| Ficus beipeiensis S.S Chang is a member of the family Ficus,that a genus of Moraceae.It is a dioecious tree.There are only 5 wild plants?4 females and 1 male?.Endemic to Chongqing,located in Beibei District of Chongqing,where is damp was born in the altitude of 300-500 m limestone and steep rock.Under natural conditions,the pollinating fig is lacking,the seed setting rate is low,and the seed is difficult to germinate,so the population is sparse and the survival situation is extremely dangerous.In 2004,it was listed in the"Red list of Chinese species"and was included in the list of key protected wild plants in Chongqing in 2015.It is evergreen,rich in crowns and strong in resistance to adversity.It is a excellent tree for landscaping and afforestation."Bei Rong No.1"is the new species by the Chongqing approved in 2014.So far,the breeding studies of Ficus beipeiensis include Cuttage Propagation,seed reproduction and tissue culture in vitro reproduction.Cuttage Propagation not only damages the mother trees,but also has a low survival rate;There are many problems in seed propagation,such as the low number of mature seeds and germination difficulties;The reproductive system built by the tissue culture method still has some problems,such as the low growth coefficient of the bud,the endophyte pollution and the dysplasia of the root system of the test tube.In order to realize the rapid propagation of Ficus beipeiensis,the main problems must be overcome.Therefore,in this experiment,"Bei Rong No.1"seedlings as materials,through the research of several key technologies,the system of in vitro Rapid Propagation of Ficus beipeiensis was established to lay the foundation for the large-scale production of seedlings.The main results are as follows:1.The screening of the superior clump subculture mediumBy increasing the range of cytokinin,auxin species and the expansion of medium components,it was selected to obtain the best and best bud induction medium and the subculture medium.The experimental results showed that the best induction medium was MS+0.02mg.L-1TDZ.After 30d,the buds induction rate was 100%,while the proliferation rate reached 23.67,than the original bud induced medium was significantly increased by61.35%,and the clump buds grow well with large leaves and thick stems.In the best clump induction medium,adventitious buds occurred around 15 days in the apical buds,then proliferated,and clustered buds formed after 30 days.The best growth medium for shoots was MS+1.0 mg·L-1 6-BA.After continuous subculture for 6 times,the average propagation coefficient of adventitious buds was 11.2 for 1-6 generations,and the propagation coefficient for the highest generation was 6.5,which was significantly higher than that of the original cluster buds by 12.07%and 34.94%,respectively,and the adventitious buds grew well,leaves are green and stem is long.2.Prevention and treatment of the Browning phenomenon of the successive generationsThe petiole of indica rice seedlings was inoculated in the medium with different concentrations of anti-fading agents Vc(0.5,1,2 mg.L-1),Na2S2O3(0.5,1,2 mg.L-1)and activated carbon(2,3,4 mg.L-1)for 30 d.The degree of browning and growth of the culture were recorded and several anti-browning mediums were initially obtained.Then adventitious buds that had been subcultured more than 3 times were selected to be cultured in several anti-browning mediums to determine the content of cluster buds?absorbance?and polyphenol oxidase activity.The best resistance to subculture shoots was obtained by screening.The best anti-browning medium for subculture buds was obtained by screening.The test results showed that the anti browning agents of different concentrations could inhibit the browning of the adventitious buds of Ficus beipeiensis to varying degrees.When the culture medium did not add anti browning agent,the subculture buds began to browning in the third generation.With the increase of subculture times,the degree of browning was deepened.Add 1 mg.L-1 VC,bud browning was improved to 5,and the 6 generation appearing slight browning,buds in quinone content and polyphenol oxidase activities were decreased by 48.65%and 57.43%,and the adventitious buds grow well.Therefore,the best anti browning medium of the fig subgroup bud is a cluster bud subculture medium with 1 mg.L-1 VC,that is,MS+1.0 mg.L-1 6-BA+1 mg.L-1 VC.3.Overcoming the problem of subcultured endophytic bacteriaThe adventitious buds were inoculated in the medium containing different concentrations(0.05?0.08?0.1 g·L-11 Yipeiling;100?200?300gL-1 Car;0.05%?0.08%?0.10%PPM)of bacteriostatic agents.After 30 d culture,the bacteriostatic effect and the growth of adventitious buds of the endophytic bacteria of the adventitious buds were counted.The experimental results showed that under general conditions,endophytes began to appear when the bud was cultured to the third generation.The growth of endophyte was inhibited after adding bacteriostatic agent.Adding 0.08%PPM had the best inhibitory effect on the growth of adventitious buds and the adventitious buds grew well.4.Two segment rooting of adventitious budsWhen adventitious buds were rooted by general methods,the resulting adventitious roots were mostly rod roots with undeveloped root systems and a low survival rate for transplanting.It adventitious buds from leaves as explants,adventitious buds were inoculated into the rooting medium,two rooting method was used?inducing adventitious roots in agar medium and completing root development in a dark,porous matrix?,and compared with general rooting methods.The test results showed that the rate of the two rooting method was 100%.Compared with the average rooting method,The root tip number,total root length,total root volume and root mean diameter of rooting seedlings increased by 301.42%,27.85%,123.53%and 9.68%,respectively.The plant height,leaf increment,the chlorophyll content and the nitrate reductase activity increased by 307.89%,83.33%,27.90%and 69.49%respectively.The survival rate of rooting seedlings?100%?,plant height and leaf increment of transplanted seedlings were increased by 15%,41.12%and23.81%respectively,and the differences among the two were significant.The experimental results show that the two-stage rooting method is significantly better than the general rooting method.In addition,the root system of the test tube was developed,the survival rate of transplanting was higher,and the growth state was better.5.Cultivation of high quality seedlingsUsing a biodegradable non-woven seedling bag nursery to study different substrates for seedling growth[Pan's Nutrient Soil,Pinshao's Nutrient Soil:Vermiculite?1:1?,Pinhao's Nutrient Soil:Vermiculite:Perlite?2:1:1?],understory humus and vermiculite:perlite?volume ratio 1:1?],different effects of fertilizing materials on seedling growth and transplanting survival rate of Ficus beipeiensis.At the same time,the effect of seedling size?plant height and leaf age?on the survival rate of seedling transplanting was studied.The experimental results showed that the optimum substrate for seedling growth was Pinkevitch nutrient soil,in which the survival rate of transplanting plantlets was the highest,reaching 96.29%;The plant height increased by 1.90 cm and the number of leaves increased by 2.75,which was increased by 65.22%and 10.00%,respectively,compared with the sub-plants?understory humus?.The optimum foliar fertilization was1%urea.After spraying 30 d,the plant height increased by 1.94 cm,and the leaves increased by 3.2.The suitable height of the nursery stock was 7-9 cm?5-8 leaf ages?.After transplanting the soil for 30 days,the survival rate was 96.29%,the plant height increased by 1.4 cm,and the number of leaves increased by 3.4.The three indicators were significantly higher than the average.Small seedlings. |
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