| As one of the third generation of small fleshy berries,blackberry has rich nutritional and economic values.In recent years,the blackberry industry is booming in China.Both the growing area and yields have been growing intensively.The spread and expansion of fungal or viral diseases have caused serious losses.At the same time,the efficiency of traditional breeding methods is low,which cannot meet the market's demand.In order to improve the efficiency of blackberry breeding and accelerate its breeding process,many researchers have devoted time and money in the fields of modern biotechnology and genetic engineering.At present,deficiency of suitable blackberry cultivars has to some extent limited the development of blackberry industry.The time for propagation based on tissue culture techniques can be short and the abtained seedlings are tidy uniform.The progress can be supervised in controlled conditions depletion of environmental factors like growing season limits.In this study,factory nursery techniques based tissue culture,basically,from explants sterilization to plantlet transplantation were studied.Using the spineless blackberry cultivar'Arapaho',we optimized the conditions of every stages including explants sterilization time,medium component for multiplication,root induction,and plantlet explanation.As well,we investigated the systems for adventitious plantlet induction and callus induction via leaf disks.Finally,protoplast isolation and purification procedures from in vitro tissues were also characterized.The main results were as follows:1.Tissue culture systems for factorial seedling-raising has be established for blackberry'Arapaho':(1)The sterilization method suitable for the meristems and stems of blackberry'Arapaho'was:70%alcohol for 10 seconds,sterilized water rinsing for three times,and then 0.1%HgCl2 sterilization fir 10 min,and finally sterile water rinsing six times.(2)The best seedling multiplication culture procedure:tissue cultured seedlings were subcultured on MS media+20 g/L sucrose for about 30 days,and then transferred to proliferation MS+1.0 mg/L 6-BA+0.1 mg/L NAA+20 g/L sucrose+6 g/L AGAR.The highest proliferation coefficient was 4.98.(3)The best rotting medium:Blackberry'Arapaho'plants get rooting easily.The rooting rate of was 93.33%when 1g/L of activated carcoal was added to 1/2 MS medium.When IBA concentration reaches 5 mg/L,100 percent plantlets produce roots in the medium.(4)Seedling transplanting:the tissue cultured seedlings with good rooting and healthy leaves were selected.The plantlet boxes were opened with half lid place on top of the box for two days before full lid open after one week.The transplanted soil matrix was peat soil,perlite and raw soil in proportion of 6:3:1.The survival rate reached 100%.(5)The best adventitious bud induction medium for leave disks:the leaves that were fully expanded in tissue culture at about 30 days of subculture were used as the material.The average adventitious bud regeneration rate was the highest when leave disks were placed on the culture medium MS+1.5mg/L TDZ+0.4mg/L IBA+20g/L sucrose+6 g/L AGAR.Lower concentrations of TDZ can still induce the regeneration of adventitious buds of leaves,but has no significant differences between concentration of 0.01,0.02,0.04,0.08 and 0.16 mg/L.(6)The best leaf callus induction medium:Fully expanded leaves at the top of the cultured seedling of about 30 days were used.Callus of'Arapaho'of blackberry was induced by the combination of low-concentration auxin NAA and cytokinin TDZ.The induction rate was up to 100%.When NAA is 0.1mg/L and TDZ is 1mg/L,the calli were mostly loose,light green or yellow-green.The growth rate is high as well.(7)In the whole process,the commercial white granulated sugar and tap water can be used as carbon source to substitute the analytically pure sucrose and distilld water used in lab.2.An protoplast isolation and purification protocol was established for blackberry.High yield and vitality of protoplast were obtained.The yield of protoplast obtained by the optimal system can be as high as 8.64×10~7 g/FW leaves,and the vitality can be as high as 94.44%.(1)The best enzymatic combination:2.5%Cellulase R-10,0.03%Pectolyase Y-23,and 1%Macerozyme R-10.(2)When fresh leaves being digested at 9 h on a constant shaker at low speed(about 80r/min),high yield and high activity of protoplast can be obtained.(3)When mannitol is used as osmotic pressure regulator,the appropriate concentration is 12%.High or low concentrations can affect the yield and activity of protoplast.(4)Dark treatment of the material for 7d before protoplast separation can improve the yields. |
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