The traditional breeding method of medicinal saffron can′t meet the demand of market,so in recent years the researchers have tried to make use of saffron as explants to explore its tissue culture method.The aim of this article is to understand systematicly and to guide scientifically the research on saffron′s different aspects including explants,components of the culture media,culture conditions,etc.

Objective: To investigate the viral pathogens in cultivated Crocus sativus. Methods: Viral pathogen identification was carried out by the observation of virus particle morphology and cytopathology as well as the detection of DAS-ELISA, RT-PCR, and sequencing. Results: Linear virus particles of 600-900 nm in length were observed in C. sativus by negative staining under transmission electron microscope (TEM). Bundles of linear virus particles, cylindrical inclusion bodies of subdivision II, and amorphous inclusion bodies were observed in the cells of C. sativus under TEM after ultrathin-section. These observations resembled the cytopathology of infectious Bean yellow mosaic virus (BYMV). Positive result of DAS-ELISA was obtained from the leaves of C. sativus by using monoclonal antiserum against BYMV capsid protein. Positive result of RT-PCR induced by the Potyvirus specific primers (Sprimer and M4) was also obtained. Sequencing after RT-PCR revealed that the viral sequence in this diseased C. sativus had a homology of 99% with the BYMV sequence. Conclusion: The pathogenic virus of this C. sativus disease is identified as BYMV.

Chlorophyll plays an important role in our everyday life.It is used not only as an annexing agent in medicinal and calleidic products but also as an unartificial food coloring agent.Chlorophyll and its ramifications have antioxidant and anti-mutation properties.China has abundant mulberry resources.The mulberry leaf is an excellent Chinese herb from ancient times to the present day and contains many kinds of bioactive components,in which the chlorophyll content is an important physiological index.However,the optimization of chlorophyll extraction from mulberry leaf by response surface methodology(RSM) has not been reported so far.Response surface methodology(RSM) is an effectively statistical technique for optimizing complex processes.The greatest advantage of RSM is that we need fewer experimental trials.It is widely used in optimizing variables of the extraction process,such as polysaccharides,alkaloids,phenolic compounds and protein from various materials.Box-Behnken design(BBD) is one of RSM,which only has three-levels(low,medium and high,coded as ?1,0 and +1),and needs fewer experiments.It's more efficient and easier to arrange and interpret experiments compared with others.This study focused on the optimal conditions of extracting mulberry leaf chlorophyll using RSM.The mixture of acetone and ethanol was used as extraction solvent.The chlorophyll content was calculated by Arnon's method.Extraction time,temperature,ratio of liquor to material and volume ratio of acetone to ethanol in extraction buffer were selected as the experiment factors according to the results of single factor experiments.The extracting parameters were optimized by RSM.Under the optimal extraction conditions described above,the confirmatory experiment was conducted.The single factor experiment results indicated that extraction time of 5 h(Fig.2),extraction temperature of 50 °C(Fig.3),ratio of liquor to material of 100∶1(Fig.4) and volume ratio of acetone to ethanol of 3∶1(Fig.1) were adopted for following experiment.Three independent variables were extraction time(C),extraction temperature(B) and ratio of liquor to material(A),according to the influences of the four factors on the chlorophyll productivity.The statistical analysis indicated that the three variables(Table 4) and the quadratic of extraction time(C) and ratio of liquor to material(A)(Fig.6) had significant effects on the yields,and there had the significant interaction effects between the variables of extraction temperature(B) and extraction time(C)(Fig.7).A high determination co-efficient mathematical model was gained that could be used to optimize chlorophyll extraction.Design Expert 7.1.6 software showed the optimal extraction condition was as follows: extraction time of 5.25 h,extraction temperature of 56.5 °C,ratio of liquor to material of 103∶1.Under the optimal extraction condition,the chlorophyll productivity was 5.376 mg/g,which was in consistent with the predicted value of 5.451 mg/g.In conclusion,the optimization of chlorophyll extraction by RSM is convenient,feasible and highly efficient.