Studies On Synchromization Of Mitosis And Induction Of Micronuclei In Suspension-cultured Cells Of Hevea Rubber

There are many advantages expected from cell suspension cultures system over the conventional cultivation of whole plants, such as faster growth rate, direct contact with the medium nutrients, high dispensability arid uniformity, independence from various environmental and seasonal factors, cell contents enrichment, good experimental reproducibility and convenient harvest. Therefore, cell suspension cultures are excellent material as protoplast culture, cell fusion, gene transformation and fast-screening of somatic mutation. It is of great significance to use cell suspension cultures as breeding material for shortening breeding cycle, improving breeding efficiency, and overcoming shortcomings of traditional breeding. In this study, the culture system of Hevea rubber suspension cells was systematically studied, and cell suspension cultures and plant regeneration system were established. Cryopreservation method, aslo, synchromization of mitosis and induction of micronuclei in suspension-cultured cells were studied. Our objectives are to provide the theoretic and pratical scientific data for cell engineering research of Hevea rubber. The main results are as follows:Friable anther callus induction: The anthers at uninulear stage were placed on modified MS calllus medium supplemented with 0.1 g/L myo-inositol, 1.5 mg/L 2,4-D (2,4-Dichlorophenoxy acetic acid), 1.5 mg/L NAA (α-naphthalene acetic acid), 1.5 mg/LKT (kinetin), 70 g/L sucrose, 2.2 g/L phytagel, and CW (coconut water) 5(v/v)% (MMCM) in 100×20 mm-Petri dish at 28±2℃in darkness. After 50 days, the calli were transferred onto fresh MMCM and subcultured every 10 days until formed a mixture of compact, semi-friable and friable calli.Establishment of cell suspension cultures: About 3 g friable calli were chopped into small pieces with 2-3 mm and transferred aseptically into liquid modified MS suspension medium (MMSM) [modified MS medium supplemented with 0.1 g/L myo-inositol, 1 mg/L 2,4-D, 2 mg/L NAA, 1.5 mg/L KT, 50 g/L sucrose, and CW 5(v/v)%] in 100 ml Erlenmeyer flasks. Cultures were maintained on a 5～7-day subculture schedule at a dilution of 1:6 (inoculum: fresh medium), until set up well growth suspension cultures.Isodiametric, thin-walled and richly cytoplasmic cells, rapid division, good dispersion and homogeneous suspension cell cultures were established. The suspension cells were composed of single cells (minority) and small cell aggregates (majority). The growth kinetics studies showed a typical sigmoidal profile including lag phase, log phase or exponential phase, linear phase and deceleration phase, and the suspension cultures growth cycle is 3⁵ cell doublings and 7¹2 days in duration. Sedimented cell volume (SCV), fresh weight (FW), growth index (GI) of FW, the decrease of electric conductivity (EC) in medium have a very good linear correlation with dry weight (DW) or GI of DW.Cell suspension cultures subculture: The cell suspension cultures were cultured optimized suspension medium (OSM) [modified MS medium supplemented with 0.1 g/L myo-inositol, 0.2 g/L L-asparagine, 0.4 g/L casein hydrolysate, 0.2 mg/L 2,4-D, 2 mg/L NAA, 1 mg/L KT, 50 g/L sucrose, and CW 5(v/v)%] have a better growth, and which is suitable for long-term culture. Furthermore, we can ruduce the frequency of subculture by changing inoculum and speed of swing bed.Suspension cells induced embryogenic callus: The callus induction rate was 100% as suspension cells inoculated into suspension callus medium (SCM) [modified MS medium supplemented with 0.1 g/L myo-inositol, 2 mg/L 2,4-D, 4 mg/L NAA, 70 g/L sucrose, 2.2 g/L phytagel, and CW 5(v/v)%]; the induction frequency of embryogenic callus was more than 30% as calli inoculated into suspension embryogenic callus medium (SECM) [modified MS medium supplemented with 0.1 g/L myo-inositol, 2 mg/L BAP (6-Benzyl amino purine), 2 mg/L NAA, 2 mg/L GA (gibberellic acid), 70 g/L sucrose, 2.2 g/L phytagel, and CW 5(v/v)%]; and the white embryogenic callus from SECM can be inoculated to suspension embryogenic callus proliferation medium (SECPM) [modified MS medium supplemented with 0.1 g/L myo-inositol, 0.5 mg/L BAP, 1 mg/L KT, 2 mg/L GA, 70 g/L sucrose, 2.2 g/L phytagel, and CW 5(v/v)%] in order to long-term subculture and proliferation.Cryopreservation of suspension cells: 3～4 mL sedimented cell volume (SCV) suspension cultures in the log phase (2～3 days after subculture) were transferred directly to 30 mL preculture medium [OSM add 200 g/L sucrose and 5% DMSO (dimethylsulfoxide)] in 100 mL flasks and precultured for 1 h. The preculture medium was replaced by 10 mL cryoprotectant solution [OSM add 200 g/L sucrose] and then aliquots of 1 mL cell suspension cultures (approximately 0.3 mL SCV) was transferred to 2 mL sterile cryotubes that had been prefrozen at -20℃for 2 h followed by submerging in LN (liquid nitrogen) for more than 1 h. Thereafter the suspension cells were thawed rapidly in a water bath at 40℃until the ice melted (approximately 2～3min). The cryotubes were removed rapidly from the water bath and washed with 70% ethanol and allowed to dry in a laminar flow cabinet. The suspension cells were transferred to SCM to recover.Suspension cells mitotic synchronization: The effect of two DNA-synthesis inhibitors [hydroxyurea (HU) and aphidicolin (APH)] and four spindle toxins [amiprophos-methyl (APM), oryzalin (ORY), colchicine (COL) and chlorpropham (CIPC)] on the synchronization and chromosome morphology in suspension cells of Hevea brasiliensis were investigated. The metaphase index (MI) increased after log phase suspension cells were treated with HU and APH, the largest MI (HU 34.9%, APH 33.8%) was obtained (HU 10 h, APH 12 h) at the end of the treatment with 4 mmol/l HU and 4μmol/l APH for 24 h. Among the four spindle toxins, CIPC induced the lowest MI (<16.7%), whereas APM, ORY and COL induced high frequency of synchronization (>80%) in cells. The most effective treatment for synchronization has MI of 88.3% achieved with 8μmol/l APM treated cells for 8 h. HU and APH had no significant effect on chromosome morphology, throughout the treatment. However, the metaphase chromosomes was condensed and gathered as cells treated with four spindle toxins at certain concentration. For APM, chromosome condensation and aggregation were positively correlated to APM concentrations. At higher concentration, chromosome aggregation became longer with higher MI.Suspension cells micronucleation: Suspension cells were incubated for 14 h, using a mixed emzyme solution [5 g/L MES, 10 g/L cellulase, 1 g/L pectinase, 5 g/L hemicellulase, 15 g/L macerozyme, 130 g/L mannitol, 27.2 mg/L KH₂PO₄, 101.0 mg/L KNO₃, 1480 mg/L CaCl₂ 2H₂O, 0.16 mg/L KI, 0.025 mg/L CuSO₄·5H₂O, pH 5.8], which condition for protoplast separation was best,'with high separation efficiency. The purified protoplasts were uniform size, richly cytoplasmic, highly viability. The MI was 39.7%, micronucleus index (MNI) was 7.3% and the average 3.8 micronucleus per cell when log phase cells were inculated with mixed emzyme solution for 10 h after 1μmol/L APM treated the cells 10 and was removed.