| Forsythia suspensa（Thunb.）Vahl is a deciduous shrub of Forsythia in Oleaceae.It blossoms in early spring with a large number of flowers and beautiful yellow color.It has strong adaptability and resistance to pests and diseases.Forsythia suspensa is not only a major and commonly used Chinese medicinal materials,but also has good ecological value and ornamental value,and is an excellent flower shrub in early spring in northern China.Genetic engineering breeding is an effective means of breeding.Due to the lack of transgenic system,genetic engineering breeding of Forsythia suspensa has been hindered,resulting in the lack of cultivars.Modern molecular biotechnology,especially the development of plant tissue culture and genetic engineering,provides technical support for genetic engineering breeding and new variety improvement of Forsythia suspensa.In this study,stem segments of Forsythia suspensa were selected as explants to explore the tissue culture regeneration system,and leaves,hypocotyls and cotyledons of sterile seedlings were used as explants to explore the receptor system for genetic transformation.The tolerance of Forsythia suspensa explants to kanamycin and hygromycin was determined.Agrobacterium tumefaciens-mediated transformation of p CAMBIA1301 vector carrying plant expression vector β-glucosidase（GUS）and hygromycin phosphotransferase（Hpt）selective marker genes into Forsythia suspensa explants was carried out,and the transformation conditions were optimized.The experimental results were as follows:1、When the stem segments of Forsythia suspensa were sterilized with 75%alcohol for 30 s and 0.1%（m/V）HgCl2 for 8 min,the contamination rate of the stem segments of Forsythia suspensa was lower（23.33%）,the browning situation was less and the survival rate was the highest（66.67%）.The optimal culture medium for stem initiation was MS（Murashige and Skoog）+ 6-BA 1.0 mg·L-1 + NAA 0.3 mg·L-1 +Sucrose 30 g·L-1+ Agar 7 g·L-1,the germination time was 3～5 days,and the induction rate was 97.00%.The optimal roots medium was 1/2 MS + IBA 0.2 mg·L-1 + NAA0.2 mg·L-1 + Sucrose 25 g·L-1 + Agar 7 g·L-1,and the rooting rate reached 73.54%.The roots were fast,long and strong.2、The best disinfection treatment of Forsythia suspensa seeds was 75% alcohol disinfection for 30 seconds,and then disinfection with 0.1% HgCl2 for 12 minutes,the contamination rate is 16.88% lower,the germination rate is higher,and the growth of sterile seedlings is good.The callus induction rate of cotyledons and hypocotyls in Woody Plant Medium（WPM）was higher than that in MS,so WPM medium was used as callus induction medium.The optimal induction medium for hypocotyl and cotyledon of Forsythia suspensa was: WPM + 6-BA 1.0 mg·L-1 + NAA 0.5 mg·L-1 +Sucrose 30 g·L-1+ Agar 7 g·L-1.The induction rates were 89.67% and 89.00%,respectively.The best differentiation medium of cotyledons callus was WPM + 6-BA1.0 mg·L-1 + NAA 0.1 mg·L-1 + KT 1.0 mg·L-1 + Sucrose 30 g·L-1 + Agar 7 g·L-1 and the induction rate was 35.56%.The adventitious shoot induction medium of hypocotyl callus was WPM + 6-BA 2.0 mg·L-1 + KT 0.3 mg·L-1 + Sucrose 30 g·L-1 +Agar 7 g·L-1.3、Forsythia suspensa explants are not sensitive to kanamycin but are sensitive to hygromycin.According to the callus induction status of explants,the lethal concentration of hygromycin was 20 mg·L-1 in hypocotyl and 15 mg·L-1 in cotyledon.The bacteriostatic agent carboxybenzillin at a concentration of 400 mg·L-1could effectively inhibit the growth of Agrobacterium tumefaciens,and had little inhibitory effect on explants.4 、 The concentration of bacterial solution OD600 = 0.8,The optimal transformation conditions of hypocotyls were vacuum-assisted infection for 3 min,then conventional infection for 20 min,co-culture for 4 days,and the GUS expression rate was the highest（52.72%）.The concentration of cotyledons in bacterial solution OD600 = 0.8.The optimal transformation conditions were vacuum-assisted infection for 1 min,followed by conventional immersion for 20 min,co-culture for 3 days,and the GUS expression rate was 33.31%.Co-culture for a long time made Agrobacterium multiply and explants die.In this study,the regeneration system and genetic transformation receptor system of Forsythia suspensa were established by using forsythia sterile material.The influence of bacteria concentration,infection time and co-culture days on genetic transformation was discussed,and the optimal conditions for transformation were screened,which laid a foundation for genetic engineering breeding and improvement of good characters of Forsythia suspensa using genetic engineering technology. |
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