Studies On In Vitro Culture Of Different Types Of Embryos And Genetic Diversity Analysis Of Regenerated Plants In Bitter Gourd (Momordica Charantia L.)

In this study, seedlings from normal and infertile seeds were obtained by in vitro culture in 'Bixiu' bitter gourd (Momordica charantia L. cv. 'Bixiu'). RAPD and morphological observation were used to evaluate these seedlings' genetic diversity. The results were as follows:1. Definition of infertile seeds and their occurrence frequencyThe occurrence rates of normal and infertile seeds in 142 mature fruit were studied. The colour of mormal seeds was brown, and average weight was 0.178g, whereas the colour of infertile seeds was white, average weight was 0.046g (about 1/3 that of normal seeds). 3071 normal seeds and 333 infertile seeds were obtained from 142 'Bixiu' bitter gourds in total. There were 21.627 normal seeds and 2.345 infertile seeds per fruit on average. The ocurrence frequency of infertile seeds was 9.1%.2. In vitro culture(1)The best inoculation medium of normal seeds was MS, which didn't contain any other components. On this medium, the percentage of germination could reach 88.9%, the percentage of seedling production could reach 83.3%. The best inoculation medium of infertile seeds was MS + 0.5mg/L 6-BA, and on this medium, the percentage of germination could reach 66.7%, the percentage of seedling production could reach 41.7%.(2)The best proliferation medium of normal seedlings was: MS+1.0mg/L 6-BA+ 0.5mg/L KT+0.1mg/L IBA. The best proliferation medium of infertile seedlings was: MS+2.0mg/L 6-BA+0.5mg/L KT+0.1mg/L IBA.(3)The best rooting medium of normal seedlings was: 1/2MS+0.2mg/L IBA+ 20g/L sucrose. The best rooting medium of infertile seedlings was: 1/2MS+0.5mg/L IBA+ 30g/L sucrose. 3. Morphological characterizationMorphological characterization showed that infertile seed seedlings differed from normal seed seedlings. The infertile seeds were hard to germinate, cotyledons were smaller, stems were thinner, euphyllas were smaller, abnormal and couldn't sprout. Normal seed seedlings had well developed root system with many rootlets, but infertile seed seedlings didn't have well developed root system with only one or a few tiny roots many of which didn't have rootlets.4. RAPD analysis(1)In this study, improved CTAB method was applied. The result showed that it was good when tender leaves from in vitro culture were used to extract DNA. The extract was not prone to browning, and the quality of DNA was good (A₂₆₀/A₂₈₀ was 1.8-2.0). It could be applied for RAPD analysis, without wiping off RNA.(2)The best reaction system was established. The total volume was 25μl, with 0.2μmol/L primer, 1.5mmol/L Mg²⁺, 0.2mmol/L dNTP, 1.0U Taq DNA polymerase, 25mmol/L buffer and 20ng templet DNA.(3)The suitable amplification procedure was that DNA was pre-denatured at 94℃for 2min, then denatured at 94℃for 1 min, primers were annealed at 36℃for 1 min and extended at 72℃for 2min, the reaction was re- extended at 72℃for 10min after 45 cycles and finally the products were stored at 4℃.(4)14 random primers were selected from 64 random primers, which had more, clearer polymorphic bands.1420 clear bands were got in total, of which 333 bands were specific, accounting for 10.3%.