| In this study, many seedlings from small seeds in Eureka lemon were obtained, by in vitro culture and direct sowing in greenhouse. RAPD, chromosome analysis and morphological observation were used to evaluate these seedlings' genetic diversity. The results were as follows:1. Definition of small seeds and their occurrence frequencyThe occurrence rates of normal and small seeds in 156 Eureka lemons' mature fruits were studied. The result showed that average weight of small seeds were just 0.034g, about 1/4 of normal seeds. Based on the data, a small seed was defined as just 1/3 or less of normal seed in weight. On occurrence frequency, there were 0.865 small seeds and 1.359 infertile seeds per fruit on average, i. e., 3.5% for small seed and 5.6% for infertile seeds, the total ocurrence frequency being 9.1%.2. In vitro culture(1)The best inoculation medium was the basic MS, which didn't contain any other components. On this medium, the percentage of germination could reach 90.0%, and 85.0%, respectively.(2)The best proliferation medium was:MS＋0.5mg/L6-BA＋0.5mg/LKT＋0.1 mg/LNAA. On this medium, average multiplication rate could reach 4.4.(3)The best rooting medium was:1/2 MS＋1.0mg/LNAA＋0.5mg/LIBA. On this mediium, average number of roots could reach 8.14 per shoot.3. RAPD analysis(1)In this study, improved CTAB method was applied. The result showed that it was good when tender in vitro culture leaves were used to extract DNA. The extract was not prone to become brown, and the production of DNA was high, (1 mg fresh leaves could produce 198-372ng DNA) and the quality of DNA was good(A260/A280 was 1.71-1.92). It could be applied for RAPD analysis, without wiping off RNA.(2)The best reaction system was established: The total volume was 25μl, with 1.5μmol/L primer, 2.5mmol/L Mg2+, 150μmol/LdNTP, 1.4U Taq DNA polymerase, 25mmol/L buffer and 40ng templet DNA.(3)The suitable amplification procedure was: 93℃for 2 min, 93℃for 1 min, 36℃for 1 min, 72℃for 2 min, for 40 cycles, followed by an extension at 72℃for 10 min. The amplified products were saved at 4℃.(4)12 random primers were selected from 74 random primers, which had more, clearer, various bands. 2032 clear bands were got in total, and especial bands were 347, accounting for 17.8%. All the 11 small seed seedlings, which were selected randomly, exhibited genetic diversity. But normal seed seedlings had none especial band at all.4. Morphological identificationMorphological identification showed that small seed seedlings differed from normal seed seedlings. Their leaves were rounder, thicker, greener, and leaf index (length/width) was small (the average leaf index was 1.53, against 2.22 for the normal seed seedlings).5. Chromosome analysisChromosome analysis was carried out on 14 in vitro cultured small seed seedlings. There were 5 seedlings haveing 27 chromosomes, but the normal diploid lemon seedlings had 18 chromosomes, indicating that the 5 small seed seedlings were triplods. The occurrence frequency of triploid was 35.7%. |
