An effective method for establishing a regeneration and genetic transformation system for Actinidia arguta

Abstract

The all-red A. arguta (Actinidia arguta) is an anthocyanin-rich and excellent hardy fruit. Many studies have focused on the green-fleshed A. arguta, and fewer studies have been conducted on the all-red A. arguta. Here we reported a regeneration and Agrobacterium-mediated transformation protocol by using leaves of all-red A. arguta as explants. Aseptic seedling leaves of A. arguta were used as callus-inducing materials. MS medium supplemented with 0.3 mg·L^-1 2,4-D and 1.0 mg·L^-1 BA was the optimal medium for callus induction of leaves, and medium supplemented with 3 mg·L^-1 tZ and 0.5 mg·L^-1 IAA was optimal for adventitious shoot regeneration. The best proliferation medium for adventitious buds was MS + 1.0 mg·L^-1 BA + 0.3 mg·L^-1 NAA. The best rooting medium was 1/2MS + 0.7 mg·L^-1 IBA with a 100% rooting rate. For the red flesh hardy kiwi variety 'Purpurna Saduwa' (A. arguta var. purpurea), leaves are receptors for Agrobacterium (EHA105)-mediated transformation. The orthogonal experiment was used for the optimization of each genetic transformation parameter and the genetic transformation of the leaves was 21% under optimal conditions. Our study provides technical parameters for applying genetic resources and molecular breeding of kiwifruit with red flesh.

Keywords: Actinidia arguta; genetic transformation; plant growth regulators; regeneration system; tissue cultures.

Figure 1

Effect of different PGRs on callus induction in ‘Purpurna Saduwa’ (A: the rate of callus formation, B: fresh weight). Normal letters in every column indicate significant differences at 0.05 level by Duncan’s multiple range test. The total number was 30 explants. Values represent the mean ± standard error of three replications (n = 10).

Figure 2

Effect of different PGRs on adventitious shoots regeneration of ‘Purpurna Saduwa’(A, C: regeneration rate, B, D: the number of adventitious shoots). Normal letters in every column indicate significant differences at 0.05 level by Duncan’s multiple range test. The total number was 30 explants. Values represent the mean ± standard error of three replications (n = 10).

Figure 3

Regeneration of adventitious shoots from ‘Purpurna Saduwa’ leaves (A: dark culture for 15 d after inoculation, B: dark culture for 30 d after inoculation, C–E: light culture for 7 d (37 d after inoculation), F: light culture for 25 d (55 d after inoculation)).

Figure 4

Rapid propagation of kiwifruit ‘Purpurna Saduwa’ (A: adventitious bud proliferation; B, C: seedling rooting; D: transplanting for 30 days).

Figure 5

Effects of PGRs on adventitious shoot proliferation of ‘Purpurna Saduwa’(A: fresh weight and dry weight, B: Proliferation rate and proliferation rate of plant height > 2cm). Normal letters in every column indicate significant differences at 0.05 level by Duncan’s multiple range test. The total number was 30 explants. Values represent the mean ± standard error of three replications (n = 10).

Figure 6

Effects of IBA on rooting of ‘Purpurna Saduwa’ plantlets (A: 0 mg·L^-1 IBA, B: 0.3 mg·L^-1 IBA, C: 0.5 mg·L^-1 IBA, D: 0.7 mg·L^-1 IBA, E: 1.0 mg·L^-1 IBA).

Figure 7

Effects of PGRs on adventitious shoot proliferation of ‘Purpurna Saduwa’(A: fresh weight and dry weight, B: the average plant height and the root length, C: Rooting rate and Number of roots per plant). Normal letters in every column indicate significant differences at 0.05 level by Duncan’s multiple range test. The total number was 30 explants. Values represent the mean ± standard error of three replications (n = 10).

Figure 8

Effect of different antibiotics on the induction of callus in leaves (A: 0 mg·L⁻¹ Kan, B: 25 mg·L⁻¹ Kan, C: 50 mg·L⁻¹ Kan, D: 75 mg·L⁻¹ Kan, E: 100 mg·L⁻¹ Kan, F: 125 mg·L⁻¹ Kan, G: 150 mg·L⁻¹ Kan, H: 175 mg·L⁻¹ Kan, I: 0 mg·L⁻¹ Hyg, J: 5 mg·L⁻¹ Hyg, K: 10 mg·L⁻¹ Hyg, L: 15 mg·L⁻¹ Hyg, M: 20 mg·L⁻¹ Hyg).

Figure 9

GUS staining of the resistant callus (A, B: all blue callus, C: partial blue callus, D: no blue callus).

Figure A1

Different PGRs combinations of leaf induced callus after 30d (A: 0.5 mg·L^-1 BA + 0.1 mg·L^-1 NAA, B: 0.5 mg·L^-1 BA + 0.2 mg·L^-1 NAA, C: 0.5 mg·L^-1 BA + 0.3 mg·L^-1 NAA, D: 0.5 mg·L^-1 BA + 0.1 mg·L^-1 2,4-D, E: 0.5 mg·L^-1 BA + 0.2 mg·L^-1 2,4-D, F: 0.5 mg·L^-1 BA + 0.3 mg·L^-1 2,4-D, G: 1 mg·L^-1 BA + 0.1 mg·L^-1 NAA, H: 1 mg·L^-1 BA + 0.2 mg·L^-1 NAA, I: 1 mg·L^-1 BA + 0.3 mg·L^-1 NAA, J: 1 mg·L^-1 BA + 0.1 mg·L^-1 2,4-D, K: 1 mg·L^-1 BA + 0.2 mg·L^-1 2,4-D, L: 1 mg·L^-1 BA + 0.3 mg·L^-1 2,4-D).

Figure A2

Adventitious shoot proliferation of 'Purpurna Saduwa' (A: treatment 1, B: treatment 2, C: treatment 3, D: treatment 4, E: treatment 5, F: treatment 6).

Figure A3

PCR analysis for GUS.