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In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation in asakura-sanshoo (Zanthoxylum piperitum (L.) DC. F. inerme Makino) an important medicinal plant.

Zeng X, Zhao D - Pharmacogn Mag (2015 Apr-Jun)

Bottom Line: The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants.Individual transgenic Asakura-sanshoo lines were obtained.In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, Institute of Agro-Bioengineering, Guiyang 550025, Guizhou Province, P.R. China ; The State Key Lab of Green Pesticide and Agricultural Biological Engineering, Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, Guizhou Province, P.R. China.

ABSTRACT

Context: Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo.

Aims: To establish a regeneration and Agrobacterium tumefaciens-mediated genetic transformation system in Asakura-sanshoo, which could be used for cultivar improvement and gene function analysis.

Settings and design: The various combinations of indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and naphthalene acetic acid (NAA) were explored for the optimal plant regeneration from petiole and stem of Asakura-sanshoo. The half-strength woody plant medium (WPM) with different concentrations of NAA and IBA was used to induce root. For genetic transformation, A. tumefaciens strain EHA-105 harboring the plasmid pBin-Ex-H-ipt which carries the isopentenyl transferase (ipt) gene, β-glucuronidase (GUS) gene and kanamycin resistance gene neomycin phosphotransferase II (NPTII) were used. The transformation efficiency was detected by the kanamycin resistant frequency.

Materials and methods: Petioles and stems were obtained from the in vitro cultured Asakura-sanshoo. The petiole and stem segments were precultured for 3 days, and then inflected using the bacterium at the concentration of OD600 0.5-0.8 for 10 min, followed by 3 days co-cultivation. Selection of the transgenic plants was carried out after 7 days the regeneration using gradient kanamycin at 30 mg/L and 50 mg/L, respectively. Successful transformed plants were confirmed by GUS histochemical assays, polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), and Southern blotting analysis.

Results: The highest shoots regeneration was obtained on WPM supplement with 0.5 mg/L BA and 0.2 mg/L NAA. The optimal rooting medium was half strength macro-element WPM. The kanamycin resistant frequency of petiole and stem was 24.66% and 25.93%, respectively. Thirty-five shoots in thousands adventitious buds were confirmed through GUS histochemical assays, PCR, RT-PCR, and Southern blotting. The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants.

Conclusions: Individual transgenic Asakura-sanshoo lines were obtained. In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants.

No MeSH data available.


Related in: MedlinePlus

Plant regeneration of Asakura-sanshoo. (a) Axillary shoots. (b-d) Callus induction from stems and petioles. (e-i) Regeneration shoots. (j-l) Roots induced from different medium. (m) Rooted plantlets transferred to pots
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Figure 2: Plant regeneration of Asakura-sanshoo. (a) Axillary shoots. (b-d) Callus induction from stems and petioles. (e-i) Regeneration shoots. (j-l) Roots induced from different medium. (m) Rooted plantlets transferred to pots

Mentions: Axillary buds sprouted after 3–5 d cultured on WPM supplemented with 1.0 mg/L BA and 0.2 mg/L IBA, and within 4 weeks, 2–3 cm sterile axillary shoots were obtained [Figure 2a]. Petiole and stem explants were excised from these axillary shoots grown in-vitro. Callus induction and differentiation were detected under different concentrations of plant growth regulators (PGRs) [Table 1]. Green callus were produced at the cutting section of the explants after 10–15 d cultured on the WPM containing BA, NAA, or IBA [Figure 2b-d]. Among all the treatments, 1.0 mg/L BA in combination with 0.1 mg/L IBA achieved maximum callus induction rate (petiole 98.18% and stem 98.81%). However, most of this callus ceased growing, and turned to brown and finally died. Only 13.64% of petiole and 29.76% of stem callus could be initiated the regeneration shoots. WPM containing 0.5 mg/L BA and 0.2 mg/L NAA was the optimal medium for shoot regeneration from petiole and stem callus, the measurements were 60.00% and 60.95%, respectively [Figure 2e-h]. After cultured on the same medium for 30 d, most of the shoots elongated to 2–3 cm [Figure 2i].


In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation in asakura-sanshoo (Zanthoxylum piperitum (L.) DC. F. inerme Makino) an important medicinal plant.

Zeng X, Zhao D - Pharmacogn Mag (2015 Apr-Jun)

Plant regeneration of Asakura-sanshoo. (a) Axillary shoots. (b-d) Callus induction from stems and petioles. (e-i) Regeneration shoots. (j-l) Roots induced from different medium. (m) Rooted plantlets transferred to pots
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4378137&req=5

Figure 2: Plant regeneration of Asakura-sanshoo. (a) Axillary shoots. (b-d) Callus induction from stems and petioles. (e-i) Regeneration shoots. (j-l) Roots induced from different medium. (m) Rooted plantlets transferred to pots
Mentions: Axillary buds sprouted after 3–5 d cultured on WPM supplemented with 1.0 mg/L BA and 0.2 mg/L IBA, and within 4 weeks, 2–3 cm sterile axillary shoots were obtained [Figure 2a]. Petiole and stem explants were excised from these axillary shoots grown in-vitro. Callus induction and differentiation were detected under different concentrations of plant growth regulators (PGRs) [Table 1]. Green callus were produced at the cutting section of the explants after 10–15 d cultured on the WPM containing BA, NAA, or IBA [Figure 2b-d]. Among all the treatments, 1.0 mg/L BA in combination with 0.1 mg/L IBA achieved maximum callus induction rate (petiole 98.18% and stem 98.81%). However, most of this callus ceased growing, and turned to brown and finally died. Only 13.64% of petiole and 29.76% of stem callus could be initiated the regeneration shoots. WPM containing 0.5 mg/L BA and 0.2 mg/L NAA was the optimal medium for shoot regeneration from petiole and stem callus, the measurements were 60.00% and 60.95%, respectively [Figure 2e-h]. After cultured on the same medium for 30 d, most of the shoots elongated to 2–3 cm [Figure 2i].

Bottom Line: The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants.Individual transgenic Asakura-sanshoo lines were obtained.In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, Institute of Agro-Bioengineering, Guiyang 550025, Guizhou Province, P.R. China ; The State Key Lab of Green Pesticide and Agricultural Biological Engineering, Center for Research and Development of Fine Chemicals, Guizhou University, Guiyang 550025, Guizhou Province, P.R. China.

ABSTRACT

Context: Asakura-sanshoo (Zanthoxylum piperitum [L.] DC. f. inerme Makino) is an important medicinal plant in East Asia. Transgenic technique could be applied to improve plant traits and analyze gene function. However, there is no report on regeneration and genetic transformation in Asakura-sanshoo.

Aims: To establish a regeneration and Agrobacterium tumefaciens-mediated genetic transformation system in Asakura-sanshoo, which could be used for cultivar improvement and gene function analysis.

Settings and design: The various combinations of indole-3-butyric acid (IBA), 6-benzylaminopurine (BA) and naphthalene acetic acid (NAA) were explored for the optimal plant regeneration from petiole and stem of Asakura-sanshoo. The half-strength woody plant medium (WPM) with different concentrations of NAA and IBA was used to induce root. For genetic transformation, A. tumefaciens strain EHA-105 harboring the plasmid pBin-Ex-H-ipt which carries the isopentenyl transferase (ipt) gene, β-glucuronidase (GUS) gene and kanamycin resistance gene neomycin phosphotransferase II (NPTII) were used. The transformation efficiency was detected by the kanamycin resistant frequency.

Materials and methods: Petioles and stems were obtained from the in vitro cultured Asakura-sanshoo. The petiole and stem segments were precultured for 3 days, and then inflected using the bacterium at the concentration of OD600 0.5-0.8 for 10 min, followed by 3 days co-cultivation. Selection of the transgenic plants was carried out after 7 days the regeneration using gradient kanamycin at 30 mg/L and 50 mg/L, respectively. Successful transformed plants were confirmed by GUS histochemical assays, polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), and Southern blotting analysis.

Results: The highest shoots regeneration was obtained on WPM supplement with 0.5 mg/L BA and 0.2 mg/L NAA. The optimal rooting medium was half strength macro-element WPM. The kanamycin resistant frequency of petiole and stem was 24.66% and 25.93%, respectively. Thirty-five shoots in thousands adventitious buds were confirmed through GUS histochemical assays, PCR, RT-PCR, and Southern blotting. The regeneration shoot per explants elevated 5.85 fold compared with the wild-type plants.

Conclusions: Individual transgenic Asakura-sanshoo lines were obtained. In this paper, it first revealed the expression of ipt gene significantly promoted the adventitious buds induction in Asakura-sanshoo as the same action as in other plants.

No MeSH data available.


Related in: MedlinePlus