| Seabuckthorn（Hippophae rhamnoides）is a class of deciduous shrubs or small trees with high ecological value and economic benefits.At present,both breeding and planting of the plants use traditional techniques,which is difficult to meet the production and market demands.Establishing an efficient and stable technique system for plant regeneration and transplantation of seabuckthorn by tissue culture is not only a necessary prerequisite for the plants’genetic transformation but also an effective means for their rapid propagation on a large scale.Chinese seabuckthorn（H.rhamnoides L.subsp.sinensis Rousi）is the largest population of seabuckthorn in the world and the differentiation frequency and transplanting survival rate of regenerated plants from the subspecies’tissue cultures are still low.Moreover,its genetic transformation has not been reported to date.In this thesis,based on previous research,major problems in the plant regeneration and transplanting of Chinese seabuckthorn tissue culture were studied and improved using the sterile seedlings directly from the seeds as explant material.As a result,a relatively high-efficient and stable system for the plant regeneration and transplanting was established.On this basis,a preliminary study was conducted to explore the conditions influencing Agrobacterium-mediated genetic transformation of this plant.The main results are as follows.1.On the WPM+0.5 mg/L 6-BA+0.2 mg/L NAA+500 mg/L CH+20 g/L sucrose medium supplemented with 100 mg/L Vc,cotyledons were the best explant material for callus induction and adventitious bud differentiation among various organs of the sterile seedlings.Adding 50 mg/L Vc and 50 mg/L Cys to this medium effectively inhibited the browning of cotyledon cultures and led to a better result that the callus induction rate,adventitious bud differentiation rate and adventitious buds per callus reached 92.9%,89.1%and 16.6,respectively.2.In vitro rooting of the regenerated shoots was very slow and had a poor root quality,with the highest rooting rate of 42.6±0.5%and a maximum average root length of 3.3±0.4cm.Rooting of the regenerated shoots under the ex vitro conditions was obviously accelerated and improved in root quality,with a rooting rate of 74.7±1.4%and an average root length of 10.4±0.5 cm.In a mixed substrate of vermiculite and nutrient soil（the volume ratio is 3:1）,survival rate and plant height of the in vitro rooted seedlings were55.5±2.4%and 11.6±0.9 cm,respectively,while those of the ex vitro rooted seedlings were71.7±4.4%and 16.4±1.4 cm;the growth of these seedlings was generally weak.In a mixture of vermiculite,nutrient soil and the rhizosphere soil of seabuckthorn adult plants（the volume ratio is 2:1:1）,the two sets of rooted seedlings had 65.0±3.9%and75.9±3.7%of survival rate as well as 16.4±0.9 cm and 23.1±0.8 cm of plant height,respectively,and the growth vigor,especially that of the ex vitro rooted seedlings,increased obviously with time.Therefore,the ex vitro rooting method is better than the in vitro rooting method,and adding seabuckthorn rhizosphere soil to transplanting substrate can promote the survival and growth of transplanted seedlings,especially their growth.3.Genetic transformation experiments were conducted on cotyledons and seedlings using A.tumefaciens（containing the plant expression vector p CAMBIA3301）as a mediator,and suitable transformation conditions were selected primarily according to the GUS staining rates,combined with the situations of bacterial contamination and browning:pre-cultured for 2 d,acetosyringone at 200μmol/L,an infection solution with 0.5 or 0.6 of OD600,infected for 10 min or 15 min,co-cultured for 2 d or 3 d.A PCR detection of the Bar gene was conducted on the infected seedlings,with a positive rate of 6.9%.This study lays a foundation for the optimization of tissue culture techniques and the establishment of Agrobacterium-mediated transformation method of Chinese seabuckthorn. |
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