Chemicals

Taq polymerase, 100 bp Plus Ladder were purchased from Bioline. BC,G were produced by Integrated DNA Technologies.

Sterilization of culture materials

Seeds of L.was locally collected in district 9, Ho Chi Minh City, Vietnam. The selected seeds had good quality and free from infection. The seeds were washed with 80% ethanol for two minutes. Then, the seeds were shaken and soaked in 2% sodium hypochlorite for 10 minutes and then washed with sterile distilled water. Seeds after sterilization were placed on the MS (Murashige and Skoog) medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) phytoagar (pH 5.8). The seeds germinated in a growth chamber at 25 ± 2C under standard cool white fluorescent tubes with a 16-h/8-h photoperiod. Plantlets were collected at different ages depending on the purpose of the experiment.

Investigating factors affecting hairy root induction in L.

Preparation of Agrobacterium rhizogenes

ATCC15834 strain was obtained from RIKEN bank (Japan) through the MEXT project. ATCC15834 cells were grown in a nutrient broth medium (beef extract 3 g/L, peptone 5 g/L, pH 7.0) for 48 hours in a shaking incubator (110 rpm, 25 ± 1C).

The original procedure for hairy root induction

The original procedure used for L. hairy root induction was as following 10: the leaves from 15-day-old plants were injured on the surface to facilitate the infection process. These segments were soaked in the ATCC15834 suspension (OD = 0.6) for 20 minutes. After four days of co-cultivation, the explants were transferred to the MS medium (3% sucrose) supplemented with cefotaxime (250 mg/L) to eliminate the remained ATCC15834. The samples were grown under dark conditions at 25C for hairy root induction. Within two weeks, numerous hairy roots emerged from the wounded sites on leaf explants. The number of responsive explants and number of hairy roots per explant were recorded 30 days after infection.

Besides the investigated factors, the remaining factors were similar to the original hairy root induction procedure. All experiments were repeated three times, with the same negative control is leaf explants from 15-day-old seedlings that were not infected with .

The effect of age and different parts of plantson hairy root induction

Roots, stems, leaves of three types of plants: 15-day-old plants, 45-day-old plants and after two subculture generations plants were infected with to induce hairy roots.

The effect of infection time on hairy root induction

Samples were infected with for 10 minutes, 20 minutes, and 30 minutes to determine the optimal infection time.

The effect of culture medium on hairy root induction

Three types of medium were used: MS, GB5 (Gamborg B5 medium) and WPM to determine the optimal culture medium.

The effect of the combined of improved factors on hairy root induction

The hairy roots were induced in two procedures: the original procedure and the optimized procedure with optimized conditions (age, plant parts, time of infection and medium of induction).

Confirmation of transgenic roots

Genomic DNA samples were extracted from hairy roots and roots (non-transgenic roots) by the CTAB method as described previously 11. The Ri-plasmid was isolated from A. rhizogenes ATCC 15834 by the method described by Curier and Nester 12. PCR reactions were performed using the genomic DNA from the hairy roots and non-transgenic roots as well as the Ri-plasmid with specific primer sets for rolB, rolC, and virG genes. The sequencing primers include F-rolB (5’- GCTCTTGCAGTGCTAGATTT-3’), R-rolB (5’-GAAGGTGCAAGCTACCTCTC-3’); F-rolC (5′-CTCCTGACATCAAACTCGTC-3’), R-rolC (5’-TGCTTCGAGTTATGGGTACA-3’); and F-virG (5’-TTATCTGAGTGAAGTCGTCTCAGG-3’), R-virG (5’-CGTCGCCTGAGATTAAGTGTC-3’).

The expected amplified fragment sizes were 423 bp for gene, 626 bp for C gene, 1030 bp for G gene 13. The PCR reactions were performed in a total volume of 25μl containing 100ng of plant genomic DNA (or 40 ng of Ri-plasmid DNA), 5 µL Taq polymerase buffer (5X), 0.5 µM of each primer and 1U Taq polymerase (Bioline). PCR steps included initial denaturation at 95C for 5 minutes, followed by 35 cycles of amplification (95C for 30s, 54C for 30s and 72C for 60s) and a final extension at 72C for 10 minutes. PCR products were visualized by agarose gel electrophoresis.

Statistical analysis

Each treatment included 20 explants in replicates of three. The transformation frequency was calculated at four weeks after infection. All data analyses were performed using the SPSS 16.0 (Copyright SPSS Inc.). Experimental results were shown as mean ± standard deviation (SD). Differences between means were evaluated by Duncan’s multiple range tests. Statistical significance was accepted at 0.05.

Sterilization of culture materials

The seeds of L. were sterilized by 2% sodium hypochlorite in 10 minutes. The germination rate was 90-100%. After two days on MS medium, germinated seeds developed into seedlings. 45-day-old plants were mature enough and suitable for subculture. Seedlings were collected at different ages depending on the specific purposes of each experiment (Figure 1).

Investigating important factors to improve the hairy root induction in

The effect of age and different parts of plants on hairy root induction

After four weeks after infection, L. leaves had the highest transformation frequency compared to the stems and roots at all ages (Table 1). The older plants had a lower transformation frequency than the younger plants. Specifically, leaves of 15-day-old plants had the highest transformation frequency (97.33%), and the second highest was stems (86.33%) of the same age plants (Figure 2). This result demonstrated that the younger tissues, especially cotyledon, positively correlate with the higher transformation frequency.

The effect of infection time on hairy root induction

The results indicated that the infection time of 10 and 20 minutes had a similar transformation frequency (97.67% and 97.33%, respectively) (Table 2). However, when the infection time increased to 30 minutes, the transformation frequency significantly decreased (93.67%) (Table 2). In addition, the longer infection time correlates with a lower recovery rate of the wounded explants, and the induced hairy roots also developed to the lesser extent because of the bacterial overgrowth during the co-cultivation. Explants infected for 10 minutes have more hairy roots than the ones infected for 20 and 30 minutes (Figure 3).

Effect of culture medium on hairy root induction

The results showed that WPM and MS medium had a similar transformation frequency four weeks after infection (100% and 97.3%, respectively). GB5 medium had the lowest transformation frequency (91.67%) four weeks after infection (Table 3). However, WPM medium had higher hairy root development than the other two media (Figure 4). This observation indicated that the composition and the mineral content in WPM medium are better in supporting the growth of the L. hairy roots than the MS and GB5 medium.

The effect of the optimized conditions on hairy root induction

After identifying the optimal conditions for the hairy root induction in L., these conditions were combined and compared to the original procedure. Specifically, 15-day-old leaves were infected for 10 minutes and cultured in WPM medium. The results in the table 4 and the figure 5 showed that the optimized conditions had the transformation frequency of 100%, while the original procedure achieved a transformation frequency of 97.33% (Table 4, Figure 5). Importantly, the fresh biomass of hairy roots per explant in the optimized procedure (0.517g) was 3.2 times higher than the one of the original procedure (0.160g). In summary, this result showed that the optimized procedure is more effective than the original procedure in inducing and supporting the growth of hairy roots in L..

Transgenic roots were confirmed by PCR

The hairy root samples were analyzed by PCR to test whether the transgenic process was successful. DNA samples from the putative hairy roots and from non-transgenic roots were isolated and subjected to PCR analysis for the presence of ,, and genes, which are present in Ri-plasmid. The -plasmid of ATCC15834 was also included to serve as a positive control. The presence of and, as well as the absence of virG from these hairy roots confirmed that -plasmid was integrated successfully into the plant genome (Figure 6).