WO2002057407A2 - Novel method for transgenic plants by transformation & regeneration of indica rice plant shoot tips - Google Patents

Abstract

The present invention deals with the technology of gene transfer into plant tissues by use of the Agrobacterium method, wherein, callus has been generated from 4 day old Indica rice shoot tips which were cocultivated with the binary vector pIG121Hm or any other binary vector.

Description

NOVEL METHOD FOR TRANSGENIC PLANTS BY TRANSFORMATION & REGENERATION OF INDICA RICE PLANT SHOOT TIPS

Field Of The Invention:

The present invention deals with generating callus from Indica rice shoot tips and opens up whole new possibilities for micro propagation and for producing genetically improved plants with the aid of novel methods of agro-biotechnology and genetic engineering. This invention covers the technology of delivery of genes into the plant tissues by using the Agrobacterium method.

Background:

The inadequacy of a routine, repetitive regeneration from plant cell culture systems for agriculturally significant crops is a major hurdle in the application of genetic engineering technology to plants. Regeneration of cells into more cells, tissues, organs and even whole organisms is a process central to life. The study of regeneration in plants has been well documented, especially, where it is vital in vegetative propagation. Plants can however be propagated in two ways, through the seeds or without use of the seeds as starting material to obtain the desired plant, but, both kinds of propagation may be undesirable or impossible under certain conditions. In cases, where propagation through seeds is not satisfactory, i.e., in cases, where, there are no seeds or only a few seeds formed or the desired seeds lose their germination viability quickly, in that event, the alternative is to take recourse to the seedless propagation route. In many instances, on account of sexual crossing, a heterogeneous progeny through seeds is often considered unsatisfactory. Undoubtedly, seedless propagation of essentially seedless starting material may at a later stage, give rise to the desired seeds, which can be then utilised further, to obtain the desired plants.

Regeneration in plants consists of the formation of new tissues having, both, root and shoot meristems, separate shoot or root meristems, plant organs or organ primordial from individual cells or groups of cells. Regeneration in general, impersonates the process of normal cellular and organ differentiation that take place during plant development and results in the formation of different plant organs.

In respect of the seedless propagation of plants, two major fields can be distinguished, namely, in vitro and in vivo. In vivo propagation, viz, cuttings, splitting or division, layering, earthing, grafting and budding are but a few examples, which, have for years, played an important role in agriculture, e.g. with potatoes, apples, pears, many ornamental crops like carnations, chrysanthemums etc. Vegetative propagation too is important in plant breeding.

In in-vivo propagation, the traditional methods often fall too short, either due to being too slow, too difficult or too expensive or impossible. It has been almost two decades since the discovery came to light that plants can be more rapidly cloned in vitro than in vivo, and ever since then, knowledge concerning vegetative propagation has grown in leaps and bounds, and the same holds good for diverse plants from diverse regions, namely, temperate, sub-tropical and tropical. In fact, it is now possible to clone species by making use of in vitro culture techniques that are impossible to clone in vivo. With regard to the generation of transformed or transgenic plants, in vitro propagation is even considered a pre-requisite, since it is the totipotency of individual plant cells that underlie most plant transformation systems.

In order to propagate plants from starting material in vitro, it is necessary that at least one cell in the starting material is capable of regeneration. The ability for regeneration is determined by the genotype, the environmental conditions, i.e. the nutrient supply, the regulators and the physical conditions, the developmental stage of the plant and/or a combination of all these. Plant cells or groups of cells under normal conditions are unable to initiate the formation of certain plant organs, meristems or organ primordial and can often be motivated by extra cellular stimuli altering the differentiation stage of the cell. It is only after the re-differentiation of a cell or tissue that regeneration is possible, for, it will result in differentiated tissue that will once again comprise the requisite three-dimensional lay out of the emerging plant, the apical-basal or shoot- root body plant from which the mature desired plant can develop.

The process of regeneration of the original starting cell into a multi cellular, totipotent tissue underlying and preceding the somatic embryogenesis or organogenesis in the cell, tissue or explant cultures can lead to a fully differentiated plant. Even though the skill of individual plants and cells to regenerate into complete plants, i.e., totipotency, is a well known phenomenon, however, each plant or part of the plant requires specialised studies to invent the conditions that permit such regeneration.

An important aspect that has to be looked into is the fact that inventing conditions for efficient regeneration of plants requires developing specialised knowledge about a given plant. Innovativeness is essential for tissue culture in identifying the plant that responds positively and efficiently to the various conditions and ultimately leads to prolific regeneration. There is no general principle that can be applied to achieve regeneration. In every case, identification of the explant and the culture conditions are by themselves innovative steps in the development of a tissue culture method for regeneration of a plant.

In shoot cultures, apart from axillary shoots, there are adventitious shoots formed directly on existing leave or stem implants and/or shoots arising indirectly from the callus at the base of the explant. Since a number of plants have been propagated commercially by the tissue culture method, by which, the tissues of the selected plant are grown on a medium, which in turn causes a multiplication of the tissues. These multiplied tissues are divided and the divisions grown on other media can cause rapid increase of other structures thereby ultimately giving rise to wholly regenerated plants. Plant regeneration can be obtained from organised cell systems, bud, stem segment, shoo tip, meristem and other organ cultures.

Plant cell cultures are a potential commercial source of medicines, dyes, enzymes, flavouring agents and aromatic oils. Plant cell production of those compounds are much sought after when they are produced by the plant in small quantities, in transitory or unharvestable developmental stages of the plant's life cycle and when produced by plants in conditions not amenable to agriculture or in inaccessible environments and also when the compounds cannot be satisfactorily synthesised in vitro or by other biosynthesis systems.

In general, plant transformation indicates the introduction of a nucleic acid segment carrying a functional gene, normally, a heterologous gene into a plant that did not earlier on possess the gene. In a triumphant transformation, a DNA construct containing a structural coding sequence is placed in the genome of a plant by one of the several known methods. The well known transformation methods include direct gene transfer into the protoplast, microprojectile bombardment, injection into the protoplast, cultured cells and tissues or meristematic tissues, electroporation and Agrobacterium-mediated transformation.

Transformation of dicotyledenous plants with Agrobacterium tumefaciens has been well established and widely used, but not so in the case of monocotyledonous plants. However in recent years, a lot of data has been unveiled on the transformation of monocots using Agrobacterium, viz, the report of T-DNA integration into genomic DNA of rice, Oryzae sativa (Raineri, D.M. et al, 1990), proved that graminaceous crop plants can be transformed by the Agrobacterium method.

The Agrobacterium-mediated transformation is a complex process involving many aspects. Gene transfer by means of engineered Agrobacterium strains has become routine for many dicotyledenous crops but considerable difficulty has been experienced using Agrobacterium to transform monocots, in particular, cereal plants like rice (Kloti Andreas S., 2001-02). Raineri et al., Bio/Technol. 8, 33-38 (1990) inoculated a supervirulent strain of Agrobacterium into eight varieties of rice consequent to injuring the scutella of rice plants. Even though, certain resistant calli were observed, transformed plants could not be obtained from the calli (Kloti Andreas S., 2001-02). Previously it was reported that potato suspension culture (PSC) is essential for the ._4grobαcterz^'«m-mediated transformation (Chan M.T. et al, 'Transformation of Indica rice (Oryza sativa L) mediated by Agrobacterium", Plant Cell Physiol, (1992), 33:577-583). PSC is rich in the phenolic compounds acetosyringone (AS) and sinapinic acid (SA). Although the role of these two compounds in the success or efficiency of transformation is not yet known, the results imply that transformation of monocots, at least rice, using Agrobacterium can be improved by the addition of certain substances. It was previously shown that young tissues of rice root have a greater potential to be transformed by Agrobacterium, if, appropriate conditions are applied (Chan, M.T. et al., 1992).

Hiei (U.S. Patent No. 5,591,616 and European Patent Application 0 897 013 Al) describes methods of transforming monocots like rice with an Agrobacterium system including the super binary vectors with the use of prepared, de-differentiated cultured tissue such as callus or adventitious embryo-like tissue in cell culture, but the whole of the rice plant is not described therein. Dong et al. (U.S. Patent No. 6,037,522) have described a method for Agrobacterium-mediated transformation of monocots, wherein, the monocot inflorescences are dissected from the plants and cultured in callus-inducing tissue culture media, where the inflorescences or the callus derived thereof, are co-cultivated with the Agrobacterium cells. But, this method includes tissue culture steps and is not an in planta transformation method. Successful transformation of rice using Agrobacterium has far more advantages over biolistics in that, the frequency of the gene transfer is higher. Furthermore, biolistics results in insertion of undersirable gene fragments. Rapid, reliable production of regeneration-competent rice callus from any genotype is still not routine (Sung Hun Park et al., 1996).

Summary:

The present invention deals with the technology of gene transfer into the plant tissues by use of the Agrobacterium method, wherein, callus has been generated from 5 day old Indica rice shoot tips which were cocultivated with the binary vector pIG121Hm.

Description:

The present invention as set forth herein below is capable of being adapted to other plant species, which are capable of regeneration from the shoot apices or axillary buds without any significant experimentation and/or deviation from the invention per se. The method as such can be applied to other monocotyledonous plant species. Certain additives like acetosyringone for example, can be added to enhance the successful implementation of the transformation of the shoot apices.

Unless otherwise defined, all technical and scientific terms used herein, possess the same meaning as is normally understood.

Transformation: The term transformation refers to the stable introduction of a nucleic acid sequence into the said host.

DNA: DNA refers to and means the deoxyribonucleic acid, considered to be substantially the same as 'DNA Sequence' or 'DNA Polynucleotide' or 'Polynucleotide' and can be used interchangeably.

Strain: Strain means and includes different forms of species belonging to the Agrobacterium group maintained in successive cultures for subsequent plant treatment contact or inoculation.

Plasmid: The term 'plasmid' refers to a small, circular, independently replicating piece of DNA, i.e., those that exist inside a bacterial cell, but, are independent of the bacteria's main DNA.

Vector: The term vector means and includes a plasmid that can be used to transfer DNA sequences from one organism to another, for e.g., in the present invention, the vector has been made use of to transfer the DNA from the Agrobacterium clone to a plant cell.

Asrobacterium: Agrobacterium is a plant pathogen that transfers a set of genes encoded in a region called T-DNA of the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes to transfer DNA into the plant chromosomes. In this invention, the transformation of the Indica rice plant, a monocotyledonous plant, has taken place by using the shoot apex tissue. The shoot apex is most favoured for this kind of technique since most monocotyledons can be regenerated into plants with the use of this source. Further, shoot cultures are known to develop directly and swiftly into rooted plants.

Germination of explant source:

IR64 and the Basmati-370 rice variety seeds were dehusked and surface sterilised with 70% ethanol for one minute. This was then soaked in 50% commercial bleach, which lasted for approximately 20 minutes. Subsequently, the seeds were rinsed five times in sterile water.

Next, the sterilised seeds were aseptically germinated on the Murashige and the Skoog media, which contained 3% sucrose in it. Four days later the germinated seed shoot tip was excised and co-cultivated together with the Agrobacterium strain, EHA- 105, possessing the binary vectors, pIG121Hm and pCAMBIA1390.

Preparation of Asrobacterium Culture:

The vector pIG121Hm was transformed into the Agrobacterium strain EHA 105, by using the Freeze-Thaw method (An et al, 1998). The transformed colonies were selected and thereupon confirmed through digestion by making use of the enzyme PvuII. The transformed colonies were then streaked onto a YEP plate and later, on to an AB plate. It was from the AB plate that one loopful of the culture was taken and inoculated in 20 ml of induction media with 100 μm acetosyringone and placed in it for 2-3 hours till the optical density displayed a reading of 0.6-1.0 at 600 nm.

The excised shoot tips were inoculated in induction media for about 10 minutes and the excess Agrobacterium was blot-dried on sterile tissue paper and later, placed on co-cultivation media for about three days. After three days, the shoot tips were washed 2-3 times in sterile water by shaking the said shoot tips vigorously. The shoot tips were then placed on 250 ppm of cefotaxime for 2-3 hours. After this, the shoot tips were callused on MS media containing 3% maltose and 5.0 mg/L 2, 4-D with 30 mg/L hygromycin for selection and 250 mg/L cefotaxime. The resultant calli obtained were subcultured on RC medium containing 5.0 mg/L 2,4-D for about 4 weeks.

Pre-Regeneration Treatment:

Prior to placing the calli for regeneration, the said calli were placed on the RC medium containing in it 2% sucrose and 2% mannitol together with the hormones ABA (5mg/L), NAA (1.0 mg/L) and Kinetin (2 mg/L) for 7 days and then incubated at 28°C in the dark.

After having subjected the said calli to pre-regeneration, the same were transferred to the regeneration medium, RC medium containing in it 3% maltose and the hormone- kinetin (2.5 mg/L) and NAA (0.1 mg/L). The calli were incubated in the dark at 28°C for three days after having been transferred onto the regeneration medium for shoot development and thereafter being subjected to light and dark cycles of 16 hours and 8 hours respectively until the sprouting of the green shootlets.

Root Induction:

The shootlets were excised from the calli and transferred onto the half strength MS medium for root induction.

Hardening:

The well-rooted shootlets were transferred to the vermiculite for hardening and then, watered with Hoaglands solution and finally transferred to the soil, where paddy is cultivated, after two weeks.

Accordingly, in the present invention, we have described the transformation of the Indica rice variety with Agrobacterium.

This is the first report for shoot tip derived calli from the Agrobacterium mediated transformation technique involving a reporter gene. Therefore the chosen foreign gene i.e., the reporter gene (β glucuronidase) uidA used in the present invention plays two important roles as a : i) foreign gene to be inserted ii) reporter gene to indicate the successful transformation of the shoot tip.

Example 1

Corn seeds (variety Ganga-11) were rinsed with distilled water for about 10 minutes and treated with 70% ethanol for 1 minute followed by 50% commercial bleach for 20 minutes and then rinsed 3-4 times with sterile water. The strerile seeds were transferred to plain MS media for germination. After 3 days, the shoot apices were isolated and co-cultivated with the Agrobacterium strain EHA-105 possessing the binary vector pIG121Hm for two days. After 2 days, the shoot tips were washed with cefotaxime and placed for callusing on MS media containing 5 mg/1 2-4, D with hygromycin for selection-25mg/l. The calli were subcultured for 15 days and the transferred onto the regeneration media to obtain multiple shootlets.

Example 2

Pearl millet (843-B) seeds were rinsed with distilled water for 10 minutes and treated with 70% ethanol for 1 minute followed by commercial bleach of 50% strength for 20 minutes and rinsed 3 to 4 times with sterile water. The sterile seeds were transferred onto plain MS media for germination. After 3 days, the shoot apices of 3mm in length were isolated and co-cultivated with the Agrobacterium strain EHA-105 containing the binary vector pIG121Hm for 2 days. Two days later the shoot tips were washed with cefotaxime and placed for callusing on LS media containing 0.2mg/L kinetin and 2.5mg/l, 2, 4-D and 30mg/l hygromycin. The calli were subcultured every 15 days and shifted to the regeneration media containing IBA, 1.5mg/l and BAP 2mg/l to procure multiple shootlets. Conclusion:

Even though many methods for transformation of rice using protoplasts or suspension cells are available, they have by and large proved to be unsuccessful. Methods relating to the use of the soil bacterium Agrobaύterium tumefaciens are still preferred since it does not require protoplasts and it generally results in higher transformation efficiency and an even more predictable pattern of external DNA integration than other transformation techniques (Czemilofsky, A.P. et al, 1986, DNA 5: 101-1 13).

In this invention, we have been able to generate 15% transformed calli from the co- cultivated shoot tips of the Indica rice variety.

References

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Claims

Claims:

1. A novel method of transforming excised shoot tip tissue of the Indica rice cultivars by using the Agrobacterium method.

2. The method of claim 1 wherein the shoot tip comprises of the meristem dome.

3. The method of claim 1 wherein the shoot tip is excised from 4 day old germinated seeds.

4. The method of claim 1 wherein the Agrobacterium tumefaciencs comprises of the strains, EHA 105 and LB A 4404, but, other strains can also be used.

5. The method of claims 1 and 4 wherein acetosyringone is used for inducing virulence genes in the Agrobacterium transformation technique.

6. The method of claims 1 and 5 wherein the Agrobacterium transformation comprises of the induction media.

7. The method of claims 1 and 6 wherein the shoot tips were co- cultivated after inoculating the same in induction media to transform them.

8. The method of claim 1 wherein the co-cultivated shoot tips were placed on MS media for callusing the same.

9. The method of claims 1 and 8 wherein the calli were placed on the regeneration media until the formation of the shootlets.

10. The method of claim 1 wherein phytohormones are induced into the media.

11. The method of claim 1 wherein the target shoot tip extends to pearl millet and corn apart from rice.

12. The method of claim 1 wherein, by making use of the Agrobacterium method, 90% of the shoot tips have revealed positive results whereas 15% were callused.