SUMMARY OF THE INVENTION:

Agrobacterium tumefaciens (C58/pMP90) harbouring binary vector (pTCS5) was used for cotton transformation. Prof. Gopi Podilla of University of Alabama, USA constructed the binary vector and transferred to JK-Agrigenetics Ltd for the improvement of cotton. Fig.l shows the T-DNA region of binary vector carrying PTM3 (Sense orientation) and uidAlnptll fusion gene. Duplicated cauliflower mosaic virus (CaMV) 35S promoter and NOS terminator drive the expression of both these gene.

Cotton transformation was performed by following the optimized protocol in our laboratory.The hypocotyl explants (5-7mm) of 4 days old seedlings were used as primary explants for developing the transgenic lines. A total of 500 hypocotyls explants were co-cultivated with Agrobacterium tumefaciens. The plants regenerated from 67 kanamycin resistant calli were established in pots and designated as individual transformation events (To Event 1-67).Also the non-transgenic plants regenerated through somatic embryogenesis were established for comparisons in our studies. The PCR amplifications of internal fragment of nptll gene and PTM3 gene (data not shown) was confirmed that the T-DNA was stably integrated into cotton genome in all the events.

Since both GUS and nptll gene were expressed from the same promoter, GUS staining was observed in all the transgenic events that were positive for nptll gene. Fig.2 shows the results of GUS assays performed during various stages of transformation and plant parts of the selected transgenic cotton event-10. The results of GUS staining of pollen (Fig.2.K) showed the segregation of transgene (T-DNA insert) close to 1:1 ratio when replicated for counting under microscope. The southern blot performed on selected transgenic events, showed the hybridization at 8.8 kb, 10.7 kb and 15.9 kb for the event-10, 24 and 48, respectively (Fig.3A). No hybridization was observed in non-transgenic control. The results indicated that these represented independent transformation events and have a single copy of PTM3 insert. All these events were analyzed by western blot analysis and a 28kd size band, expected of PTM3 protein, was detected (Fig.3B). Non-transgenic control plant did not show any band

Agronomical characters of five randomly selected plants of each To transgenic lines (Event 1-67) were evaluated and compared with equal number of non-transgenic plants produced from the same experiments. All the transgenic events showed similar morphology at early growth period as compared to control plants. All the transgenic events except event-24 showed earliness in flowering and maturity that varied from 4-15 days as compared to the respective non-transgenic counterparts. In addition, some events showed no square dropping. The abnormal growth and absence of flowering in event-24 might be due to the positional effect of the PTM3 insert. Among all transgenic lines, the event-10 was found to be more significant in terms of early flowering and maturity and its potential value in breeding. The data generated on agronomic characters of To event-10 is presented in Table 1. From the data it is evident that event-10 showed significant difference in earliness by 12 days in flowering and 15days in maturity as compared to control.

Cseke et al. (2005) reported earliness in flowering and maturity in Arabidopsis and tobacco when the same PTM3 gene was ectopically expressed The event-10 showed no square dropping throughout the crop cycle and it would be interesting to know how PTM3 gene is involved in the development of boll in cotton and its dropping behaviour. A similar result was reported by Mao et al. (2000) that the deletion mutant of JOINTLESS (MADS-box gene) tomato failed to develop the abscission zones on their pedicels and observed no normal abscission of flowers or fruits. Since no square dropping was observed in event-10, the early squares flowered early and matured 15 days earlier leading to a significant yield increase by about 27% as compared to control (Table 1).

The results of event 10 clearly indicated that it is superior to non-PTM3 counterparts. Similar to PTM3 gene, significant early flowering phenotype was reported in various crop species expressing other SEP-c\ass genes (Jang et al., 1999,2002; Pelaz et al., 2001; Tzeng et al., 2003; Ferrario et al., 2003; Lemmetyinen et al., 2004). Similar to To generation, the Ti generation plants were evaluated in the greenhouse and exhibited less than 10% square dropping, 13 days early flowering, 12-15 days early maturity and 22% increased yield as compared to control. The average data generated on randomly selected 10 plants of Ti generation of event-10 are presented in Table -2. Since To and Ti generations were evaluated in the pots, transferred T2 transgenic plants directly on soil in the contained green house and evaluated. The T2 plants also showed similar characteristics of minimal square dropping, 8-15 days early flowering, 12-15 days early maturity (Fig.4) and enhanced yield of 17%. The average data obtained from 63 plants of T2 generation in comparison with equal population of control is presented in Table-3. These results confirmed stable inheritance of PTM3 induced characters in cotton for two successive generations and clearly indicated that it is superior to non-PTM3 counterparts.

Reference

1. Cseke, L.J., Cseke, S.B., Namritha, R., Taylor, L.C., Anupama, S., Banalata, S., Ramesh, T., David, F.K., Podila, G.K., 2005. SEP-class genes in Populus tremuloides and their likely role in reproductive survival of poplar trees. Gene 358,1-16.

2. Jang. S., An, K., Lee, S., An, G., 2002. Characterization of tobacco MADS-box genes involved in floral initiation. Plant Cell Physiol. 43, 230-238.

3. Jang, S., Hong, M.Y., Chung, Y.Y., An, G., 1999. Ectopic expression of tobacco MADS genes modulates flowering time and plant architecture. Mol. Cells 9,576-586.

4. Mao, L., Begum, D., Chuang, H.W., Budiman, M,A., Szymkowiak, E.J., Irish, E.E., Wing, R.A., 2000. JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development. Nature 406, 910-913.

5. Pelaz, S., Gustafson Brown, C, Kohalmi, S.E., Crosby, W.L., Yanofsky, M.F., 2001. APETALA1 and SEPALLATA3 interact to promote flower development. Plant J. 26, 385-394

6. Prakash, A.P., Kumar, P.P., 2002. PkMADSl is a novel MADS-box gene regulating adventitious shoot induction and vegetative shoot development in Paulownia kawakamii. Plant J. 29,141-151.

7. Tzeng, T.Y., Hsiao, C.C., Chi, P.J., Yang, C.H., 2003. Two lily SEPALLATA-like genes cause different effects on floral formation and floral transition in Arabidopsis. Plant Physiol. 133,1091-1101.

8. Vrebalov, J., Ruezinsky, D., Padmanabhan, V., White, R., Medrano, D., Drake, R., Schuch, W., Giovannon, J.A., 2002. MADS-box gene necessary for fruit ripening at the tomato Ripening-inhibitor (Rin) locus. Science 296,343-346

9. Zhang, H., Forde, B.G., 1998. An Arabidopsis MADS-box gene that controls nutrient-induced changes in root architecture. Science 279, 407-409

Table 1 Agronomic data generated from To generation plants of PTM3 event -10 and its non-PTM3 counterpart.

Line Days to
50%
flowering Square dropping No of Sympodia Total No of bolls Yield/ plant (g> Increased yield Maturity over
-Non-PTM3 cotton

PTM3 Cotton Event-10
Non-PTM3 Cotton 51±1.14 63±1.30 No Yes 18.2 13.9 42 31 185.4 ±4.82 145.7 ±4.13 27% 15 days

Table 2: Agronomic data generated from Ti plants of event-10 and control.
Line Days to 50% Square Number of Total bolls Yield Increased Maturity flowering dropping Sympodia yield over control

Event-10
Control

52 65

No Yes

18.2 14.4

96 422.6 g 22% 12-15 days
74 345.4 g

Table 3: Agronomic data generated fromT2 plants of event-10 and control directly on soil at Contained Green House

Characters

Event-10

Control


Square dropping No Yes
Days to 50% flowering 52 (8 days early) 60
Boll size Medium big Small
Boll weight (Average of 20) 5.35g 4.6g
Yield :1st picking 6.40 kg 2.90 kg
2nd Picking (after 25 days) 0.82 kg 3.27 kg
Total fiber from two picking 7.22 kg 6.17 kg
Yield Increase 17% -
Maturity 12 days early
Note Presented data of 63 plants


Fig.l T-DNA region of PTCS5 binary vector: RB: Right border; LB: Left border; nptll: neomycin phosphotransferase II; GUS: P -glucuronidase; NOS- 3': Signal of Nopaline Synthase terminator; 35/35S: Cauliflower mosaic virus 35s promoter with double enhancer; AMV: untranslated leader sequence from Alfalfa Mosaic Virus RNA4; PTM3 cDNA: Populus tremuloides MADS-box gene 3 in sense orientation

Fig.2 GUS assay for various developmental stages of PTM3 event-10. A: Embryogenic calluses, B: Embryo's, C: Developing embryo's, D: Plantlet with roots, E: Leaf, F: Leaf petiole junction, G: Petiole, H: Cross section of petiole, I: Anthers, J: Embryo of non - transgenic cotton, K: Pollen of To transgenic cotton

Fig.3. A. Southern hybridization showing single copy insert in selected transgenic events. Mw: Molecular weight marker (Lambda Hindlll marker), Lane 1: Event 48 (15.9 kb size), Lane 2: blank, Lane 3: Event 24 (10.7 kb size), Lane 4: Event 10 (8.8 kb size), Lane 5: Negative control. B. Western blot analysis. Lane 1: Event 48, Lane 2: Event 24, Lane 3: Event 10, Lane 4: Negative control

FIg.4 Agronomic evaluation in Polyhouse: T2 plants of event-10 and control transplanted directly in soil. A - Plant vigour; B - Early maturity; C- Enlarged picture of early maturity.

In plants, MADS-box genes form a large family of transcription factors that is involved in various aspects of development including flowering, floral meristem identity, floral organogenesis, fruit formation, seed pigmentation, endothelium development, control of root structure (Zhang, 1998) and fruit ripening (Vrebalov et al., 2002). Mutations in these genes usually result in homeotic transformation of floral organs. The developmental process in higher plants is a complex phenomenon in which extrinsic and intrinsic factors plays important roles (Prakash and Kumar, 2002).Currently the available genetic and molecular information in Arabidopsis, Antirrhinum and Oryza sativa has let to better understanding of these processes. The expression or suppression of MADS-box gene in transgenic plants and analyzing loss or enhanced function mutants would reveal the function of MADS-box genes in that particular plant species. The ectopic expression of MADS-box genes in tobacco or Arabidopsis under the control of constitutive promoters has become a useful tool in the analysis of gene functions (Mandel et al., 1992; Kempin et al., 1995; Davies et al., 1996; Rutledge et al., 1998; Cseke et al., 2005). PTM3 is a MADS-box gene identified from Aspen tree (Populus tremuloides). Transgenic Arabidopsis expressing PTM3 gene in sense orientation resulted in early flowering phenotype (Cseke et al., 2005). At the same time, transgenic Arabidopsis expressing inverted repeat constructs of PTM3 gene resulted in dwarfism with delayed flowering. Also Mao et al. (2000) reported that the deletion mutant of JOINTLESS (MADS-box gene) tomato failed to develop the abscission zones on their pedicels and observed no normal abscission of flowers or fruits. Similar to PTM3 gene, significant early flowering phenotype was reported in various crop species expressing other S£P-class genes (Jang et al., 1999, 2002; Pelaz et al., 2001; Tzeng et al., 2003).

Brief Description;

The PTM3 gene of Aspen was ectopically expressed in cotton and produced several transgenic lines via. Agrobacterium tumefaciens mediated genetic transformation. The transgenic cotton lines expressing PTM3 gene showed earliness in flowering and maturity. Among all the events, the event-10 was found to be phenotypically significant with earliness in flowering and maturity and yield enhancement. In addition, the event-10 showed no square dropping and allowed the plants to bear more number of bolls.

Executive Summary;

The PTM3 (MADS box) gene of Aspen was ectopically expressed in cotton via genetic manipulation to explore the opportunity to introduce desirable agronomic traits with the potential to improve yield and modify the duration of the parent cotton variety. Several transgenic cotton lines expressing PTM3 gene were developed and exhibited earliness of 4 to 15 days variations in flowering and maturity. Among all the events, the event-10 was found to be phenotypically significant with earliness of 12 days in flowering and 15 days in maturity and yield enhancement of 27%. In addition, the event-10 showed no square dropping

Background of the Inventions;

The searches were focused based on the following

1. Ectopic expression of MADS-box gene from any plant system to model system to understand the gene functions in developmental processes.

2. Identified functional mutant due to over-expression of MADS-box gene
Differences;

To the best of our knowledge, this is the first report of transgenic cotton expressing PTM3, a MADS-box gene from Aspen tree {Populus tremuloides). PTM3 expression in some events led to superior agronomic characters that are of interest to breeding shorter duration varieties with improved yield. Among all the events, variations of 4 - 15days were found for earliness in flowering. In addition, some events including event-10 showed no square dropping. The analysis of event-10 revealed that the plants matured 15 days earlier with increased yield (27%) as compared to control.

Advantages;

Heterologous expression of PTM3 gene in cotton event-10 exhibited significant difference with earliness in flowering and maturity and enhancement of yield. In addition, the event-10 showed no square dropping and allowed the plants to bear more number of bolls over Non-PTM3 plant

Claims;

1). I/we claim the The PTM3 gene of Aspen was ectopically expressed in cotton to explore the opportunity to introduce desirable agronomic traits with the potential to improve yield and modify the duration of the parent variety