Establishment Of Regeneration System Of Rose And Genetic Transformation Of RrDFR Gene

Rose(Rosa chinensis)is a kind of perennial evergreen woody plant in the Rosaceae family. Rose known as the Queen of Flowers, is one of the ten traditional flowers, with a high practical and commercial value.It has the following characters which is upright posture, rich color, flavor pleasant, long flowering period, three quarters of the year flowering, drought-resistant barren resistant ability.However, because of Rosa plant genetic background is very complex, after repeated hybridization, it will become highly sterile. Thus, the traditional Rose plant breeding methods for genetic improvement has great limitations. The transgenic plant technology can take advantage of exogenous gene targeting improving its ornamental characteristics, becoming a new way to cultivate new varieties of rose. The experiment has the following results:1、To establish rose x micro propagation systemThere is a new line of diploid rose in our laboratory. It is very similar with Rosa chinensis ‘Slater’ s crimson China’ and Rosa chinensis cv. Old Blush, with fast-growing,flowering month and producing fertile seeds advantage.Because it has not yet officially named, so call it x.In Rosa x stems with buds as explants to explore the effects of different hormone combinations on its micro propagation plants growth, to filter out the best medium. The experimental results show that the optimum medium is MS+0.5mg/L BA +0.01mg/L NAA +0.1mg/L GA3+30g/L Sucrose+7.5g/L Agar.2、To establish rose x regeneration systemUse leaves to establish Rosa x regeneration system. Research shows that: The types and sources of explants, the light condition, the following generation period, the combination of hormone content have a vital influence on the somatic embryo induction.In dark conditions, culturing leaf and petiole tissue in medium(MS + 5.0mg / L2,4-D + 10 mg / L AgNO3 + 400 mg / L L-p + 30 g / L Glucose + 3g / L GEL) has the highest induction rate which will be 69.25% and 84.00%.In this culture medium every 4weeks to do subculture cultivation, about 8 weeks later it will produce somatic embryos,the induction rate is 2.67%. When The callus is transferred to the light conditions with the medium(MS + 1.0mg / L TDZ + 0.01 mg / L NAA + 0.1mg / L GA3 + 30 g / L Glucose +3.0g / L GEL), callus renewable induction rate was 3.18%.In 1 / 2MS + 1.0mg / L BA +0.01 mg / L NAA + 30 g / L Glucose + 3.0g / L GEL conditions, Callus and somatic embryos renewable can quickly germinate, the germination rate was 100%.After the buds sprouted, transferred into medium(MS + 0.5mg / L BA + 0.01 mg / L NAA + 0.1mg / LGA3 + 30 g / L Sucrose + 7.5g / L Agar) can quickly germinate. When the plants grow to about 4cm, it can be transferred to the rooting medium(1/2MS+0.5mg/L IBA+30g/L Sucrose+7.5g/L Agar)culture medium can normally take root, rooting rate was 100%.3、Genetic transformation of RrDFR geneDFR( dihydroflavonol 4-reductase) is an important enzyme in the anthocyanin biosynthesis pathway and a key regulatory point. RrDFR gene was cloned from rose. It can promote the increase of anthocyanin content in petals, so that the color can be deepened. Previous experiments in our laboratory have successfully transferred the RrDFR gene into the model plant tobacco, tobacco red petals significantly deepened.With Laboratory preserved somatic embryos of R. hybrida ‘Samantha’ as acceptor material, By Agrobacterium mediated transformation, RrDFR gene was transferred to the somatic embryo of R. hybrida ‘Samantha’, and finally transgenic plants of RrDFR were obtained successfully. when detecting Transgenic plants by PCR analysis, RrDFR gene was successfully introduced into the genome of R. hybrida ‘Samantha’. Because the positive plant has not yet flowering, whether the color deepened is still unknown, other tissues had no difference with the control plants.