| The Chinese medicinal Forsythiae Fructus is the dried fruit of Forsythia suspensa（Thunb.）Vahl of Oleaceae family,with the effects of clearing heat and detoxicating,detumescence and dissipating mass,and dispelling wind-heat.Forsythiae Fructus is one of the main Chinese herbal medicines in China.It is often used to treat various infectious diseases,such as acute respiratory tract infection,pneumonia,dysentery,scrofula and ulcers.It is known as the"holy medicine for sores"and has played an important role during the SARS,avian influenza and COVID-19 epidemics.In recent years,due to the influence of late spring cold and the ravages of COVID-19,Forsythiae Fructus as one of the Chinese herbal medicines for epidemic prevention and control,has a high demand and its price has doubled.F.suspensa has the advantages of low planting cost,simple management,low requirements for land,and large market demand,showing good economic and ecological benefits.It has become the main project of helping the poor and enriching the people and greening barren hills,and the artificial cultivation area has gradually increased.The breeding of some new varieties of Chinese herbal medicines in China started late,the breeding level was relatively weak,and the management of seeds and seedlings was relatively backward.In the cultivation of F.suspensa,there are some phenomena,such as only planting,not selecting,hybrid degeneration and poor stress resistance,which make the yield and quality not guaranteed.Therefore,the economic and ecological benefits of artificial large-scale planting of F.suspensa can be improved by breeding high yield and high quality varieties and promoting them.In present study,after selecting the dominant single plants from hundreds of acres of F.suspensa,according to the results of phenotypic characteristics detection and effective component content determination of F.suspensa branches,leaves and fruits,the excellent plants were selected to prepare for further application of varieties.In order to promote the rapid application of high-quality F.suspensa plants in production,a complete in vitro rapid propagation system was established through tissue culture technology for the selected dominant plants.However,in the process of forsythia tissue culture,it was found that part of F.suspensa tissue culture seedlings showed no increase,low proliferation coefficient,leaf yellowing and other bad growth state.Therefore,by changing the time and mode of illumination,adding gibberellin,measuring the content of internal and external hormones,and detecting viruses,we hope to find out the cause of the poor growth state and solve this problem.The main research achievements are as follows:1、Through the detection of leaf phenotypic characteristics,it was found that the common plant formed a significant difference from FRL-4 in the condition of single leaf thickness,but there was no significant difference from other plants.In the other conditions,the common plants did not show any significant advantage.The results of fruit phenotypic characteristics showed that the fruit setting rate of common individual plant was 62.67%,and that of excellent individual plant was 89.32～91.60%.The 100-grain weight of common individual plant was 75.08g,and the 100-grain weight of excellent individual plant ranged from 117.91～152.40 g.The common individual plant fruit length,transverse and longitudinal diameter were 19.55mm,7.69mm and 9.59 mm,while the excellent individual plant fruit length,transverse and longitudinal diameter were 24.42～29.40mm,10.15～11.36mm and 11.53～14.03 mm,respectively.The phenotypic data of the excellent individual plants were better than those of the common individual plants.The content of essential oil of FRL-1 was 1.4%,which did not meet the standard of pharmacopoeia（≥2%）,while the content of essential oil of other plants met the standard of pharmacopoeia.The results of forsythin content detection showed that each strain met the standard of pharmacopoeia（≥0.15%）.The results of forsythoside A content detection showed that the content of forsythoside A in FRL-1 was 1.51%,which did not meet the standard of pharmacopoeia（≥3.5%）,and the content of forsythoside A in other strains met the standard of pharmacopoeia.It was preliminarily believed that FRL-2,FRL-3,FRL-4 and FRS-2 were excellent plants.2、The rapid propagation system of FRL-2 in vitro was established and put into production practice.The best medium for the first generation of FRL-2 was MS medium+3.0 mg·L-16-BA+0.1 mg·L-1IBA.The optimum subculture medium for FRL-2 was MS+3.0 mg·L-16-BA+0.1 mg·L-1IBA.The optimum rooting medium of FRL-2 was 1/2 MS+1.0 mg·L-1IBA,in which 30g·L-1sucrose and 7g·L-1agar were added.The optimum acclimation time of FRL-2 was 3 d,and the air humidity during transplanting was90%.3、In the establishment of in vitro rapid propagation system,it was found that the leaves of tissue culture seedlings of FRL-1,FRL-3,FRL-4 and FRS-2 were yellowed and could not grow to a high level.By changing the illumination time and mode and adding gibberellin with different concentrations,the poor growth of the tissue culture seedlings has not been improved obviously.The contents of endogenous and exogenous hormones on the 10th day,20th day and 30th day of the subculture seedlings were determined,and the results showed that high concentration of 6-BA promoted the growth of plants,and FRL-1 and FRL-3 showed a downward trend of 6-BA from the 10th day to the 20th day;High concentration of IBA inhibited the growth of plants,while FRL-3 had higher IBA concentration on the 10th day;High concentration of ABA promoted the growth of plants,and the ABA concentration of FRL-3 was relatively low from the 10th to the 20th day;High concentration of ABA promoted the growth of plants,and the ABA concentration of FRL-3 was relatively low from the 10th to the 20th day.4、After optimization of the conditions,the poor growth state was not improved,and the existence of the virus was preliminarily speculated.The small RNA sequencing indicated the possible existence of pepper chlorotic spot virus（Gen Bank accession number:KX 247379）.Primers were designed by NCBI,and virus primers belonging to the same genus were found in the literature.Through RT-PCR verification,FRL-1,FRL-3,FRL-4 and FRS-2 showed a single bright spot on 2%agarose gel.After being sent to the company for sequencing,the construction of the phylogenetic tree could not be completed,so questions about whether the virus exists still exists.The results of shoot tip detoxification,heat treatment detoxification and exogenous additive detoxification showed that significant differences were formed in the proliferation coefficients of melatonin concentration of 20 mg·L-1,30 mg·L-1and ribavirin concentration of20 mg·L-1with other concentrations,and with the subculture tissue culture seedlings without any detoxification treatment.The growth effect of the tissue culture seedlings was significantly improved,and the proliferation coefficient reached 4.27. |
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