Effects Of Carbon Nanotubes Treatment On Leaf Regeneration And Rooting In Gala Apple(Malus X Domestica Borkh)

Apple(Malus x domestica Borkh),rose family,kernel fruit subfamily,apple genus,deciduous perennial tree,cross-pollinated,prefers to dry and cool.Apple industry plays an important role in China’s agricultural production and national economy.Apple tissue culture technology is an important way to obtain apple virus-free seedlings.Although this technology is relatively mature,there are still many problems,such as low rooting rate,pollution,browning,vitrification and so on.In terms of plant tissue culture,it has been found that basic medium,plant growth regulator and organic additives have important effects on the culture effect of explant.Studies have shown that carbon nanotubes(CNTs),as a new nanomaterial,can affect plant growth and root development.There are no studies on the genetic transformation of CNTs in apples.To explore the potential physiological effects of CNTs in gala tissue culture,the effects of CNTs at different concentrations on callus proliferation and differentiation in gala leaves were studied in this paper.The effect of CNTs on the rooting of aseptic seedling(stem segment explants)was analyzed by detecting the rooting status of aseptic seedling under the treatment of CNTs at different concentrations,as well as the physiological and biochemical indicators of root tissues and the expression of rooting related genes.The main research results are as follows:1.Effects of CNTs on leaf callus proliferation in gala tissue culture seedlings.Gala tissue culture seedling leaves were used as explants,MS+2 mg/L 6-BA+1 mg/L IAA+0.1 mg/L GA3+25 g/L sucrose+6 g/L AGAR was used as callus proliferation medium,and CNTs at different concentrations were added.The treatment time of gala leaf callus proliferation test was 20 days.The results showed that the proliferation coefficient and fresh weight of all the treatment groups treated with CNTs were higher than that of the control group(0 mg/L),and the differences were significant.Among them,CNTs treated with 10 mg/L had the best effect on the callus induction and proliferation of the leaves of gala.The effect intensity of CNTs was closely related to its concentration,and CNTs at low concentration had a promoting effect on the proliferation of callus in gala leaves,and the promoting effect decreased with the increase of the concentration beyond a certain concentration range.2.Effects of CNTs on leaf regeneration of gala tissue culture seedlings.In this experiment,CNTs at different concentrations were used to treat the leaves of tissue culture seedling,and the inoculation and culture time was 40 days.The effects of CNTs treatment at different concentrations on MDA content and the activities of SOD,CAT and POD antioxidant enzymes in the regeneration process of gala leaves were studied.The experimental results showed that CNTs treated with 20 mg/L had the best effect on the regeneration of gala leaves,and the content of antioxidant enzymes SOD and CAT were significantly higher than that of the control group,and the content of MDA was significantly lower than that of the control group.The addition of CNTs stimulated the activity of antioxidant enzymes,and reduced the damage caused by adversity stress and promote the growth and development of explants.3.Effect of CNTs on rooting of gala aseptic seedling.In this study,the effects of CNTs treatment on the rooting of aseptic seedling were studied by comparing the rooting states of aseptic seedling treated with CNTs at different concentrations,measuring the growth and development indicators of adventitious roots,and detecting the expression levels of rooting related genes.The results showed that CNTs treatment shortened the time needed for the formation of adventitious root and changed the way of the formation of adventitious root.Among them,the treatment of CNTs at 10 days,1 mg/L,10 mg/L and 20 mg/L induced the formation of root primordium at the base of the stem of gala aseptic seedling.On the 12th day,at 1 mg/L,5 mg/L,10 mg/L,and 20 mg/L CNTs,there was obvious adventitious root formation at the base of the stem of gala aseptic seedling.At 19 days,the control began to form adventitious roots.The addition of CNTs in each treatment(except at the concentration of CNTs at 15 mg/L)significantly increased the rooting rate of gala aseptic seedling,and the number of adventitious roots and root length of the gala aseptic seedling treated with CNTs at 10 mg/L were the highest and the root length was relatively long.CNTs also altered the expression of genes related to root development and induced oxidative stress,compared with control.When CNTs concentration was 10 mg/L,the activities of SOD,CAT and POD were higher than those of the control,and the content of MDA was lower than that of the control.Moreover,at CNTs concentration of 10 mg/L,the expression levels of rooting related genes such as PIN7,ABCB1,LAX2,LAX3 ARR3,CKI1,ARRO1,ARF7 and TTG1 were all higher than those of the control group.Among them,PIN7 and ABCB1 are auxin outflow genes,LAX2 and LAX3 are auxin inflow genes,ARR3 gene is involved in cytokinin synthesis,and ARRO1,ARF7 and TTG1 genes are involved in the formation of advection roots,lateral roots and root hairs,respectively.The comprehensive analysis showed that the treatment of CNTs at 10 mg/L was most conducive to the formation and growth of adventitious roots of gala aseptic seedling.In addition,the effect of CNTs on the rooting of gala aseptic seedling was related to its concentration.The addition of CNTs could affect the content of MDA and stimulate the activity of antioxidant enzymes in adventitious root,as well as promote or inhibit the expression of rooting related genes,thus affecting the rooting of gala aseptic seedling.