Applying Biotechnology in the Propagation and Further Selection of Vaccinium uliginosum × (V. corymbosum × V. angustifolium) Hybrids

Abstract

The most serious problem of intergeneric and interspecific hybridization is related to overcoming the reproductive isolation of different species. We assessed the efficiency of reproduction under in vitro conditions and the ex vitro growth capacity of interspecific hybrids of Vaccinium uliginosum × (V. corymbosum × V. angustifolium). The percentage of seed germination in in vitro culture was 88% for V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), SC5-8, while it was 42% for V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), 'Northcountry'. The analysis of mean value showed that the multiplication rate increased and the shoot height decreased as the 2-isopentenyl adenine (2iP) concentration was increased in the nutrient medium of the studied hybrids. The maximum rate was achieved using 15 μM 2iP. A detailed analysis of the hybrids indicated that the hybrid variant reliably affected growth and development indicators. Inter simple sequence repeat analysis demonstrated that all analyzed hybrids inherited DNA fragments of the parent plants in various combinations, confirming their hybrid nature. Thus, the use of in vitro methods for the propagation and further selection of genotypes is demonstrated as being an effective approach for developing interspecific hybrids of V. uliginosum × (V. corymbosum × V. angustifolium).

Keywords: ISSR analysis; berry crops; in vitro culture; interspecific hybrids.

Figure 1

V. uliginosum × (V. corymbosum × V. angustifolium) hybrids in in vitro culture: (A) seedlings on 0.6% water agar; (B,C) microshoots of variant No. 4-1 and variant No. 4-5 on Anderson medium, supplemented 5 µM 2iP; (D‒J) rooted plants of variant Nos. 1-1, 1-2, 1-5, 2-2, 4-2, 4-8, 7-9 on 1/2 Anderson medium. Bar: 1 cm.

Figure 2

Schematic depiction of the experiment showing stages of in vitro propagation and ex vitro acclimatization, conditions and cultivation periods. Note: V.ul.8—V. uliginosum, form No. 8; ‘NC’—(V. corymbosum × V. angustifolium) ‘Northcountry’; ‘SC 5-8’—(V. corymbosum × V. angustifolium) ‘SC 5-8’; A—Anderson nutrient medium.

Figure 3

The multiplication rate of variants V.ul.8 × ‘NC’ (A) and V.ul.8 × ‘SC 5-8’ (B) on Anderson medium, supplemented 5 µM 2iP at the 2nd passage. Data are presented as mean values with confidence intervals (p ≤ 0.05). N = 1–6. Note: *—callusogenesis of variant No. 7-11.

Figure 4

Effect of 2iP concentration on the multiplication rate of V.ul.8 × ‘NC’: (A) detailed analysis of values of hybrid variants; (B) average values of hybrid variants. Means followed by the same letter are not significantly different according to the LSD at p ≤ 0.05. Note: *—conglomerate of shoots variant No. 4-6; 1-1, 1-2, 1-5, 2-2, 4-2, 4-6, 4-8, 5-3—variant Nos. 1-1, 1-2, 1-5, 2-2, 4-2, 4-6, 4-8, 5-3.

Figure 5

Effect of 2iP concentration on the multiplication rate of V.ul.8 x ‘SC 5-8’: (A) detailed analysis of values of hybrid variants; (B) average values of hybrid variants. Means followed by the same letter are not significantly different according to the LSD at p ≤ 0.05. Note: 6-2, 7-3, 7-9—variant Nos. 6-2, 7-3, 7-9.

Figure 6

Effect of 2iP concentration on shoot height of V.ul.8 × ‘NC’: (A) detailed analysis of values of hybrid variants; (B) average values of hybrid variants. Means followed by the same letter are not significantly different according to the LSD at p ≤ 0.05. Note: 1-1, 1-2, 1-5, 2-2, 4-2, 4-6, 4-8, 5-3—variant Nos. 1-1, 1-2, 1-5, 2-2, 4-2, 4-6, 4-8, 5-3.

Figure 7

Effect of 2iP concentration on shoot height of V.ul.8 × ‘SC 5-8’: (A) detailed analysis of values of hybrid variants; (B) average values of hybrid variants. Means followed by the same letter are not significantly different according to the LSD at p ≤ 0.05. Note: 6-2, 7-3, 7-9—variant Nos. 6-2, 7-3, 7-9.

Figure 8

V. uliginosum × (V. corymbosum × V. angustifolium) hybrids after 2 years of cultivation: (A) V.ul.8 × ‘NC’ No. 1-1, 1-2, 1-5, 2-2, 4-2, 4-8; (B) V.ul.8 × ‘SC 5-8’ No. 7-9; (C) annual shoots of No. 1-1, 1-2, 1-5, 2-2, 4-2, 4-8, 7-9. Bar: 10 cm.

Figure 9

ISSR-PCR eletrophoregrams of genomic DNA with primers UBC825 (left) and UBC811 (right). 1–6—V.ul.8 × ‘NC’ No. 1-1, No. 1-2, No. 1-5, No. 2-2, No. 4-2, No. 4-8; 7—V.ul.8 × ‘SC 5-8’ No. 7-9; 8—‘SC 5-8 (♂1)’; 9 -’NC’ (♂2); 10—V. ul.8 (♀ 1), 11—V. ul. 8 (♀ 2). Note: red arrows—♀ 1-specific markers; yellow arrows—♀ 2-specific markers; green arrows—♂ 1-specific markers; blue arrows—♂ 2-specific markers.