Micropropagation of Apple Cultivars 'Golden Delicious' and 'Royal Gala' in Bioreactors

Abstract

This study aimed to investigate culture conditions for the efficient micropropagation of apple cultivars 'Golden Delicious' and 'Royal Gala' in liquid medium by temporary immersion. RITA^® bioreactors were used for the multiplication stage whereas RITA^® or Plantform™ were used for the rooting stage. Murashige and Skoog media (MS) with N⁶-benzyladenine (BA) was used for shoot multiplication and indole-3-butyric acid (IBA) for root induction. During the multiplication phase, we evaluated the mineral medium, BA concentration, immersion frequency, silver nitrate and activated charcoal supplementation and the use of physical supports to hold explants in an upright position. The results demonstrated that longer incubation periods (10 weeks) were better than shorter periods (6 weeks) for decreasing hyperhydricity and increasing the multiplication coefficient (MC). For 'Golden Delicious', the highest MC were obtained either with explants placed directly on the bioreactor basket and immersed six times per day for 60 s in MS with 2.2 µM BA or explants placed between rockwool cubes cultivated with 4.4 µM BA (both yielding MC of 8.9 and 5-10% hyperhydricity). These results were superior to 'Royal Gala', which showed a MC of 7.3 and 23% of hyperhydricity when cultivated in MS with half nitrates, 1.55 µM BA and rockwool cubes. Both varieties rooted efficiently (96-100%), and resulting plantlets were successfully acclimated. This is the first report in the micropropagation of these two commercial fruiting cultivars in temporary immersion, demonstrating the potential of this technology to enhance large-scale plant production for the apple nursery industry.

Keywords: Malus domestica; hyperhydricity; in vitro; multiplication; rooting; temporary immersion.

Figure 1

Initial experiments of apple in bioreactors. (a,b) Normal (a) and partially hyperhydric (b) shoots of ‘Golden Delicious’ after 6 weeks of culture. (c) Hyperhydric shoots of ‘Royal Gala’ after 6 weeks of culture. Bars = 1 cm.

Figure 2

Initial explants of apple in bioreactors with or without supports in the moment of inoculation in the bioreactors. (a) Apical sections of ‘Royal Gala’ placed between 1 cm³ rockwool cubes. (b) Apical sections of ‘Golden Delicious’ inserted between plastic slots. (c) Apical sections of ‘Royal Gala’ without support. Bars = 1 cm.

Figure 3

Effect of cytokinin concentration and subculture duration on the growth parameters of ‘Golden Delicious’ shoots cultured in bioreactors with or without rockwool cubes. Shoots were cultured in MS with BA 2.2 or 4.4 µM and immersed 6 times per day for 60 s. Data were recorded 6 and 10 weeks after starting the experiment. (a) Number of total shoots per explant. (b) Number of normal shoots per explant. (c) Percentage of hyperhydric shoots per explant. (d) Multiplication coefficient. (e) Length of the longest shoot per explant. (f) Number of rootable shoots per explant. Means ± standard error were calculated from 3 replicates, each with 8 shoots per treatment (n = 24). For each subculture duration, different uppercase letters indicate significant differences between BA supplementation and different lowercase letters indicate significant differences regarding the use of a support (p ≤ 0.05). A significant interaction between factors in sections (a–d,f), required a Bonferroni adjustment to detect simple main effects between means.

Figure 4

Effect of activated charcoal in shoot quality of ‘Golden Delicious’ cultured for 10 weeks in RITA^® bioreactors. (a) Shoot rooted spontaneously in the multiplication medium. (b,c) Aspect of leaves cultured with (b) or without activated charcoal. Bars = 1 cm.

Figure 5

Effect of subculture duration on the growth parameters of Royal Gala shoots cultured in bioreactors with or without cubes. Shoots were immersed 6 times per day for 60 s, and data were recorded 6 and 10 weeks after starting the experiment. (a) Number of total shoots per explant. (b) Number of normal shoots per explant. (c) Percentage of hyperhydric shoots per explant. (d) Multiplication coefficient. (e) Length of the longest shoot per explant. (f) Number of rootable shoots per explant. Means ± standard error were calculated from 3 replicates, each with 8 shoots per treatment (n = 24). Different uppercase letters indicate significant differences regarding the duration of subculture, and different lowercase letters indicate significant differences regarding the use of support (p ≤ 0.05).

Figure 6

Effect of silver nitrate (SN) and activated charcoal (AC) on ‘Royal Gala’ cultured for 10 weeks in RITA^® bioreactors. (a) Hyperhydric (left) and normal shoots (right) developed in medium with SN. (b). Shoot rooted spontaneously in the multiplication medium with AC.

Figure 7

Effect of the culture system on the root formation of ‘Golden Delicious’ (a) and ‘Royal Gala’ (b). Shoots were treated for 5 weeks with MS ½ macronutrients, 3% sucrose and 4.9 µM indole-3-butyric acid, either in jars with agar or in bioreactors with liquid medium. RITA^® bioreactors were operated by TIS (6 immersions per day) and Plantform™ (PF) either by TIS (6 immersions plus 9 aerations) or CIS (15 aerations). Duration of immersions and aerations were 60 s. Shoots were placed between plastic slots (Figure 2) or 1 cm³ rockwool cubes or inserted in 2 cm³ rockwool cubes. Means ± standard error were calculated from 3 replicates, each with 7 shoots per treatment (n = 21). Different letters indicate significant differences (p ≤ 0.05).

Figure 8

(a) Shoots of ‘Golden Delicious’ (GD) used for rooting experiments after 6 weeks in multiplication medium. (b) Shoots of ‘Royal Gala’ (RG) used for rooting experiments after 6 weeks in multiplication medium. (c–i) Rooted shoots 6 weeks after root induction. (c,d) Shoots of GD (c) and RG (d) rooted in RITA^® with slots. (e–g) Shoots of GD rooted in Plantform™ by TIS (e) and CIS (f) with 1 cm³ cubes and by CIS inserted in 2 cm³ cubes (g). (h,i) Shoots of RG rooted by TIS with 1 cm³ (h) and 2 cm³ cubes (i). (j,k) Rooted shoots of GD (j) and RG (k) 2 months after transfer to pots. Bars = 1 cm.

Figure 8

(a) Shoots of ‘Golden Delicious’ (GD) used for rooting experiments after 6 weeks in multiplication medium. (b) Shoots of ‘Royal Gala’ (RG) used for rooting experiments after 6 weeks in multiplication medium. (c–i) Rooted shoots 6 weeks after root induction. (c,d) Shoots of GD (c) and RG (d) rooted in RITA^® with slots. (e–g) Shoots of GD rooted in Plantform™ by TIS (e) and CIS (f) with 1 cm³ cubes and by CIS inserted in 2 cm³ cubes (g). (h,i) Shoots of RG rooted by TIS with 1 cm³ (h) and 2 cm³ cubes (i). (j,k) Rooted shoots of GD (j) and RG (k) 2 months after transfer to pots. Bars = 1 cm.