Title of Invention

A METHOD OF TREATING SUNFLOWER PLANTS

Abstract

Disclosed herein a method of treating sunflower plants to introduce foreign genes into their genomes, which comprises the steps of: a. Inoculating the seedlings of sunflower plant with one cotyledon detached, the inoculum being an Agrobacterium tumefaciens strain carrying marker genes on a binary plasmid; b. Following cocultivation, the germination process of the seedlings being continued for a definite period by planting them upright in Murashige and Skoog medium containing 250p,g/ml cefotaxime for 5 days; c. Subjecting the shoot portion of these seedlings to selection and rooting; d. Putatively transformed plantlets (To) being adapted to greenhouse conditions; e. Ascertaining by molecular analysis the integration and expression of the introduced genes in the primary transformants (To) and in the progeny (T1), the process being such that the transformation procedure facilitates rapid generation of transgenics containing functional foreign genes.

Full Text

This invention relates to the field of biotechnology. The invention further relates to a simple process of producing transgenic plants of sunflower (Helianthus annuns L.) Wherein the component of tissue culture-based regeneration has been substantially eliminated. Two-day old seedling of the sunflower cultivar KBSH-1 with one cotyledon detached were inoculated with an Agrobacterium tumefaciens strain carrying marker genes on a binary plasmid. Following cocultivation, the germination process of the seedling was continued for 5 days by planting them upright in Murashige and Skoog Medium containing 250 ng/ml cefotaxime. Later the shoot portion of these seedling were subjected to selection and rooting. The putatively transformed plantlets (To) were adapted to greenhouse conditions. They were advanced to produce Ti generation plants. The integration and expression of the introduced genes viz., the uid A and npt II marker genes in the primary transformants (To), and in the progeny (Ti) was ascertained by molecular analysis. The transformation procedure facilitated rapid generation of up to 2% phenotypically normal fertile plants containing functional transgenes. It is well known in the art that the use of tissue culture methods to faciUtate the transfer of genetic information into crop species has become increasingly important with most transformation procedures. Agrobacterium tumefaciens can readily infect several dicotyledonous crops. Nevertheless, the delay in obtaining transformed plants in these crops can be attributed to the difficulty of having the transformation and regeneration events occur in the same cell/tissue types. Further, regeneration in vitro of plants for many crops has been difficult as it is the case in many elite sunflower (Helianthus annuus L.) lines. Some potential goals of sunflower biotechnology are to improve the overall yield, increase resistance to insects and pathogens and to alter the oil composition. One route to accomplish these goals involves transfer of appropriate foreign genetic material into sunflower tissue and regeneration of whole plants from this tissues. Development of methods to obtain transformed sunflower plants which are independent of problems inherent to tissue culture of the genus has been the endeavor of many laboratories. Protocols involving minimized tissue culture or no tissue culture for obtaining transgenic sunflower plants indeed represent major accomplishments. Grayburn and Vick (1995) obtained Agrobacterium tumefaciens- transformed plants following wounding with glass beads and cocultivation. Knittel etal. (1994) recovered transgenic plants by microprojectile bombardment of half shoot apices in combination with Agrobacterium cocultivation. These methods are either cumbersome or require an equipment that can control the acceleration of microprojectiles. A transformation process that is simple, rapid, independent of large tissue culture effort is developed for sunflower. Two-days old seedlings with one of the cotyledons detached to create a wound site adjacent to the plumule, cotyledonary node and the hypocotyl were inoculated with an Agrobacterium tumefaciens strain LB A 4404/pKIWI105. Following cocultivation, the seedlings were allowed to grow up right on Murashige & Skoog gelled medium containing 250-jig/ml cefotaxime for 5 days. Later the shoot portions of the seedling were subjected to selection and rooting. Plantlets obtained were adapted to growth in the greenhouse where they flowered and set seed. Results of molecular analyses carried out confirmed the transgenic nature of the primary transformats and their progeny for the marker genes introduced. The transformation protocol invented is radically different in that it is independent of large tissue culture effort. The procedure becomes applicable to all those sunflower lines that show susceptibility to Agrobacterium Infection. The process developed deals with an Agrobacterium tumefaciens-msdiated gene transfer into sunflower {Healianthus annuus L.) cv. KBSH-1 in which the in vitro regeneration component has been substantially eliminated. Foreign genes were introduced with relative ease. The procedure is simple and reproducible. It is the primary objective to invent a novel method of treating sunflower plants to introduce foreign genes into their genomes. Further objectives of the invention will be clear from the following description. This invention thus provides a method of treating sunflower plants to introduce foreign genes into their genomes, which comprises the steps of: (a) Inoculating the seedlings of sunflower plant with one cotyledon detached, the inoculum being an Agrobacterium tumefaciens strain carrying marker genes on a binary plasmid; (b) Following cocultivation, the germination process of the seedlings being continued for a definite period by planting them upright in Murashige and Skoog medium containing 250p.g/ml cefotaxime for 5 days; (c) Subjecting the shoot portion of these seedlings to selection and rooting; (d) Putatively transformed plantlets (To) being adapted to greenhouse conditions; (e) Ascertaining by molecular analysis the integration and expression of the introduced genes in the primary transformants (To) and in the progeny (Ti), the process being such that the transformation procedure facilitates rapid generation of transgenics containing functional foreign genes. Now the invention will be described with reference to drawings accompanying the specification. Fig 1. of the drawings shows the stages of the sequential operation leading to the production of transgenic plants of sunflower (Helianthus annuus.L) cv.KBSH-1; Fig 2 of the drawings shows the expression of neomycin phosphotransferase II gene; Fig 3 of the drawings shows the western blot analysis of 3 -glucuronidase; Fig 4 shows the southern blot of PCR products of primary transformants probed with 2.1 kb uid A gene fragment; and Fig 5 shows the genomic southern analysis. Two-day old aseptically germinated seedling with one cotyledon freshly removed were inoculated for 5 minutes with over night culture of Agrobacterium tumefaciens strain LBA 4404 that contained the p-glucuronidase {uid A) and neomycin phosphotransferase (npt) II genes on the binary plasmid pKIWI105. Inoculation was carried out by immersion of seedling in the bacterial suspension in Mxirashige & Skoog (MS) medium adjusted to pH 5.6. The seedlings were blot dried and cocultivated for 48 hours and later washed for 30 minutes by gentle agitation in MS medium added with cefotaxime at 250 fig/ml. They were then planted upright in growth regulator-free MS medium containing cefotaxime at 250µg/ml for five days. Later, only the shoot axis (axis cut below the hypocotyl and the primary root portion discarded) of each seedling was subjected to kanamycin (50 µg/ml) selection for seven days and then transferred to MS medium with 250µg/ml of cefotaxime for root formation. The selection process was repeated with shoots that developed roots. Following a second round of selection, the plantlets were transferred to soilrite and later shifted to greenhouse. They were screend for the presence of the introduced genes in their vegetative and floral tissues. Molecular analysis of the primary transformants and their progeny confirmed the presence and inheritance of the transgenes. The transformation procedure facilitated rapid recovery of upto 2% phenotypically normal fertile transformants containing functional transgenes. A small number of selected plants from a batch of primary transformants (To) were not taken to the greenhouse and instead were cut back into the medium for axillary shoot multiplication. Also, tissue homogenates from individual plants of this batch were inoculated into Luria broth (LB medium) to check for the presence of residual Agrobacterium. Post-inoculation incubation of seedlings and shoot axes was carried out in a growth room maintained at 26-28°C under a 14 hours day white fluorescent light of intensity 35 µmolm"2 s"1. Preliminary experiments of tissue inoculation, coculture and transient GUS assays had shown that the sunflower cultivar KBSH-1 is susceptible to Agrobacterium tumefaciens infection. Detachment of one cotyledon at the plane of the embryonal axis produced a clear wound site for inoculation of the plumule, cotyledonary node and adjacent hypocotyledonary region. Of the different strains of Agrobacterium tested, LBA 4404 harboring the binary vector pKIWI105 was found to be the most effective vector for the transformation of KBSH-1. Also the uid A gene construct in pKIWI105 provided an additional advantage by eliminating expression of bacterial P-glucuronidase in the transformed tissues. The shoot axes of the cocultivated seedlings formed roots after kanamycin treatment whereas those of unifected (control) plants did not form root and showed sensitivity to kanamycin-induced bleaching. The rooted shoots established well in the greenhouse, flowered and set seed. Seeds of the To plants and tissues Ti plants were analyzed for the presence of the introduced genes. Transgene expression assays: the method of Jefferson (1987) was used to assess uid A gene expression at different time points beginning day 4 post-inoculation. Entire seedlings, leaf segments, florets and seeds of green house established plants and Ti plant tissues were assayed. The assay allowed determination of the frequency and degree of chimerism from the protocol. The inheritance of the uid A gene was evident in the randomly selected seeds as well as in the leaves of a few Ti generation plants. Western blot analysis also confirmed the integration of the uid A gene into sunflower genome as a band at 74 kDa position appeared in the protein samples of one month old plants when probed with GUS antibody. The assay to detect npt H expression was performed according to Reiss et al (1984). Protein extracts from leaves of both primary transformants and of Ti progeny gave signal at the expected 12-14kDa position indicating co-transformation. DNA Analysis: DNA was isolated from To plants which are verified as bacteria-free and from T12 progeny using the procedure of Dellaporta etal. (1983). Plasmid DNA was isolated from Agrobacterium and E. coli following the modified method of sambrook et al(1989). Polymerase chain reaction was performed using 200 ng of genomic DNA from primary transformants as well as from seeds of Ti generation to check for the presence of uid A sequence in and vir C sequences in the transformats. PCR results have shown the uid A sequence in the DNA of one-month old primary transformants and their seeds. When the vir C specific primers were used, no amplification was detected in the transgenic material indicating that the amplication obtained for uid A was not due to the presence of bacteria. DNA from selected primary transformants showing consistent GUS expression was used for southern analysis. A 2. Ikb uid A gene fragment was used as probe. The hybridization pattern of the To plant DNA is represented in Fig. 5a. All DNA samples showed signal at the expected fragment size of 3.4 kb. In the case of DNA from one of the plants (Fig. 5a, Lane 2), another higher molecular weight band was seen which more likely indicates a rearrangement. The integration of the uid A transgene in the genome was observed for one of the Ti plants also. The data demonstrated that our procedure indeed allowed recovery of transformed plants. The mature plants were characterized for phenotypic expression of the marker genes. The early indication of their stability in the progeny was available in the floral tissue, especially, in the ovule and pollen. The transformation regime facilitated rapid generation of up to 2% phentotypically normal fertile plants containing functional transgenes. The integration and transmission of marker genes to the progeny was demonstrated. Despite low frequency, the method described here offers certain advantages. It is not dependent on a large tissue culture effort and therefore can be utilized for the transformation of all those sunflower genotypes, that are susceptible to Agrobacterium infection but respond poorly to tissue culture manipulations. The transformed plants could be recovered rapidly without the possibility of genetic alterations imposed by tissues culture steps. Further, the method is technically simple and rapid. Sunflower genotypes/cultivars are difficult-to-regenerate plants in tissue culture, a limitation that hitherto hindered the application of genetic engineering methods for crop improvement. The procedure invented offers a means of introducing useful agronomical traits into sunflower genotypes/cultivars which are susceptible to Arobacterium infection but respond poorly to manipulations for plant regeneration in tissue cultures. Further, the method is technically simple, less labour-intensive and is not dependent on a large tissue culture effort. Transformed plants can be recovered rapidly without the possible risk of genetic alterations imposed by tissue culture steps. Fig 1. Stages in the production of transgenic plants of sunflower {Helianthus annuus L.) cv.KBSH-1. A, GUS expression in 4-day old seedlings infected with LBA 4404/pKIWI105 (bar = 4 mm). B, Kanamycin-selected plant four weeks after infection, (bar = 5 mm) C, GUS expression in the pollen of primary transformants. (bar = 30 µm) D, GUS expression in the seeds of transformed plants and lack of expression in the seed of uninfected plant. (bar= 1 mm) E, T1 plants established in the greenhouse. Fig 2 : Expression of neomycin phosphotransferase II gene. A. Expression in primary transformants. Lane 1 and 2: total protein extracts from one- month-old sunflower plants from uninfected with LBA 4404/ PKIWII05; Lane 3; total protein extracts from uninfected sunflower plants (negative control). B. Expression in Ti plants. Lane 1 and 2: total protein extracts from two GUS positive Ti plants; Lane 3: total protein extracts from uninfected sunflower plant (negative control). Fig 3: Western blot analysis of p-glucuronidase. Lanes 1, 2 and 3: total protein extracts (50 ng) from one month old sunflower plants infected with LBA 4404/ pKIWI105; Lane 4: total protein extract (50µg) from uninfected sunflower plant (negative control); Lane 5: purified GUS protein (20µg) (Clonetech) - positive control. Fig 4: primary transformants probed with 2.1 kb uid A gene fragment A. Southern blot of PCR products to show the presence of uid A gene in the primary transformants probed with 2.1 kb uid A gene fragment. Lane 1: marker, Lanes 2-7: amplified product size of 514 bp. Lanes 10-15: amplified product size of 667 bp; Lanes 2and 10: LBA 4404/pKIWI105 DNA (positive control); Lanes 4-7 and 12-15: sunflower plant DNA infected with LBA 4404/pKIWI105; Lanes 3 and 11: uninfected sunflower plant DNA (negative control). B. PCR to show the inheritence of the uid A transgene in the Ti generation (seeds). Lane 1 : marker; Lane 2: LBA4404/pKIWI105 DNA (positive control; Lane 3: DNA of seeds from uninfected sunflower plant (negative control); Lanes 4 -7: DNA from seeds of primary transformats. Fig 5: genomic southern analysis A. Southern blot of primary transformants. 10 fig DNA from the primary transformants was digested with EcoRI and probed with a 2.1 kb uid A gene fragment. Lane 1: DNA of uninfected sunflower plant (negative control); Lanes 2, 3 and 4: DNA of putative transformants - arrows at 3.4 kb position indicate the expected band; arrow at 6.6 kb indicates either a second site of integration or a rearrangement of the uid A transgene; undigested DNA at the major band position (Lane 2). B. Southern blot showing the transmission of the uid A gene to Ti generation. Undigested DNA and 10 jig of Xba I-digested DNA from one of the Ti plant was probed with a 2.1 kb uid A gene fragment. Lane 1: DNA from uninfected plant (negative control). Lane 2: 2.1 kb uid A gene fragment (positive control). Lane 3: Digested DNA of T1plant. Lane 4: undigested DNA from the same plant. It is to be noted that the aforesaid description is intended to explain the salient features of the invention and it is not intended to limit the scope of the invention. It is to be further noted that within the scope of the invention, various modifications are permissible. The scope of the invention is defined in the above descriptions. We Claim; 1. A method of treating sunflower plants to introduce foreign genes into their genomes, which comprises the steps of: (a) Inoculating the seedlings of sunflower plant with one cotyledon detached, the inoculum being an Agrobacterium tumefaciens strain carrying marker genes on a binary plasmid; (b) Following cocultivation, the germination process of the seedlings being continued for a definite period by planting them upright in Murashige and Skoog medium containing 250p,g/ml cefotaxime for 5 days; (c) Subjecting the shoot portion of these seedlings to selection and rooting; (d) Putatively transformed plantlets (To) being adapted to greenhouse conditions; (e) Ascertaining by molecular analysis the integration and expression of the introduced genes in the primary transformants (To) and in the progeny (Ti), the process being such that the transformation procedure facilitates rapid generation of transgenics containing functional foreign genes. 2. A method as claimed in claim 1, wherein the transformed plants are recovered rapidly without the possibility of genetic alterations imposed by tissue culture steps. 3. A method of treating sunflower plants to introduce foreign genes into their genomes substantially as herein before described and illustrated in the accompanying drawings.

Documents:

168-mas-1999 abstract duplicate.pdf

168-mas-1999 claims duplicate.pdf

168-mas-1999 description (compelet) duplicate.pdf

168-mas-1999 drawings duplicate.pdf

168-mas-1999-abstract.pdf

168-mas-1999-claims.pdf

168-mas-1999-correspondence others.pdf

168-mas-1999-correspondence po.pdf

168-mas-1999-description complete.pdf

168-mas-1999-description provisinol.pdf

168-mas-1999-drawings.pdf

168-mas-1999-form 1.pdf

168-mas-1999-form 19.pdf

168-mas-1999-form 26.pdf

168-mas-1999-form 5.pdf