| Isatis indigotica Fortune, a kind of biennial herb, belongs to Isatis genera, Cruciferae family. Both the isatis roots and indigowood leaves are used as traditional Chinese medicine. The quantity and quality of Isatis have reduced greatly in recent years because of planting without screening the excellent species. Therefore, there is an urgent demand to develop new species with better yields, qualities and genetic stability. The medicinal plant polyploidy posses manifold merits, such as improved biomass accumulation and pharmaceutical ingredients, as well as enhanced stress resistance which offers an alternative strategy for the development and utilization of traditional medicinal plants. Focused on the polyploidy breeding of isatis, in succession, the rapid propagation system of isatis was established, the isatis polyploidy was induced and identified, the inheritance stability was analyzed. Ultimately, the excellent tetraploid strains were screened. Following was the key results in this work.Firstly the rapid propagation and culture technique system of isatis was established. It shows that hypocotyl is easier to induce callus by studies of inducing callus from explant of isatis and rooting of test-tube plantlet. The optimum medium recipe for inducing callus, differentiation and rooting is respectively MS+6-BA1.0mg/L+2,4-D0.8mg/L, MS+6-BA2.0mg/L+NAA0.2mg/L. and 1/2MS+NAA 0.2mg/L+IBA 0.2mg/L. The best time for hardening off is 4 days, and the best ground substance recipe for transplanting is vermiculite:perlite=1:2. The nutrient solution consisting of Ca(NO3)2 945mg/L+KNO3 607mg/L+(NH4)2HPO4 115mg/L+MgSO4 493mg/L can boost the growth of transplant significantly. The planting density of 30cm×10-20cm is suitable for isatis. The optimized harvest period is between October 15 and October 20.Secondly, the 16 autoteraploid strains of isatis were screened by inducing and identifying poplyploid. It comes to the conclusion that the culture medium added with 90 mg/L colchicines for 28d is the best for polyploidy induction.Compared to the corresponding plantlets of diploid, the test-tube plantlets and the transplants of polyploidy showed a lot of different situation:the seed germination was slowly, the seedling is stubby, also with incrassate hypocotyl; the leaves of differentiation seedlings are plumpy, with dark green color; the leaf veins are thick and obvious; the roots of the petiole expand; in comparison to the diploid, the leaves of polyploidy have more drape, the leafedge is zigzag. The transplants of polyploidy with vigorous growth are thick and strong, with strong tillering ability and winterness. It was found that the stomas of polyploidy were bigger significantly than diploid. Big stoma and the number of chloroplast in guard cell are important index for polyploidy identification.Then the inheritance stability of isatis teraploid strains was analyzed with ISSR. The modified CTAB method is screened as the optimum method for extracting isatis DNA. The optimized ISSR reaction system is 1×buffer 2.5(not including MgCl2), MgCl2 2.0mmol·L-1,primer 0.76μmol·L-1, dNTPs 190μmol·L-1, Taq DNA polymerase 1.5U in 25μl system.5 polymorphic primers were chosen for ISSR analysis to explore the genetic stability of subcultrued test-tube plantlets from three tetraploid strains. Electrophoresis illustrated that there was no obvious difference within the plantlets and 5 polymorphic primers, which indicated that the sub-culture of isatis can keep genetic stability within this range.Finally, the economical character, quality and stress resistance were evaluated. By comparison of economical characters between different strains, the results show that tetraploid and diploid exist difference, moreover, the indigowoad leaf and isatis root output are different between different tetraploid strains. Different strains can be choosed according to different harvest target. If the indigowoad leaf is needed, DB3, DB5, DB7 and DB10 are the best; if the isatis root is wanted, DB3, DB7, DB10 and DB16 are the best.Though comparing the content of indigotin and indirubin in different strains, more than 90% of 16 tetraploid strains in root contain high content than that in diploid. While in the leaves, the content of both indigotin and indirubin is just the opposite.As respect to the content of polysaccharide in isatis root, tetraploid is lower than diploid, among the strains determined, DB1, DB10 and DB15, contained more polysaccharide. While in the indigowoad leaves, the content of polysaccharide of 70% of the 16 strains is higher than that of diploid. The content in DB6, DB13 and DB15 is the highest.Amino acid analysis showed that the content of arginine both in indigowoad leaves and isatis roots of strain DB7 and DB8 is very high; The DB1, DB7 and DB12 strains contain high praline content, which can be drought resisting materials. As respect to the content of total amino acid, the strains of DB7, DB9 and DB12 are the best.PEG and NaCl were used as stress to screen the stress resistant abilities based on their physiology index such as the activity of SOD, POD, the praline content and electrical conductivity as well as the content of important metabolites such as indigotin and indirubin. It illustrated that DB3, DB5 and DB6 were the best strains based on all indexes. As far as the saltresistance is concerned, DB2, DB5, DB6, DB8, DB10, DB12 and DB14 showed an advantage over other strains, but further studies was needed to confirm.The pathogenic bacteria were obtained from disease plants, and identified as F. solan, F.oxysporum and R.cerealis respectively. The disease resistance of tetraploid strains was compared by the means of root segment inoculation. Most strains appeared the disease symptom from the third day. By the seventh day, the disease rates of DB1, DB3, DB6, DB9 and DB14 get higher, while that of DB5 and DB11 were lower, and they possess high resistance to root rot disease.On the base of the comprehensive evaluation of 16 tetraploid strains of isatis, all of which, except DB2, DB4, DB5, DB6 and DB13 are superior to the diploid. Among them, DB3, DB10 and DB16 are the optimum and can be further studied as reproduction materials. |
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