Cotton is one of the most important fiber r and food crop in the world and is a unique species, which has both genetic and commercial value Cotton belongs to the genus gossypium and has genetic resources both in domesticated and wild forms (Bhale, 1999). Cultivated Asiatic species, G.arboreum L. and G.herbaceum L., are diploids with 26 chromosomes and the cultivated New Word species, G.hirsutum and G.barbadense are allotetraploids with 52 chromosomes (Stewart and Hsu, 1977).

The world's commercial cotton production is dominated by tetraploids, there by driving a constant need to broaden its available genetic base (Moradi, 1997).

Resistance to certain pathogens and insects, male sterility and certain plant morphological traits possessed by Old World cotton (diploids) are potentially useful for incorporation in tetraploid cotton for higher production (Stewart and Hsu, 1978).

For a long time, cotton breeders have attempted crosses between diploid and tetraploid (2n=52) species (Gill and Bajaj, 1987). However many, of the hybrids are difficult or impossible to obtain in situ because of several incompatibility factors. Ovule-embryo cultures, application of Gibberellic Acid, and naphthalene Acetic Acid have been suggested to improve distance hybridization in cotton (Amalraj, 1989; Mofidabadi and Modir-Rahmati, 2000). Many attempts have been done for in vitro nutrition of immature embryo but due to cross pollinated flower abscission and long term embryo development in cotton, embryo rescue was not a reliable method to capture hybrid plant. Therefore in this study, Gibberellic was used to overcome flower abscission of pollinated tetraploid and diploid seed parents to allow hybrid embryo development and seed setting.

1 Results and discussion
Out of 400 flowers witch have been treated with 100 ppm, 234 bolls were retained (58.5%)  45 days after pollination (DAP), While only 30 bolls were developed  from 400 pollinated flowers (7.5%) which was not treated with GA3.In spite of a few boll developments, in control treatment, no seed was observed after 45 DAP (Day after pollination).Comparison between mean effects of hormone growth regulator for boll retention indicated highly significant differences between control and GA for hybrid boll setting at α=0.05 level using Chi-square test. Therefore application of GA3 is necessary and recommended to overcome pollinated flower abscission. Mean effect of parent for cross ability indicated that Siokra has more potential for crossing with two different diploid parent then the Sahel (Table 1). Maximum boll development (64%) observed in Sephid as tetraploid seed parent in crosses with Hasham Abad (as diploid pollen donor parent) while low level of boll and seeds development was observed in Hashem-abad×Sephid (18%) crosses (Table 1). The same result for cross ability of Siokra reported by Bhale 1999. He stated that Okra leaf type of cotton has higher potential for cross ability than other commercial cotton varieties. Therefore at least, 50 meters isolation distance has to be considered for seed production of Okra leaf type variety in seed production nursery. Highly Significant differences were observed between species with different level of ploidy for boll retention and hybrid seed setting at α=1%. (Table 1 and Table 2).  Comparison between tetraploid and idploid as seeds parents, through chi-squares indicated that highest boll retention was observed for tetraploid in crosses with diploid as donor pollen parent and showed significant difference at α=5% level  using chi-squares (Table 2). In contrast to Stewart and Hsu (1977; 1987); Moradi (1997) and  Gill and Bajaj (1981), whose were successful in distance hybridization of cotton through ovule-embryo culture, Gibberellic Acid, and napthaline Acetic Acid, we were able to produce hybrid seeds and obtaining sterile F1 triploid plants by conventional crossing between 2x and 4x in two dirction . The number of seeds per boll varied between 0~1 seed (0~2.78) per boll. The bolls and seeds were not as well developed as those from the self pollinated female parents (Figure 1).The hybrid plant was more vigorous than either parent and showed highly sterile (Figure 2).

Figure 1 Developmental comparison between inter specific (2x × 4x) and intra specific (4x × 4x) bolls

Figure 2 Sterile F1 hybrid plant developed from seeds Tetraploid and diploid cotton species

Table 1 Mean effect of parents and GA3 on boll and seed development

Table 2 Mean effect of 4n x 2n and reciprocal crosses on boll development using 100 ppm

2 Materials and Methods
Field crosses were used to produce F1 interspecific hybrid between the selected parent species with different ploidy level in cotton. Emasculated flowers of two tetraploid commercial veraities (Sahel and sephid) have been pollinated with pollen grain of Hashem Abad and Kashmer diploid plant (G. arboretum) in two directions. 

The seeds of selected parent were planted in rows spaced at 90 cm and plant to plant spaced at 90 cm.  Ten rows for seed parent and two for pollen parent have been planted in each experimental unit. Hybridization was done by following hand emasculation and pollination under field condition. Emasculation carried out at appropriate stage of development of flower buds in the evening and pollinated the next morning. Totally 100 flowers were emasculated and pollinated for each cross combination involving one tetraploid and one diploid parents (Table 1).  Fifty flowers from each cross have been treated for each GA3 treatment (0, and 100 ppm).

All the recommended cultural practices like weeding, fertilizers and irrigation were done properly at the required time. Giberlic-acid with two different concentrations (0 and 100 ppm) was than sprayed at base of the pedicel for 7 days after pollination to encourage boll retention and prevent flowers abscission. The numbers of boll and developed hybrid seeds counted and analyzed using Chi-square test. Harvested seeds were planted in next growing season and sterile F1 hybrid plants have been observed among the progenies.

References
Amalraj S.F.A., 1989, Combining ability studies on G.hirsutum and G.barbadens hybrids, Indian J. Agric. Res., 23(2): 65-69

Bhale N.L., 1999, Heterosis breeding, In: Sundaram V., Bass A.K., Narayanam S.S. and Rafendran T.P. (eds.), Handbook of Cotton In India, Ind. Soc. Cotton Improv., Muymbai, pp.57-78

Gill M.S., and Bajaj Y.P.S., 1987, Hybridization between diploid (G. arboreum) and Tetraploid (G. hirsutum) cotton through ovule culture, Euphytica, 36(2): 625-630 http://dx.doi.org/10.1007/BF00041512

Mofidabadi A.J., and Modir-Rahmati A.R., 2000, Production of Populus euphratica Oliv. X P. alba L. hybrid poplars through ovary and ovule culture, Plant Genetic Resources Newsletter., 122 (13-15)

Moradi A.R., 1997, Hybridization of diploid and tetraploid cottons through in vitro embryo culture, Journal of Heredity, 69(6): 404-408

Stewart J.M., and Hsu C.L. 1978, Hybridization of diploid and tetraploid cottons through in-ovule embryo culture, Journal of heredity, 69(6): 404-408

Stewart J.M.D., and Hsu C.L., 1977, In-ovule embryo culture and seedling development of cotton (Gossypium hirsutum L.), Planta, 137(2): 113-117 http://dx.doi.org/10.1007/BF00387547