| Impatiens balsamine L. is a Balsaminaceae Impatiens L. It is a perennial evergreen herb flowers, located in various parts of China. Common for monochrome, single impatiens,double pattern is simple, and spend more bears for axils, greatly limits the conventional ornamental flowers impatiens as fully exploited. Ion implantation technology has been successful used as a mutation through appropriate ion implantation of different doses in different organs of plant, can be bred offspring from new mutations strain. Through the proper dose of low-energy N~+ ion implantation Impatiens balsamine L., can be selectively new strain from different organs of the mutant descendants. But the tissue culture,rapid propagation system and the analysis of the mutation mechanism report the low-energy ion mutagenesis Impatiens balsamine L.are rarely. The project materials is stabilization strin of pink and red Impatiens balsamine L.by low-energy N~+ ion beam induced, established rapid propagation system of mutantion Impatiens balsamine L. preliminarily through tissue culture. Meanwhile, cloning and sequencing the marker genomic DNA of Impatiens balsamine L. by ISSR and RAPD, through the analysis of sequence revealed low-energy N~+ ion implantation cause genomic variation mechanism of Impatiens balsamine L. .The main results were as follows:1. Initially established tissue culture system of Impatiens balsamine L.. The medium for hycopotyl callus formation and subculture: MS+ 6-BA2.5 mg/L+ NAA2.0 mg/L+3% sucrose + 0.5% agar; The medium for callus differentiation buds:1/2MS + 6-BA 0.2mg/L+ NAA 0.05mg/L; The medium for callus differentiation roots: MS+ NAA0.5 mg/L+ 2.5% sucrose+ 0.5% agar; After the transplant, refined seedlings,rapid propagation seedlings has been obtained.2. Established the ISSR reaction system, ISSR technique was employed with the comparative study of the mutants and controls Impatiens balsamine L., then analysis the cloning and sequencing amplification peculiar patterns. The results show that from 121 ISSR primer have 6 can steady expansion peculiar patterns, polymorphism of primer is 4.96%, get 46 specific amplification altogether, including 18 red mutagenesis and 28 pink mutagenesis. In cloning the ISSR red mutant Impatiens balsamine L., 55 bases have mutated; In cloning in the ISSR pink mutant Impatiens balsamine L., 71 bases have mutated. Analysis peculiar patterns by cloning and sequencing found that:red mutant and pink mutant base variable type all including four types ,which is transition, transversion, deletion and insertion. In the red mutants, the frequency of bases substitution (70.91%) is more than bases insert and deletion (29.09%), and in the bases substitution the frequency of transversion (45.45%) is about 1.79 times the transition (25.45%); In the pink mutants, the frequency of bases substitution (76.05%) is also more than bases insert and deletion ( 23.95%), and in the bases substitution the frequency of transition( 39.44%) is closed to the frequency of transversion (36.62%).According to the number of base mutation found that adenine (A) of red mutants there were 22 times mutations, 40.00% of the total number of mutations, it has high radiation sensitivity to low-energy N~+ ions; and thymine (T) of pink mutants there were 27 times mutations, 38.03% of the total number of mutations, it has high radiation sensitivity to low-energy N~+ ions.3. Established the RAPD reaction system, RAPD technique was employed with the comparative study of the mutants and controls Impatiens balsamine L., then analysis the cloning and sequencing amplification peculiar patterns. The results show that from 208 RAPD primer have 6 can steady expansion peculiar patterns, polymorphism of primer is 2.89%, get 35 specific amplification altogether, including 16 red mutagenesis and 19 pink mutagenesis. In cloning the RAPD red mutant Impatiens balsamine L., 60 bases have mutated; In cloning in the RAPD pink mutant Impatiens balsamine L., 53 bases have mutated. Analysis peculiar patterns by cloning and sequencing found that:red mutant and pink mutant base variable type all including four types ,which is transition, transversion, deletion and insertion. In the red mutants, the frequency of bases substitution (73.33%) is more than bases insert and deletion (26.67%), and in the bases substitution the frequency of transition (40.00%) is closed to the transversion (33.33%); In the pink mutants, the frequency of bases substitution (90.57%) is also more than bases insert and deletion ( 9.43%), and in the bases substitution the frequency of transition( 58.49%) is about 1.79 times the frequency of transversion (32.08%).According to the number of base mutation found that adenine (A) of red mutants there were 26 times mutations, 43.33% of the total number of mutations, it has high radiation sensitivity to low-energy N~+ ions; and thymine (T) of pink mutants there were 20 times mutations, 37.74% of the total number of mutations, it has high radiation sensitivity to low-energy N~+ ions. This study consistent with the reslut by ISSR. 4. Analysising genome variations of low-energy N~+ ion implantation mutant Impatiens balsamine L., in the mutations, low-energy N~+ ion implantation cause Impatiens balsamine L. genome had base transition, transversion, deletion and insertion. The rate of total mutate bases is 0.63%, in the 239 total mutate base, single base substitution for 77.40%, significant higher deletion(13.81%) and insertion (8.79%). In the base substitution, transition (100times) and transversion (85times) no significant difference. A/T transversion account 47.06% for the frequency of total transversion, significantly higher than other transversion type. In insert and deletion mutation, four bases all have lower frequency of mutate. Therefore, low-energy N~+ ion implantation mainly cause singel base substitution of mutate Impatiens balsamine L.genome. |
PDF Full Text Request