Genotype Variations In Cd And Pb Accumulations And Rapid Screening Methods For Low Cd Accumulative Cultivars In Two Leaf Using Vegetables

This study take celery （Apium graveolens L.） and lettuce （Lactuca dolichophylla Kitam.） that are vulnerable to contamination by soil Cd and Pb as the object. Two pot experiments were conducted to investigate the variations, transfer potential, and stability of Cd and Pb accumulations in 27 celery genotypes and 28 lettuce genotypes respectively, and to screen for Cd+Pb-PSC （pollution-safe cultivar , which edible parts accumulate certain pollutant at low enough level for safe consumption while grown in contaminated soil）. Using vegetable typical Cd accumulative genotypes that got from this study and preliminary experiments, variations of their Cd concentration of commercial seed, NaCl extractable or HCl extractable Cd concentration in their plant seedlings in early development were investigated. Using gene cloning and gene sequencing technology,the research of single nucleotide polymorphism in LdPCS1 gene in 10 typical Cd accumulative genotypes of lettuce were carried out. On this basis, we set up the rapid screening methods for low Cd accumulative genotype of leaf vegetables. Main results gained are summarized as follows: 1. In order to investigate the genotypic variations in accumulation of Cd in celery,a pot experiment was conducted on 27 celery cultivars under three soil Cd levels (L: 0.142 mg kg^-1,M: 0.431 mg kg^-1;H: 0.631 mg kg^-1). Results of two-way ANOVA for the variations of Cd concentrations and biomass in shoots showed that variations from genotypes, soil Cd and interaction of genotype×soil Cd were all significant （p < 0.01）, the Cd concentration （Fresh basis）in the shoot ranged from 0.046 to 0.218 mg kg^-1, 0.270 to 0.633 mg kg^-1 and 0.208 to 0.686 mg kg^-1, their coefficient of variation （CV） were 26.9%, 20.9% and 24.5% under soil Cd treatments L, M and H, respectively. In treatment L (soil Cd 0.142 mg kg^-1), except for one, shoot Cd concentrations in almost all tested cultivars in treatment L were lower than the ML of Cd allowed by CAC standards (0.2 mg kg^-1 FW), 3.7% tested cultivars being higher than the limitation. However, none of the tested cultivars measured up to the standard in treatment M and treatment H (soil Cd 0.431 mg kg^-1 and 0.631 mg kg^-1, respectively). that was to say, there was no Cd-PSC among the tested cultivars when soil Cd concentration > 0.431 mg kg^-1. This suggests that celery is a vegetable being easily polluted by Cd in soil. These eight genotypes （No.7、9、20、22、24、25、26、28） with lower shoot Cd concentrations and higher shoot biomass, could be treated as typical low-Cd accumulating cultivars. The genotype （No.2） could be treated as a typical high-Cd accumulating cultivar.2. The other pot experiment was carried out to study the differences of accumulation of Cd and Pb by these typical Cd accumulative cultivars. variations of Cd and Pb concentrations in shoot from genotypes, soil Cd or Pb level and genotype×soil Cd or Pb level were all significant at p < 0.05 level, In treatment LCdLPb (soil Cd 0.142 mg kg^-1, Pb 49.89 mg kg^-1), five cultivars （No.7、9、22、24、25） can be used as Cd-PSCs, Pb-PSCs and Cd+Pb-PSCs. Among them, shoot Cd concentration of No.25 is only 41.93%⁵9.70% of No.2 under high Cd exposure (soil Cd 0.599⁰.667 mg kg^-1), and shoot Pb concentration of No.25 near the ML for Pb allowed by the CAC standards. The genotype No.25 is identified as adoptable Cd+Pb-PSC which is worth to recommend to growers, and adopting PSC strategy could help reduce the risk of Cd, Pb pollution in celery.3. A pot experiment was carried out to investigate the genotypic variations in accumulation of Cd in lettuce under three soil Cd treatment (L: 0.129 mg kg^-1,M: 0.352 mg kg^-1;H: 1.253 mg kg^-1). Results of two-way ANOVA for the variations of Cd concentrations and biomass in shoots showed that variations from genotypes, soil Cd and interaction of genotype×soil Cd were all significant （p < 0.01）, the Cd concentration （Fresh basis）in the shoot ranged from 0.027 to 0.140 mg kg^-1, 0.084 to 0.400 mg kg^-1 and 0.358 to 1.715 mg kg^-1, their coefficient of variation （CV） were 38.5%, 36.1% and 46.5% under soil Cd treatments L, M and H, respectively. In treatment L (soil Cd 0.129 mg kg^-1), shoot Cd concentrations in all tested cultivars were lower than the ML of Cd allowed by CAC standards (0.2 mg kg^-1 FW). Thirteen of the tested cultivars measured up to the standard in treatment M (soil Cd 0.352 mg kg^-1), 53.6% tested cultivars being higher than the limitation. However, none of the tested cultivars measured up to the standard in treatment treatment H (soil Cd 1.253 mg kg^-1). Thus, no Cd-PSC could be found when soil Cd concentration higher than 0.352 mg kg^-1. only 6 cultivars（No.3、6、7、8、10、11）with lower shoot Cd concentrations and higher shoot biomasses, could be treated as Cd-PSCs, and 3 cultivars（No.19、23、24）with higher shoot Cd concentrations and higher shoot biomasses, could be treated as non-Cd-PSC in the follow-up study.4. The other pot experiment was carried out to study the variations of accumulation of Cd and Pb by six Cd-PSCs and one non-Cd-PSC. Variations of Cd and Pb concentrations in shoot from genotypes, soil Cd or Pb level and genotype×soil Cd or Pb level were all significant at p < 0.01 level, under lower 0.352 mg kg^-1soil Cd level and lower 247 mg kg^-1soil Pb level, 4 cultivars（No.7、8、10、11）could be treated as Cd+Pb-PSCs.5. Detected the seeds Cd concentration in typical Cd accumulation cultivars of celery, lettuce, water spinach, amaranth and Chinese flowering cabbage that screened in early experiments, the results show that, the significant differences were observed between low-Cd Cultivars and high-Cd cultivars of celery, and the seeds Cd concentration of the tested cultivars of celery was significantly correlated with those shoot Cd concentration in pot experiment. So we can use the seed Cd concentration to rapid identify the low-Cd cultivars of celery.6. Sequential extraction method was used to analyze and distinguish NaCl extractable forms and HCl extractable forms of Cd in seedlings of typical Cd accumulation cultivars of lettuce, water spinach, Chinese cabbage and Chinese flowering cabbage that screened in early experiments, exposed to Cd. The studies showed that, it can be very effective distinguish low-Cd and high-Cd accumulative cultivars for Chinese cabbage and water spinach by using the ratio value of NaCl extractable Cd : HCl extractable Cd. But for the other vegetable species, it should be studied further.7. In an effort to understand the single nucleotide polymorphisms （SNP） in LdPSC1 gene in 10 typical Cd accumulative cultivars of lettuce, we discovered the specific mutations existed between low-Cd cultivars and high-Cd cultivars in LdPSC1 gene. The test of departure of neutral mutation of nucleotide variation in LdPSC1 gene showed there was a tendency to divide in low-Cd cultivars and high-Cd cultivars, the LdPSC1 gene experiencing strong negative selection under Cd stress. The results could provide a theoretical basis for using the molecular markers in breeding of low-Cd cultivars.