suitable for sorghum transformation using Agrobacterium, whereby the method employs one day pre-treatment of immature embryos at 4°C.
Lincoln et al. (Lincoln C, Long J, Yamaguchi T, Serikawa K, and Hake S (2005) Optimization of sorghum transformation parameters using genes for green fluorescent. In Vitro Cellular and Developmental Biology - Plant 41(3): 187-200) and Carlos et al. (Carlos HS, SciELO B. CARVALHO (2004), vol 27, 2: 259-269) Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency. Genet. Mol. Biol., vol. 21, no. 2. ISSN 0102-0536) illustrate sorghum transformation, using immature embryos and super binary vector respectively.
In the prior art, use of super binary vector and other enhancers to increase infection of explants was undertaken because of the failure to obtain efficient transformation using ordinary binary vectors. Therefore, the study of prior art shows that till date, an efficient method of sorghum transformation using binary vector has not yet proved successful. The use of binary vectors in Agrobacterium-mediated transformation is well known in the art for transformation of other plants, but has been unsuccessful in sorghum.
A binary vector was invented soon after it had been elucidated that crown gall tumorigenesis was caused by genetic transformation of plant cells with a piece of DNA, T-DNA for transferred DNA, from a Ti plasmid (tumor-inducing plasmid) harboured by the soil bacterium Agrobacterium tumefaciens (Farrandl SK and Dessaux Y (1986) Proline Biosynthesis Encoded by the noc and occ Loci of Agrobacterium Ti Plasmids. Journal of Bacteriology 167 (2): 732-734). A key finding was that the virulence genes, which are involved in the transfer of T-DNA, could be placed on a replicon separate from the one with T-DNA (Hoekema, A, Hirsch, PR, Hooykaas, PJJ, and Schilperoort, RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti plasmid. Nature 303: 179-180.). Thus, combination of a "disarmed" strain, which carries a Ti plasmid without the wild-type T-DNA, and an artificial T-DNA within a plasmid that can be replicated both in Escherichia coli and A. tumefaciens, turned out to be fully functional in plant transformation.
Until the early 1990s, Agrobacterium-mediated transformation had been used mainly in dicotyledonous and it had been difficult to apply the method to cereals. Later, a super binary vector was developed and successfully used for the transformation of monocotyledons, such as rice and maize (Hiei et al.., 1994 Hiei Y, Ohta S, Komari T and Kumasho T (1994) Efficient transformation of rice mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J.6: 271-282; Ishida Y., Saiti H., Ohta S, Hiei Y, Komari T and Kumashiro T (1996) High efficiency transformation of Maize mediated by Agrobacterium tumefaciens. Nature Biotechnol.14: 745-750). A super binary vector is an improved version of a binary vector and carries the 14.8-kb KpnI
fragment that contains the virB, virG, and virC genes derived from pTiBo542, which is responsible for the supervirulence phenotype of an A. tumefaciens strain, A281 (Jin L, Komari T, Gordon MP, Nester EW (1987) J Bacteriol 169: 441; Komari T, Das A, Stachel S, Ebert P, Allenza P, Montoya A, Nester E (1990) Transformation of cultured-cells of Chenopodium quinoa by binary vectors from phytopathogenic Agrobacterium strains. Plant J 2: 275-281). Since the total size of vector components is relatively large in the super binary system, it is not a realistic choice to introduce additional genes of interest into a super binary vector by ordinary subcloning methods. Therefore, co-integration of an intermediate vector such as pSB11 and an acceptor vector such as pSBl via homologous recombination between the shared DNA segments in A. tumefaciens is employed in the final construction step of a super binary vector.
Reported protocols for Agrobacterium tumefaciens mediated sorghum transformation have used super binary vectors, in which Agrobacterium tumefaciens strain carries extra copies of virB, virC and virG (Komari et ah, 1990) to infect explants. Most of the super binary vectors and super binary system is a proprietary technology. Thus, the cost of licensing the super binary technology for use on a broader scale is a prohibitive step (Frame et ah, 2002, Plant Physiol, 13-22). To overcome this, binary system is easily accessible to everybody.
Plant transformation techniques are of great interest and importance for inserting the gene(s) of interest to the species of interest. A variety of techniques have been used to introduce foreign genes into plant cells. Agrobacterium-mediated transformation has been described by Murai et ah (Science (1983) 222:476-482); Direct DNA uptake method has been described by Lorz et ah, (Mol. Gen. Genet.,
(1985) 199:178-182), Potrykus et ah, (Mol. Gen. Genet, (1985) 199:183-188);
Microinjection method has been described by Crossway et ah, (Mol. Gen. Genet.,
(1986) 202:179-185); High velocity micro-projectile method has been described by
Klein et ah, (Nature (1987) 327:70-73) and Electroporation method has been
described by Fromm et al (Proc. Natl. Acad. Sci. USA (1985) 82:5824-5828,
Fromm et al. (Nature (1986) 319:791-793).
Agrobacterium tumefaciens is a common soil bacterium that naturally inserts its genes into plants and uses the machinery of plants to express those genes in the form of compounds that the bacterium uses as nutrients. In the process, Agrobacterium causes plant tumours commonly seen near the junction of the root and the stem, deriving from it the name of crown gall disease. Agrobacterium-mediated transformation is one of the most common methods of introducing foreign genes into plant cells. This bacterium, being a natural plant pathogen, mediates genetic transformation as a part of the natural process when it infects a plant cell. During the process of transformation a specific segment of the vector, which is known as T-DNA, is transferred into the cells. This region (T-DNA) of the plasmid of Agrobacterium can be engineered to contain gene/s or DNA sequences of interest that can be transferred into the host plant cells and inserted into the plant genome. Agrobacterium-mediated transformation is attractive because of the ease of the protocol coupled with minimal equipment costs. Moreover, transgenic plants obtained by this method often contain a single copy of T-DNA insert.
SUMMARY OF THE INVENTION
One aspect of the present invention provides a process for producing a transgenic sorghum plant, the process comprises transforming 14 to 30 days old callus obtained from immature embryo with a recombinant DNA construct comprising one or more target heterologous DNA sequence; culturing the callus in a first culture medium comprising an effective concentration of auxin to promote development of regenerable structures capable of shoot and/or root formation; and culturing the callus thus obtained in at least a second and/or third culture medium comprising effective ratio of auxin and/or cytokinin that supports the differentiation of the callus into shoot and/or root tissues, to regenerate transgenic sorghum plant; wherein the regenerated transformed sorghum plant is produced within about 6-9 weeks of transforming the callus.
Another aspect of the present invention provides a process of regeneration a sorghum plant, said process comprising culturing an explant for 7 to 30 days in a
first culture medium comprising an effective concentration of auxin to obtain embryogenic callus; and culturing the callus in a second culture medium comprising effective ratio of auxin and/or cytokinin that supports the differentiation of the callus into shoot and/or root tissues, to regenerate sorghum plant; wherein the regenerated sorghum plant is produced within about 5-7 weeks of culturing the callus.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Figure 1 shows PCR gel amplifying the hpt gene; , HindIII cut  DNA; 1-16,
DNA from T0 putative transgenic events; w, no DNA; pc, sorghum non-transgenic
plant DNA; pl, plasmid DNA
Figure 2 a-h shows various stages of regeneration of transgenic sorghum plants; i-k
shows expression of GUS gene in transgenic sorghum calli and shoot
Figure 3 shows regeneration of transgenic sorghum seeds on selection medium.
Figure 4 shows Southern blot analysis of transgenic sorghum lines carrying the
cry1Ac gene. M: Marker; 1- 4: Samples from transgenic plant and NBT: sample
from non-transformed sorghum plant.
DETAILED DESCRIPTION OF THE INVENTION
The term "binary vector" refers to the entire combination, but the plasmid that carries the artificial T-DNA is usually called a binary vector. Binary vectors are the most popular tools in the plant science. Binary vector systems include the most commonly used vectors devised for Agrobacterium mediated genetic transformation of plant. In binary vector system, the T-DNA region comprising a desired gene is located in one plasmid and the vir region is located in a separate disarmed (without tumor-genes) Ti plasmid. The plasmids co-reside in Agrobacterium and remain independent.
The present invention provides a process for regeneration of sorghum plant from calli derived from the immature embryo from immature seeds of Sorghum bicolor and other species of the same genus. Further the invention provides Agrobacterium-mediated transformation of sorghum plant, plant cells and tissues using binary vector comprising the heterologous gene. The present invention also
provides transgenic sorghum plant comprising the crylAc gene. The process of transformation of sorghum as disclosed in the present invention focuses on use of binary plasmid as a vector to deliver a gene of interest. In addition, the present invention provides a composition of medium formulated for callus induction, regeneration, shoot multiplication and elongation, selection of transgenic calli and shoots of sorghum plant during transformation.
Immature embryos from sorghum plants were used as the target explants. The Agrobacterium strain LBA4404 carrying a binary vector comprising the crylAc gene along with hpt and GUS genes was used in the process for Agrobacterium-mediated sorghum transformation.
The process of regeneration and transformation of sorghum disclosed in the present invention is simple, efficient and reproducible. Surprisingly, high transformation frequency ranging from 4 % to 4.67 % was achieved by the process of sorghum regeneration and transformation as disclosed in the present invention (Table 2: B).
The process of regeneration and transformation of sorghum disclosed in the present invention employs use of marker-linked transformation systems.
The process of regeneration and transformation of sorghum disclosed in the present invention employs use of co-transformation system to get marker-free transgenic plants.
A variety of processes for introducing the nucleic acid into plants are known to the person skilled in the art. For example, Agrobacterium-mediated plant transformation, particle bombardment, microparticle bombardment, protoplast transformation, pollen transformation, injection into reproductive organs and injection into immature embryos can be used. The exogenous nucleic acid can be introduced into any suitable cells(s) of the plant, such as root cell(s), stem cell(s) and/or leaf cell(s), embryo, immature embryo of sorghum plant.
Various prior art describes a process for sorghum regeneration and/or transformation for example Nguyen et. al, (Nguyen Tuong-Van, Thu TT, Claeys M and Angenon G (2007) Agrobacterium-mediated transformation of sorghum
(Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system. Plant cell, Tissue and Organ culture, vol.91 (2): 155-166) describes improved regeneration process suitable for sorghum transformation using Agrobacterium, whereby the process employs one day pre-treatment of immature embryos at 4°C. The process described by Nguyen et. al did not give results in our laboratory. Only 10% of these embryos were found to show transient GUS expression after co-cultivating with Agrobacterium. Later on these explants were turned necrotic and subsequently died, giving no transformation, as against 5% reported by Nguyen et. al. Cold treatment to embryos, as mentioned gave 85% callus induction from embryos, as against 81% in case of untreated (without cold treatment) embryos. Whereas, we could get 85-90%) of callus induction using untreated explants without cold treatment. An optical density of 0.6 at 600 nm, as mentioned in the paper, was used by us, but we could not achieve the reported transformation efficiency of 5%. We observed that this O.D. gives increased necrosis of the explants during co-cultivation, and this was observed to increase as the O.D increased. An O.D. of 0.4, used in the present study by us was found to be optimum for Agrobacterium infection (Table 1: B).
To our knowledge and experience the use of immature embryos leads to bacterial overgrowth and necrosis of explants that resulted in low or no plant regenerated. The Pluronic F-68, which was used by Carlos et al. to enhance the efficiency of Agrobacterium infection, was also tried by us, however the efficiency of Agrobacterium infection was not found to be exceptionally high i.e. 100-fold as reported. We have found out that, 65% of the explants were found to show transient GUS expression without treatment with Pluronic F-68 as against 52.5%) explants showing transient GUS expression on Pluronic F-68 treatment (Table 2: A).
The present invention discloses a reproducible and efficient process of sorghum regeneration and/ or transformation using binary vector that overcomes the shortcomings of the prior art.
Development of a reproducible and efficient process for transforming sorghum using binary vector provides many benefits like easy vector construction as compared to the super binary vector. Assembly of a super binary vector system involves co-integration of the gene of interest into a large plasmid (pSBl) in Agrobacterium tumefaciens strain via homologous recombination (Ishida Y, Saito H, Ohta S, Hiei Y, Komari T and Kumashiro T, 1996, High efficincy transformation of maize (Zea maize, L.) mediated by Agrobacterium tumefaciens. Nat Biotech, 14: 745-750). The assembly of binary vector does not require this additional step, making it a more efficient way to introduce a gene of interest into an Agrobacterium tumefaciens strain. Considering the numerous benefits that it could deliver to the science and scientific community, Agrobacterium-mediated sorghum transformation using binary vectors was a point of focus in present invention.
For the generation of marker-free transgenic plants, various processes have been employed. In the marker linked system, the gene of interest is linked to a selectable marker, in the T-DNA region. This selectable marker is coupled with the gene of interest. These processes include marker gene excision (transposable elements, site specific recombination systems, intra chromosomal recombination) gene replacement and transformation with multiple T-DNAs (Abolade S. Afolabi, 2007 Status of clean gene (selection marker-free) technology, African Journal of Biotechnology Vol. 6 (25), pp. 2910-2923). Co-transformation with multiple T-DNAs, using two separate plasmids in a single Agrobacterium, i.e. one vector carrying the selectable marker gene in one T- DNA and the other vector carrying the gene of interest in another T-DNA, are used. This results in unlinked and/or linked co-integration of transgenes. During meiosis, unlinked transgenes are then segregated.
One embodiment of the present invention provides a process of regeneration of Sorghum plant(s), the process comprises obtaining callus from an explant on callus induction medium comprising N6 salts, maltose and 2.5 mg/1. of 2,4-D; and
transferring the callus onto a differentiating medium comprising MS salts, 1 mg/1 IAA and 0.5 mg/1 Kinetin to obtain shoots (Table 7: C).
Another embodiment provides a process of regeneration of Sorghum plants, the process comprising obtaining callus from an explant on callus induction medium comprising N6 salts and 2.5 mg/1. of 2, 4-D; transferring the callus onto a differentiating medium comprising MS salts, 1 mg/1 IAA and 0.5 mg/1 Kinetin to obtain shoots and transferring the shoots on a rooting medium to obtain Sorghum plant (Table 7: C).
One embodiment of the present invention provides a binary vector comprising the gene of interest; a polynucleotide encoding a selectable marker protein and/or a polynucleotide encoding a reporter protein, wherein the gene of interest: any gene that, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability. The "gene of interest" may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.
One embodiment of the present invention provides a binary vector comprising the heterologous polynucleotide operably linked to a plant expressible promoter; a polynucleotide encoding a selectable marker protein and/or a polynucleotide encoding a reporter protein.
Another embodiment of the present invention provides a binary vector comprising the heterologous polynucleotide encoding insecticidal protein, wherein the polynucleotide is operably linked to a plant expressible promoter; a polynucleotide encoding a selectable marker protein and/ or a polynucleotide encoding a reporter protein.
Another embodiment of the present invention provides a binary vector comprising the heterologous polynucleotide encoding insecticidal protein, wherein the polynucleotide is operably linked to a plant expressible promoter and a polynucleotide encoding a reporter protein.
Another embodiment of the present invention provides a binary vector comprising the heterologous polynucleotide encoding insecticidal protein, wherein the polynucleotide is operably linked to a plant expressible promoter.
Another embodiment of the present invention provides a binary vector comprising the heterologous polynucleotide encoding insecticidal protein, wherein the polynucleotide is operably linked to a plant expressible promoter, wherein the insecticidal protein is Bacillus thuringiensis crystal protein.
Another embodiment of the present invention provides a binary vector comprising the heterologous polynucleotide encoding insecticidal protein, wherein the polynucleotide is operably linked to a plant expressible promoter, wherein the insecticidal protein is selected from a group consisting of CrylAc or Cry2Ab protein from Bacillus thuringiensis.
In accordance with the present invention there is provided a process for producing a transgenic Sorghum plant with one or more heterologous nucleic acid coding sequences capable of producing heterologous proteins in the sorghum plant, the process comprising transforming sorghum callus with a binary vector comprising the heterologous gene to obtain transformed callus; culturing the transformed callus on selection medium comprising selection marker; selecting transformed callus; and regenerating selected callus on a differentiation medium comprising MS-salts, selection marker and growth regulators to obtain transformed shoots.
The process of transformation of sorghum plant disclosed in the present invention facilitates production of transgenic sorghum having the desired traits. The transformation process as disclosed in the present invention can be used to produce abiotic stress tolerant transgenic plant, wherein the abiotic stress is selected from a group consisting of heat, cold; frost, and drought, wherein the transgenic sorghum plant are tolerant to said stress as compared to an untransformed sorghum plant.
In one embodiment of the present invention there is provided a process for producing a transgenic Sorghum plant with one or more heterologous nucleic acid coding sequences capable of producing heterologous proteins in the sorghum plant,
the process comprising: transforming sorghum callus with a binary vector comprising the heterologous gene to obtain transformed callus; culturing the transformed callus on selection medium comprising selection marker; selecting transformed callus; and regenerating selected callus on a differentiation medium comprising MS-salts, selection marker and growth regulators to obtain transformed shoots; culturing the shoots on a rooting medium to obtain transgenic sorghum plant.
In another embodiment of the present invention there is provided a process for producing a transgenic Sorghum plant with one or more heterologous polynucleotide sequence encoding heterologous proteins in the sorghum plant, wherein the heterologous polynucleotide is selected from a group consisting of a polynucleotide encoding Cry1Ac, GUS, and Cry2Ab protein.
In one embodiment of the present invention there is provided a process for producing a transgenic Sorghum plant with one or more heterologous nucleic acid coding sequences capable of producing heterologous proteins in the sorghum plant, the process comprising: transforming sorghum callus with a binary vector comprising the heterologous gene to obtain transformed callus; culturing the transformed callus on selection medium comprising selection marker; selecting transformed callus; and regenerating selected callus on a differentiation medium comprising MS-salts, selection marker and growth regulators to obtain transformed shoots, wherein the callus is obtained from an explant selected from a group consisting of immature embryos, immature inflorescence, calli derived from immature embryos and immature inflorescence, mature embryos, shoot node mesocotyl, and leaf.
In another embodiment of the present invention there is provided a process for producing a transgenic Sorghum plant with one or more heterologous nucleic acid coding sequences capable of producing heterologous proteins in the sorghum plant, the process comprising transforming sorghum callus with a binary vector comprising the heterologous gene to obtain transformed callus; culturing the transformed callus on selection medium comprising selection marker; selecting
transformed callus; and regenerating selected callus on a differentiation medium comprising MS-salts, selection marker and growth regulators to obtain transformed shoots, wherein the callus is obtained on a medium comprising N6 salts, maltose and 2.5mg/l. of 2,4-D.
In yet another embodiment of the present invention there is provided a process for producing a transgenic Sorghum plant with one or more heterologous nucleic acid coding sequences capable of producing heterologous proteins in the sorghum plant, the process comprising: transforming sorghum callus with a binary vector comprising the heterologous gene to obtain transformed callus; culturing the transformed callus on selection medium comprising selection marker; selecting transformed callus; and regenerating selected callus on a differentiation medium comprising MS-salts, selection marker and growth regulators to obtain transformed shoots, wherein the callus is transformed by using transformation process selected from a group consisting of Agrobacterium-mediated transformation, particle bombardment, electroporation and in-planta transformation.
In yet another embodiment of the present invention there is provided a process for producing a transgenic Sorghum plant with one or more heterologous nucleic acid coding sequences capable of producing heterologous proteins in the sorghum plant, the process comprises transforming sorghum callus with a binary vector comprising the heterologous gene to obtain transformed callus; culturing the transformed callus on selection medium comprising selection marker; selecting transformed callus; and regenerating selected callus on a differentiation medium comprising MS-salts, selection marker and growth regulators to obtain transformed shoots, wherein the binary vector is a plant transformation binary vector comprising the heterologous gene.
In one embodiment of the present invention there is provided calli produced from immature embryos of sorghum. These calli can be produced by isolating and culturing immature embryos on a nutrient media comprising plant growth regulators.
In one embodiment of the present invention there is provided a binary vector, wherein the binary vector comprises crylAc polynucleotide under the control of e35S promoter sequence.
In another embodiment of the present invention there is provided a binary vector, wherein the binary vector comprises crylAc polynucleotide under the control of e35S promoter sequence, hpt and GUS genes under the control of 35S promoter sequence
In another embodiment of the present invention there is provided a binary vector, wherein the binary vector comprises hpt and GUS genes under the control of 35S promoter sequence.
In accordance with the present invention in one embodiment of the present invention there is provided a process for producing a transgenic sorghum plant, the process comprises transforming 14 to 30 days old callus obtained from immature embryo with a recombinant DNA construct comprising one or more target heterologous DNA sequence; culturing the callus in a first culture medium comprising an effective concentration of auxin to promote development of regenerable structures capable of shoot and/or root formation; and culturing the callus thus obtained in at least a second and/or third culture medium comprising effective ratio of auxin and/or cytokinin that supports the differentiation of the callus into shoot and/or root tissues, to regenerate transgenic sorghum plant; wherein the regenerated transformed sorghum plant is produced within about 6-9 weeks of transforming the callus.
In another embodiment of the present invention there is provided a process for producing a transgenic sorghum plant, the process comprises transforming 14 to 30 days old callus obtained from immature embryo with a recombinant DNA construct comprising one or more target heterologous DNA sequence; culturing the callus in a first culture medium comprising an effective concentration of auxin to promote development of regenerable structures capable of shoot and/or root formation; and culturing the callus thus obtained in a second culture medium comprising effective ratio of auxin and cytokinin to obtain shoots and transferring
the shoots to third culture medium comprising cytokinin to obtain regenerated transgenic sorghum plant; wherein the regenerated transformed sorghum plant is produced within about 6-9 weeks of transforming the callus.
Another embodiment of the present invention provides a process for producing a transgenic sorghum plant, the process comprises transforming 14 to 30 days old callus obtained from immature embryo with a recombinant DNA construct comprising one or more target heterologous DNA sequence; culturing the callus in a first culture medium comprising N6 medium, 1.5 to 3.5 mg/1 2, 4-D and a selection marker; and culturing the callus thus obtained in at least a second medium comprising MS salts, 0.25 to 2 mg/1 IAA, 0.1 to 1 mg/1 kinetin and a bactericidal compound to obtain shoots and transferring the shoots on third culture medium comprising MS salts, 0.1 to 1 mg/1 NAA, and 0.1 to 1 mg/1 IB A to obtain rooted shoots; wherein the regenerated transformed sorghum plant is produced within about 6-9 weeks of transforming the callus.
Yet another embodiment of the present invention provides, auxins selected from the group consisting of IAA, 2,4-D, NAA, IBA, and dicamba; and the cytokinins selected from the group consisting of BAP, zeatin, kinetin, and TDZ.
Further embodiment of the present invention provides transformation process selected from a group consisting of Agrobacterium-mediated transformation, particle bombardment, electroporation and in-planta transformation.
In another embodiment of the present invention there is provided a process for regeneration of a sorghum plant, said process comprising culturing an explant for 7 to 30 days in a first culture medium comprising an effective concentration of auxin to obtain embryogenic callus; culturing the callus in a second culture medium comprising effective ratio of auxin and/or cytokinin that supports the differentiation of the callus into shoot and/or root tissues, to regenerate sorghum plant; wherein the regenerated sorghum plant is produced within about 5-7 weeks of culturing the callus.
In another embodiment of the present invention there is provided a process for regeneration of a sorghum plant, said process comprising culturing an explant for
7 to 30 days in a first culture medium comprising an effective concentration of auxin to obtain embryogenic callus; culturing the callus in a second culture medium comprising effective ratio of auxin and/or cytokinin that supports the differentiation of the callus into shoot and/or root tissues to regenerate sorghum plant; wherein the regenerated sorghum plant is produced within about 5-7 weeks of culturing the callus, wherein the callus is obtained from an explant selected from a group consisting of immature embryos, immature inflorescence, mature embryos, shoot node, mesocotyl, shoot apex and leaf.
In another embodiment of the present invention there is provided a process for regeneration of a sorghum plant, said process comprising culturing an explant for 7 to 30 days in a first culture medium comprising N6 medium, and 1.5 to 3.5 mg/1 2, 4-D; culturing the callus in a second culture medium comprising MS salts 0.25 to 2 mg / IAA, and 0.1 to 1 mg/1 kinetin to obtain shoots, and transferring shoots to third culture medium comprises MS salts, 0.1 to lmg/1 mg/1 NAA, and 0.1 to lmg/1 mg/1 IBA to regenerate sorghum plant; wherein the regenerated sorghum plant is produced within about 5-7 weeks of culturing the callus, wherein the callus is obtained from an explant selected from a group consisting of immature embryos, immature inflorescence, mature embryos, shoot node, mesocotyl, shoot apex and leaf.
In another embodiment of the present invention there is provided a transgenic sorghum plant comprising the heterologous gene, wherein the transgenic sorghum plant is obtained using the process of transformation as disclosed in the present invention.
One embodiment of the present invention provides a transgenic sorghum seed obtained from the transgenic plant produced using the transformation disclosed in the present invention.
Another embodiment of the present invention provides a transgenic sorghum plant comprising the crylAc gene obtained from the process as disclosed in the present invention.
Yet another embodiment of the present invention provides a transgenic sorghum seed comprising the cry 1Ac gene obtained from the transgenic plant as disclosed in the present invention.
In another embodiment of the present invention there is provided a marker-free transgenic sorghum plant comprising the heterologous gene, wherein the transgenic sorghum plant is obtained using the process of transformation as disclosed in the present invention.
In one embodiment of the present invention there is provided a process for producing an insect resistant transgenic Sorghum plant comprising a crylAc gene, the process comprising: transforming sorghum callus with a binary vector comprising crylAc gene to obtain transformed callus; culturing the transformed callus on selection medium comprising N6 salts, selection marker and 2.5 mg/1. of 2,4-D; selecting transformed callus; and regenerating selected callus on a differentiation medium comprising MS-salts selection marker, 1 mg/1 IAA and 0.5 mg/1 Kinetin to obtain transformed shoots.
In another embodiment of the present invention there is provided a marker free transgenic sorghum plant comprising the crylAc gene, wherein the transgenic sorghum plant is obtained using the process of transformation as disclosed in the present invention.
Optimization of transformation conditions
Several parameters for Agrobacterium-mediated sorghum transformation were compared and evaluated for their effectiveness. On the basis of the parameters optimized, further transformations were carried out. Different parameters optimized are described below.
Explants
Earlier, different groups have used immature embryos or immature inflorescence for direct regeneration of sorghum transformants. The present invention is based on regeneration of transgenic sorghum plant, cell or tissue via somatic embryogenesis. Calli was derived from immature embryos isolated from immature
seeds collected from field or greenhouse-grown sorghum plants. Immature seeds of sorghum (Sorghum bicolor L. Moench) were collected 15-20 days after pollination. Immature embryos (1-1.5 mm in length) were isolated from these seeds.
Twenty one days and 28 days old calli developed from immature embryos were found to be amenable for Agrobacterium infection; however 28 days old calli showed better response (Table 1: A). Various medium compositions were tested for callus formation. On the basis of quality callus was categorised in three grades, Grade A, B, and C. Grade A calli were embryogenic, golden yellow coloured and were secreting less phenolic compounds. The calli falling under the other two grades were less or non-embryogenic, soft and watery.
Also, we developed calli from nodal regions of sorghum and tried for transformation. Explants after co-cultivation were showing Agrobacterium infection in terms of transient GUS expression, but transgenic plants were not recovered.
Other explant types (immature embryos) were used to compare the efficiency of infection in terms of transient GUS expression. We found that 60-70% of the calli (derived from immature embryo) showed transient GUS expression whereas other explant types showed no transient GUS expression. During co-cultivation period, other explant types showed necrosis and subsequently died.
Effect of Pre-culture on Transformation Efficiency
The experiments were carried out to see the effect of different pre-culture period i.e. 7, 8, 9, and 14 days. But none of the treatments was able to recover transformants. Hence explants were subjected to Agrobacterium infection directly, without pre-culture, which was found to be better in terms of transient GUS expression. This was continued in further experiments.
Effect of Age of Explants on Transformation Efficiency
To optimize the age of explants, different time points were compared. We studied 7 days, 14 days and 28 days old calli for infection. Twenty eight days old calli
showed higher efficiency of infection (60-70% calli showed GUS expression) in terms of transient GUS expression as compared to other treatments studied (7 and 14 days old calli) (Table 1: A).
Agrobacterium Carrying Plasmid Constructs
Marker-linked construct:
Agrobacterium tumefaciens strain LBA4404 containing plasmid pCAMBIA1301 harbouring hpt and GUS genes was used for sorghum transformation. Plasmid pCAMBIA1301 contains hpt and GUS genes, each driven by CaMV35S promoter.
To develop marker-free transgenic sorghum plants, Agrobacterium tumefaciens strain LBA4404 containing two different binary plasmids, plasmid pMH0102 carrying the cry1Ac gene driven by e35S promoter and plasmid pCAMBIA1301 harbouring hpt and GUS genes was used for transformation. Plasmid pCAMBIA1301 contains hpt and GUS genes, each driven by CaMV35S promoter.
Binary vectors plasmid pMH0102 and pCAMBIA1301 were used for transformation of Sorghum and proved its efficient infectivity to sorghum explants. Carlos et al. (2003) reported 0.8 to 3.5 percent transformation efficiency by using the super binary vector for transformation. Zhao et al. (Zhao Zuo-yu, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J and Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant mol. Biology. 44: 789-798)2000) reported 131 events from 6175 immature embryos giving 2.12% transformation efficiency. Howe et al. (Howe A, Sato S, Dweikat I, Fromm M, and Clemente T (2005) Rapid and reproducible Agrobacterium-mediated transformation of sorghum. Plant cell report. vol.25(8): 784-791) reported 1% average transformation efficiency using immature embryos as explants.
Surprisingly, the process for Agrobacterium-mediated transformation of sorghum using binary vectors as disclosed in the present invention resulted in enhanced transformation efficiency ranging from 4% to 4.67% which is significantly higher than the transformation frequency as described in the prior art which is 2.1%
(Zhao et al . 2000) and 3.5% (Carlos et al ,2004). Further, the process of Agrobacterium-mediated transformation of sorghum as disclosed resulted in enhanced average transformation frequency in the range of 3.16 to 3.33%, which is unexpected as none of the prior art describes such high average transformation frequency in sorghum.
Concentration of Aerobacterium and duration of infection
The concentration of Agrobacterium was measured in terms of optical density (O.D.) using spectrophotometer. Different O.D.s (0.2, 0.4, 0.6 and 1.6) at 600nm was compared for infectivity. We found that Agrobacterium culture having 0.4 O.D. at 600nm showed highest GUS expression as compared to other concentrations studied (Table 1: B). Agrobacterium cultures with O.Ds less than 0.4 did not show GUS expression, whereas Agrobacterium cultures having O.Ds higher than 0.4 showed necrotic effects on calli during co-cultivation period, finally resulting in calli mortality on post-culture media.
This study showed that infection of Agrobacterium culture having 0.4 O.D. for 15 minutes resulted in higher infection of the explants.
Different treatments of co-cultivation period (3, 4 and 5 days) were compared for transformation. A co-cultivation period of 5 days in dark at 25°C±2 showed higher percent infectivity (70%) as compared to other treatments of co-cultivation.
Effect of Media Used for Diluting Agrobacterium Culture
Different liquid media were used to dilute grown overnight Agrobacterium culture before infection to explants to get required concentration of Agrobacterium. The Agrobacterium culture diluted with liquid LS media showed significant increase in GUS expression as compared to liquid MS media used for dilution. The calli infected by Agrobacterium diluted with liquid LS media showed GUS expression with prominent spots as visualised by histochemical GUS assay. The calli infected by Agrobacterium diluted with liquid MS media showed GUS expression with smeary and faint spots as visualised by histochemical GUS assay. These results
showed that the dilution media plays an important role in increasing the transformation frequency.
Effect of Antibiotics to Control Overgrowth of Bacteria
The calli were co-cultivated for 5 days and then transferred on to post-culture media for recovery and to kill excess of Agrobacterium culture. Cefotaxime (250mg/lit) did not control the overgrowth of bacteria. Carbenicillin (200mg/lit) controlled the overgrowth of bacteria. There was no negative effect of carbenicillin on calli quality. So on the basis of these results, carbenicillin (200mg/lit) was used to kill excess of bacteria in further experiments.
Effect of Light on Regeneration of Putative Transgenic sorghum calli
The sorghum calli after co-cultivation were selected on selection media and regenerated on differentiation/regeneration media. The calli maintained continuously in the dark up to shoot formation, were able to regenerate faster as compared to the cultures kept in 16 hrs photoperiod, after two selection steps in dark. On the basis of these results, it was concluded that shoots grow faster (6-7 weeks) and healthier if the cultures are maintained in dark up to shoot regeneration, and then transferred to 16 hours photoperiod for the multiplication, elongation and rooting.
EXAMPLES
Example 1
Regeneration of Sorghum plant
Explant Preparation and Callus induction
Immature seeds of sorghum {Sorghum bicolor L. Moench) were collected 15-20 days after pollination. A drought tolerant genotype P898012 was used as a source genotype in the experiments. Immature seeds collected from field as well as greenhouse grown plants were surface sterilised with 95% ethanol for 5 minutes in a 250 ml conical flask followed by three washes with sterile distilled water. These seeds were subsequently treated with 1.2 % NaOCl for 15 minutes with vigorous shaking (50 ml NaOCl for 1000 seeds) followed by five to six washes with sterile
distilled water. Immature embryos (1-1.5 mm in length) were isolated from these seeds. The immature embryos were cultured on N6 medium (Table 6) in Petri dish. These cultures were incubated in dark at 25 C for 7, 14 and 28 days for calli development.
Regeneration of sorghum plant
The calli obtained on N6 medium were further subcultured to differentiation medium containing MS salts, IAA-lmg/lit, B5 vit, kinetin-0.5 mg/lit (Table 6). The cultures were incubated in the dark at 25°C±2 for 2-3 weeks. The cultures were subcultured repeatedly until the shoots were regenerated. The regenerated shoots were then transferred to photoperiod regime of 16 hrs light: 8 hrs dark at 25°C±2. After the shoots turned green, they were sub-cultured to same regeneration media for shoot multiplication and elongation. The shoots of 5-6 cm length were transferred on to rooting media comprising ½ MS salts, sucrose 2%, 0.5 mg/1 NAA, 0.5 mg/1 IBA and phytagel 0.3% (Table 6).
The rooted shoots were washed with sterile distilled water thoroughly to remove the gelling agent. These plants were transferred to Hoagland's liquid medium (Table 6) for 24 hours and then transferred to cups containing mixture of promix and soil (1:1 proportion). After hardening for 1 month in cups, plants were transferred to soil in greenhouse.
Example 2
Sorghum Transformation
Agrobacterium Culture
Agrobacterium tumefaciens strain LBA4404 containing binary plasmid pCAMBIA1301 harbouring hpt and GUS genes was used for sorghum transformation. Plasmid pCAMBIA1301 contains hpt and GUS genes, each driven by CaMV35S promoter.
To develop marker-free transgenic sorghum plants Agrobacterium tumefaciens strain LBA4404 containing two different binary plasmids, plasmid pMH0102 carrying the cry1Ac gene driven by e35S promoter and plasmid pCAMBIA1301
harbouring hpt and GUS genes were used for transformation. Plasmid pCAMBIA1301 contains hpt and GUS genes each driven by CaMV35S promoter.
In each of the transformation systems (marker-linked and marker-free), a day before co-cultivation was to be done, the culture of the above mentioned Agrobacterium was grown overnight in liquid LB medium (Table 6) on rotary shaker (150-180 rpm) at 28°C.
Example 3
Calli Isolation
Seven and 14 days old calli were isolated from the immature embryos derived from immature seeds and cut into pieces of size 3-4 mm2 and used for co-cultivation.
For a treatment wherein 28 days old calli were used for Agrobacterium infection, 14 days old calli were isolated from the immature embryos derived from immature seeds and sub-cultured on N6 media again. The cultures were incubated for another 14 days, in the dark, at 25°C. After 14 days, calli were cut into pieces of size 3-4 mm2 and used for co-cultivation.
Agrobacterium Infection and Co-Cultivation
The sorghum calli (7, 14 and 28 days old), isolated as mentioned above, were collected in sterile petriplates. The immature embryos derived from immature seeds were also taken for infection and co-cultivation. Before infection these calli were blotted well to remove excess of media and moisture. The Agrobacterium suspension of required concentration (0.2, 0.4, 0.6, 1.0 and 1.6 O.D. at 600 nm) was prepared by diluting it with liquid LS medium. The calli were immersed in required concentration of Agrobacterium for 5, 10, 15 and 20 minutes. After Agrobacterium infection, calli were blotted well on sterile filter paper and transferred on to co-cultivation media N6AS (Table 6) containing 100µM Acetosyringone. The cultures were incubated in the dark at 25°C±2 for 3, 4 and 5 days.
Post-Culture and Selection
After 5 days of co-cultivation, the calli were transferred onto post-culture medium, N6C (Table 6). The cultures were incubated in dark at 25°C±2 for a period of 3 days.
After 3 days of post-culture, the calli were transferred on to selection medium N6H10 (Table 6) containing hygromycin 10 mg/1. The cultures were incubated in dark at 25°C±2 for 2 to 3 weeks.
Example 4
Regeneration of Transgenic Shoots
The calli from selection medium were subcultured to differentiation medium SNR2H10 (Table 6) containing MS salts, hygromycin 10 mg/1., IAA-lmg/1, B5 vit, kinetin-0.5 mg/1. The cultures were incubated in the dark at 25°C±2 for 2-3 weeks. This step was repeated once to twice until the shoots were regenerated. The regenerated shoots were then transferred to photoperiod regime of 16 hrs light: 8 hrs dark at 25°C±2. After the shoots turned green, they were sub-cultured to same regeneration media for shoot multiplication and elongation. The shoots of 5-6 cm height were sub-cultured on to rooting medium comprising ½ MS salts, sucrose 2%, 10 mg/1 hygromycin , carbenicillin 100 mg/1, 0.5 mg/1 NAA, 0.5 mg/1 IBA and phytagel 0.3%.
Hardening
The rooted plants were washed with sterile distilled water thoroughly to remove the gelling agent (agar or phytagel). These plants were transferred to Hoagland's liquid medium (Table 6) for 24 hours and then transferred to cups containing mixture of promix and soil (1:1 proportion). After hardening for 1 month in cups, plants were transferred to soil in greenhouse.
Example 5
Molecular characterization
The established shoots were assayed for the expression of GUS gene with histochemical GUS assay. The young leaves were used for GUS assay. For PCR analysis DNA from the leaf samples of GUS expressing transformants (To plants)
was isolated. PCR was carried out to amplify the hpt gene fragment. The amplified PCR products were analyzed on 1% agarose gel (Figure 1) by electrophoresis at 80V for 45 minutes.
Also putative transgenic plants regenerated deploying marker-free system were subjected to PCR analysis. The young leaves were used for DNA extraction. The PCR was carried out to amplify the crylAc and GUS gene fragments. The amplified PCR products were analyzed on 1% agarose gel by electrophoresis at 80V for 45 minutes.
It was concluded from the data (Table 1: A) that, 28 days old calli were optimum for Agrobacterium infection and subsequent transient GUS expression. Thus 28 days old calli were used for further experiments.
Example 6
Confirmation of putative Transgenic Plants
Putative transgenic shoots were analysed by GUS histochemical assay to confirm expression of the transgene. All of the independent events generated using marker-linked construct (10 events) tested showed GUS expression. Average transformation frequency was observed to be 3.33 %. These independent events were also tested for presence of hpt gene in genomic DNA, using PCR. An expected fragment was amplified showing the presence of the hpt gene in the events tested.
Putative transgenic shoots generated by co-transformation using marker-free construct were analysed by PCR to amplify the cry 1 Ac and GUS genes. Out of 51 putative transformants tested, 19 events were found to contain the cry 1 Ac as well as GUS genes.
The overall transformation frequency was found to be 3.33% in case of marker linked construct and. 3.16 % in case of marker free construct (Table 3 and 4).
Transgene integration was also confirmed by Southern Blot analysis. Southern blot of transgenic Sorghum lines carrying the cry 1 Ac gene was carried out. The probe used was a PCR amplified crylAc gene fragment. Genomic DNA was digested
with Hind III, which is absent within the cry I Ac gene cassette. It was found that the crylAc gene was stably integrated into transgenic Sorghum lines (Figure 4) and showed a single copy insertion of the cry 1 Ac gene for a majority of the events.
Southern blot of transgenic Sorghum lines carrying the cry 1 Ac gene. The probe used was a PCR amplified crylAc gene fragment. Genomic DNA was digested with Hind III. NBT, non-transformed Sorghum line; 1-4, transgenic Sorghum lines; M, molecular marker. Approximately 10- 12ug of DNA was loaded for each plant sample.
Example 7
Segregation analysis and stable inheritance of the transgene
Stability of transgenic plants in terms of gene stability in subsequent generations was also tested in T1 generation. The T1 seeds were germinated and seedlings were tested histochemically for the expression of GUS gene. Three transgenic sorghum events carrying the GUS gene were analysed for stable inheritance of the transgene (GUS). The GUS gene was found to be segregated as per Mendelian single dominant gene pattern of segregation (3:1) in Tl generation. The seedlings were scored on the basis of histochemical assay of GUS protein expression. The data is shown in the Table 5.
Table 1: A) Comparative analysis of age of explants (calli derived from immature embryos) used for transformation
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Table 1: B) Comparative analysis of concentration of Agrobacterium used for transformation (calli derived from immature embryos)

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* Concentration of Agrobacterium was measured by optical density (O. D.)
Table 2: A) Effect of Pluronic F-68 on transformation efficiency

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Table 2:B) Overall Transformation frequency Without Pluronic F-68
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Table 3: Summary of transformation experiments using marker-linked construct
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Table 4: Summary of transformation experiments using marker-free construct

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Table 5: Segregation analysis in T1 generation of Sorghum transgenic events produced using marker-linked construct
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Table 6: Media composition
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Table 7 A) Medium composition 1 for transformation and regeneration of sorghum plants from calli derived from immature embryos

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Table 7 B) Medium composition 2 for transformation and regeneration of sorghum
plants from calli derived from immature embryos

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Table 7 C) Medium composition 3 for transformation and regeneration of sorghum
plants from calli derived from immature embryos

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Table 7 D) Medium composition 4 for transformation and regeneration of sorghum
plants from calli derived from immature embryos

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Table 7 E) Medium composition 5 for transformation and regeneration of sorghum plants from calli derived from immature embryos

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Table 8 Medium composition for transformation and regeneration of sorghum plants from Immature Inflorescence

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Table 9 Medium composition for transformation and regeneration of sorghum plants from nodal region of immature seedlings
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I/We Claim:
1. A process for producing a transgenic sorghum plant, said process comprises
a. transforming 14 to 30 days old callus obtained from immature embryo with
a recombinant DNA construct comprising one or more target heterologous
DNA sequence;
b. culturing the callus in a first culture medium comprising an effective
concentration of auxin to promote development of regenerable structures
capable of shoot and/or root formation; and
c. culturing the callus from step (b) in at least a second and/or third culture
medium comprising effective ratio of auxin and/or cytokinin that supports
the differentiation of the callus into shoot and/or root tissues, to regenerate
transgenic sorghum plant; wherein the regenerated transformed sorghum
plant is produced within about 6-9 weeks of transforming the callus.
2. The process as claimed in claim 1, wherein the first culture medium comprises N6 medium, 1.5 to 3.5 mg/1 2, 4-D and a selection marker.
3. The process as claimed in claim 1, wherein the second culture medium comprises MS salts, 0.25 to 2 mg/1 IAA, 0.1 to 1 mg/1 kinetin and a bactericidal compound.
4. The process as claimed in claim 1, wherein the third culture medium comprises MS salts, 0.1 to 1 mg/1 NAA, and 0.1 to 1 mg/1 IBA.
5. The process as claimed in claim 1, wherein the auxin is selected from the group consisting of IAA, 2,4-D, NAA, IBA, and dicamba; and the cytokinin is selected from the group consisting of BAP, zeatin, kinetin, and TDZ.
6. The process as claimed in claim 1, wherein transforming the callus is transformed by using transformation method selected from a group consisting of Agrobacterium-mediated transformation, particle bombardment, electroporation and in-planta transformation.
7. A process of regeneration of a sorghum plant, said process comprising
a. culturing an explant for 7 to 30 days in a first culture medium comprising
an effective auxin to obtain embryogenic callus;
b. culturing the callus in a second and/or third culture medium comprising
effective ratio of auxin and/or cytokinin that supports the differentiation of
the callus into shoot and/or root tissues, to regenerate sorghum plant;
wherein the regenerated sorghum plant is produced within about 5-7 weeks
of culturing the callus.
8. The process as claimed in claim 7, wherein the callus is obtained from an explant selected from a group consisting of immature embryos, immature inflorescence, mature embryos, shoot node, mesocotyl, shoot apex and leaf.
9. The process as claimed in claim 7, wherein the first culture medium comprises N6 medium, and 1.5 to 3.5 mg/1 2, 4-D.
10. The process as claimed in claim 7, wherein the second culture medium comprises MS salts 0.25 to 2 mg / IAA, and 0.1 to 1 mg/1 kinetin.
11. The process as claimed in claim 7, wherein the third culture medium comprises MS salts, 0.1 to lmg/1 mg/1 NAA, and 0.1 to lmg/1 mg/1 IB A.