Study On In Vitro Tissue Culture System From Stem Segment Of Persea Americana Mill ’Duke7’

Avocado(Persea americana Mill.)is an emerging tropical fruit tree,which fruit are rich in protein,unsaturated fatty acids,mineral elements and other nutrients.The peel,pulp,and seeds all have medical and health care functions,deeply favored by consumers in developed countries in Europe and the United States.In recent years,it has become more and more popular among consumers in China,with huge consumption potential and market prospects.However,avocado trees are susceptible to diseases and insect pests.Root rot caused by Phytophthora is one of the key limiting factors affecting the safe and rapid development of the entire avocado industry,in severe cases,it’ll destroy the garden and trees.Screening root rot resistant rootstocks and breeding root rot resistant seedlings is the fundamental way to solve this production problem.‘Duke7’ is currently recognized in the world as being resistant to Phytophthora and can be used as a disease-resistant rootstock for a good varieties of avocados.However,avocados are typical cross-pollinated plants.In fact,the offspring have large variations and it is difficult to maintain the good characters of the female parent.Although the breeding techniques such as air layering and cutting can maintain the good maternal characters,the reproduction coefficient is low,and it is difficult to meet the production needs for a large number of seedlings.Tissue culture technology can not only maintain the excellent maternal characters,but also achieve batch breeding,but so far no technical report on the in vitro tissue culture of ‘Duke7’ avocado variety has been seen.Therefore,this study used ‘Duke7’ avocado stem segments as explants and Design Expert V8.0.6 as design software,using randomized block and the orthogonal rotational combination design.The combination design of random block and orthogonal rotation was used to explore the technical system of tissue culture of ‘Duke7’ avocado sterile explant,such as disinfection technology,bud induction technique,shoot regeneration induction technology,root generation technology and seedling transplanting technology,the results show that:(1)Sample in the indoor cultivation is easier to obtain sterile explant than in field,mercuric chloride disinfection effect is better than that of sodium hypochlorite.The grafted seedlings of 1～2 year old ‘Duke7’ avocado were moved back to the room for cultivation,regularly disinfected and fertilized.After 7days of cultivation,the young stems were washed with 75% alcohol 30 s + sterile water for 2～3 times + 0.1% mercury 10 min +sterile water for 4～5 times,and connect to WPM medium,the survival rate can reach88.33%;direct sampling in the field is not easy to be completely disinfected.Due to the restrictions of temperature,season and rainfall,under the same disinfection conditions,the survival rate in March is only 25%,10% in May,and 0% in August and November.(2)When the axillary buds of stem segment are induced to start growing,the petiole part will fall off.Under the same conditions,NAA and TDZ are more effective than IBA in inducing ‘Duke7’avocado stem buds,and TDZ can reduce the amount of 6-BA.Orthogonal combination test speculates that the optimal medium for ‘Duke7’ avocado shoot bud induction is: WPM + 0.13m/L TDZ + 1.72mg/L 6-BA + 0.1mg/L NAA,the theoretical induction rate is 98.94%.After verification,the actual shoot bud induction rate of this combination was 95%.(3)Within a certain concentration range(≦2.0mg/L),the higher the concentration of6-BA,the more favorable it is for the induction of cluster buds.When the concentration is too low,cluster buds will not be induced.Adding a suitable concentration of NAA on the basis of 6-BA can conductive to increasing the effect of cluster bud induction.Orthogonal experiments suggest that the optimal medium for cluster bud induction is WPM +1.73mg/L 6-BA + 0.2mg/L NAA,the theoretical value-added multiple can reach 2.96,and the actual value-added multiple has been verified to be 3.18.The combination contains callus from the bottom of cluster buds,and the longer the time,the greater the value-added multiple will be.But,if the medium is not replaced in time and the nutrition supply is insufficient,which is likely to cause part of the value-added shoot died.(4)Using 1/2MS medium as the base medium,adding appropriate concentrations of IBA or NAA alone can effectively induce adventitious roots in the stem of Avocado.The roots induction rate of IBA is higher than that of NAA,and the roots induced by IBA are relatively smaller,the plants grow slower;which the roots induced by NAA are thicker and the plants grow faster,but there are callus at the bottom.The suitable IBA and NAA concentration mixed treatment had better induction effect.Orthogonal experiments suggested that the optimal induction medium for adventitious roots of avocado stem segments was 1/2MS+ 0.5mg/L IBA + 0.34mg/L NAA + 0.1% activated carbon.The theoretical rooting rate was 59.39%,and the actual rooting rate was verified to be66.67%.did not survive(5)‘Duke7’avocado tissue culture seedlings are not survive to transplant after refining.This technique has not been successful during this trial period,and further exploration is needed.After 7days of loosening and unsealing the indoor seedlings refining,the plantlets grew normally,and the survival rate of indoor seedlings was 100%,and there was no pollution.However,after refining the seedlings,the plants were transplanted outdoors,and after 10 days of routine management such as shading and rehydration,the roots of the stems of the plants were rotten,leading to the gradual death of the plants.