| Passiflora caerulea L.,also known as passionfruit,is a perennial evergreen vine of the genus Passiflora.The fruit of P.caerulea is rich in nutrients,delicious in taste and unique in flavor.It is known as the“King of Drinks”.P.caerulea plays an important role in the international beverage market.In recent years,with the introduction and cultivation of P.caerulea,the virus of P.caerulea is seriously infected,the germplasm is degraded,and the yield and quality are greatly reduced.It has seriously hindered the development of the P.caerulea industry in China and even the world.Therefore,the rapid propagation of high-quality seedlings and the detoxification of virus seedlings are particularly important for P.caerulea.In this study,the in vitro rapid propagation system of P.caerulea was established and optimized,and the tissue culture seedlings were transplanted and domesticated.The dual RT-PCR virus detection system of TeMV and CMV was established for the two viruses infected with P.caerulea.And the detection conditions of the virus detection system were optimized.The subcellular localization of TeMV virus coat protein was studied by constructing the expression vector of CP-GFP,and the expression characteristics of TeMV in the host were quantitatively analyzed.Using the tip of P.caerulea as the material,the ultra-low temperature detoxification treatment was carried out by the established microtiter vitrification method,and the influencing factors of the detoxification system were explored.The main findings are as follows:1.Establishment and optimization of P.caerulea rapid propagation system.The stem segment of the P.caerulea bud is the explant material,and the disinfection system is optimized by screening the disinfectant and disinfection time.The results showed that the best disinfection method for P.caerulea explants was to use 75%ethanol for 30s and then 0.1%HgCl2for 12min,which could achieve lower pollution rate and browning rate,and higher survival rate.The effects of 6-BA and NAA on the proliferation of P.caerulea were studied by orthogonal experiment with sterile seedlings obtained from axillary bud development.The results showed that there was a significant interaction between the two hormones.When 3.0 mg/L 6-BA and 0.6 mg/L NAA were added to the MS medium,the proliferation coefficient of P.caerulea was the highest at 5.09.When the rooting induction test of four-week-old P.caerulea tissue culture seedlings was carried out.The effects of two hormones,IBA and NAA,on the rooting effect were explored.It is known from the test results that a lower concentration of NAA is suitable for inducing rooting.According to the average number of roots and the quality of the roots,1.5 mg/L of IBA and0.5 mg/L of NAA were selected as the best induction rooting medium.The tissue culture seedlings with strong root growth and reaching 34 main roots were transplanted and domesticated,and different types of mixed substrates were set in the transplanting substrate.The seedlings were transplanted at room temperature for 7 days.According to the growth condition of transplanted seedlings,the optimal transplanting substrate was peat soil:perlite=4:1 mixed matrix,the survival rate of transplanting was93%,and the growth potential of transplanted seedlings was also better than other test groups.2.Detection technology of P.caerulea related virusUsing the pathogen of P.caerulea infected with viral disease as a test material,molecular biology tests were carried out on four common passionflower viruses.The results showed that Cucumber mosaic virus?CMV?,Telosma mosaic virus?TeMV?,East Asian Passiflora virus?EAPV?and Passion fruit woodiness virus?PWV?were detected in the collected Passiflora leaves.On this basis,a dual RT-PCR detection system was established for P.caerulea complexed with TeMV and CMV,and the primer concentration,annealing temperature and cycle number in the detection conditions were optimized.The optimal reaction conditions of the optimized dual RT-PCR detection system are:optimal primer concentration is TeMV:CMV=1:4,final concentration of TeMV reaction is 2.0mmol/L,final concentration of CMV is 8.0mmol/L;optimal annealing temperature is 57?;the optimal number of cycles is 35 times.Sensitivity test was performed on the optimized dual RT-PCR detection system.The results showed that when the sample leaf cDNA was diluted to 10-5 of the stock solution,the specific band could still be amplified,and the detection sensitivity was equivalent to 10-5 mg tissue amount.The optimized dual RT-PCR system was applied to 36 P.caerulea leaves tests.The results showed that 8 samples from different regions were combined to infect both TeMV and CMV viruses,and 18 single-sensitive TeMV or CMV.In this study,a micro-direct RT-PCR detection system of P.caerulea was also established,which was tested for different sampling sites and needles of different sizes.According to the test results,the syringe needle with the size of 23G?0.6×25TW LB?can be selected,and the robust stem segment or blade can be used as the detection site to achieve a better detection effect on the PWV virus.This study facilitated the detection of P.caerulea diseases in the field.3.Subcellular localization and expression characteristics of TeMV coat proteinThe sub-cellular localization analysis of TeMV coat protein was carried out by using P.caerulea plants infected with TeMV as experimental materials.Transformation of tobacco leaves by constructing a CP-GFP expression vector for localization experiments.The results of the localization indicated that the CP protein of TeMV was localized in the chloroplast and was consistent with the predicted results of the website.It was speculated that the site of TeMV infection after P.caerulea was at the chloroplast of the leaf.In order to study the expression characteristics of TeMV in the host of P.caerulea,RNA extraction and reverse transcription were performed on different P.caerulea varieties and different tissue parts,and eIF-5A was used as the internal reference gene for expression pattern analysis.Quantitative results showed that the expression levels of TeMV in different parts of P.caerulea were significantly different,and the expression level was the highest in leaf parts.TeMV has significant tissue-specific expression.Quantitative expression analysis of the two varieties of“Fujian Passion fruit No.1”and“Fujian Passion fruit No.3”found that the former had a significantly higher expression than the latter,and the expression level was about three times that of the latter.It is speculated that“Fujian Passion fruit No.3”has Strong disease resistance.4.Ultra-low temperature detoxification technology of P.caerulea stem tip and detection of genetic stability of virus-free seedlingsThe shoot tip of P.caerulea seedlings carrying TeMV virus was used as the material,and the tip of P.caerulea was treated by ultra-low temperature treatment by droplet vitrification.Optimized the two factors of pre-incubation time and dehydration time which affect the vitrification method of droplets.The optimized operation procedure of droplet vitrification method is:stripping 1mm of passion fruit stem tip into liquid pre-culture medium for pre-culture 24h,then transferred to the PVS2dehydrating agent for 50min for dehydration treatment.The PVS2 droplets were prepared on a 1×4 cm aluminum foil paper and transferred to the dehydrated stem tips.The aluminum foil paper was placed in liquid nitrogen for 1h,and after unfinishing,the unloading treatment was carried out and the shoot tips were transferred to the regeneration medium to resume the culture.The ultra-low temperature detoxification study of the shoot tip of P.caerulea was carried out according to the optimized method of droplet vitrification.Different stem tip sizes were set to study the effects of stem tips of different sizes on the ultra-low temperature detoxification effect.The test results show that when the length of the stripped tip is0.81.0mm,the survival rate and regeneration rate of the shoot tip after detoxification by ultra-low temperature are the highest,they were 83.3%and 60%respectively,and the rate of detoxification was 100%.The total DNA of the leaves was extracted from the detoxified seedlings regenerated after ultra-low temperature treatment,and 17 random primers were obtained to perform ISSR amplification on the virus-free seedlings.The amplification results showed that 2,550 bands were amplified by the test,and each group of primers could amplify 4 to 9 bands.Compared with the parental control group,the test group had no specific band produced by the virus-free seedlings.There was no genetic variation in the regenerated seedlings after ultra-low temperature detoxification. |
PDF Full Text Request