Abstract
The juvenile period of blueberry seedlings typically lasts ≈3 to 4 years. To shorten this period and facilitate FasTrack breeding, we developed transgenic ‘Aurora’ blueberry plants with constitutive expression of the blueberry FLOWERING LOCUS T gene, enabling flowering of T0 transformants within just 1 year. To evaluate the potential of these transgenic lines in accelerating breeding cycles, we crossed transgenic ‘Aurora’ with transgenic southern highbush blueberry ‘Legacy’, referred to as Mu-Legacy. Mu-Legacy also exhibited early flowering mainly as a result of a transgene insertion, making it suitable for FasTrack breeding. Over 2 years of phenotyping, we observed that transgenic seedlings flowered consistently each year, whereas nontransgenic seedlings did not produce any flowers. These results suggest that either the constitutive expression of the blueberry FLOWERING LOCUS T gene or the specific transgene insertion site in transgenic ‘Legacy’ can effectively shorten the juvenile phase of blueberry plants. Given the significance of ‘Aurora’ and ‘Legacy’ in blueberry production, these transgenic lines emerge as a valuable tool for accelerating blueberry breeding programs.
Blueberry is one of the most important fruit crops within the Vaccinium genus (Song 2020; Song and Hancock 2011). Its high nutritional value has driven a rapid global expansion in blueberry production during the past two decades. This growth has led to significant advancements in various areas of blueberry research, including breeding, cultivation, genetics, genomics, physiological analysis, nutritional analysis, and marketing strategies, laying a foundation for further blueberry improvement through various approaches (Edger et al. 2022).
Blueberry breeding is a lengthy process that typically takes 5 to 10 years to develop and release a new cultivar, depending on the genotype. This extended timeline is largely a result of the 2- to 3-year juvenile period from seed germination to flowering under natural conditions. Although optimizing cultivation conditions has helped promote earlier flowering (Ohishi-Yamazaki et al. 2018), a genetic solution to shorten juvenile periods remains essential and highly desirable. To address this, we cloned the FLOWERING LOCUS T (VcFT) gene from the northern highbush cultivar Bluecrop and transformed it to another new northern highbush cultivar, Aurora, for constitutive expression, and the transgenic ‘Aurora’ plants showed precocious flowering (Song et al. 2013). In addition, transcriptomic analysis and transgrafting experiments with these transgenic ‘Aurora’ plants suggest that the constitutive expression of VcFT (CX-VcFT) could facilitate FasTrack breeding by reducing the juvenile period in blueberry (Song et al. 2019; Walworth et al. 2016). However, further evidence is needed from transgenic blueberry seedlings with CX-VcFT to confirm these findings.
In addition, we identified a unique transgenic blueberry line derived from the cultivar Legacy, referred to as Mu-Legacy (Surridge 2019). The transgenes and their insertion location in Mu-Legacy led to early flower bud formation and flowering. Both Mu-Legacy and its self-pollinated T1 transgenic offspring exhibited early flowering under nonchilling conditions (Lin et al. 2019b; Song and Walworth 2018), suggesting its potential for FasTrack breeding. Furthermore, Mu-Legacy has shown increased yield potential, reduced chilling requirements, and may have better winterhardiness compared with many low-chilling cultivars grown in southern regions with warm winters (Song and Walworth 2018).
In this study, we conducted experiments to evaluate further the potential of using CX-VcFT ‘Aurora’ or Mu-Legacy to shorten the juvenile period in blueberry seedlings.
Materials and Methods
Plant materials.
‘Aurora’ is a late-ripening northern highbush blueberry (Vaccinium corymbosum L.) variety developed from a cross between the Australian highbush ‘Brigitta’ and the northern highbush variety Elliott. Transgenic ‘Aurora’ plants contain a VcFT from ‘Bluecrop’. This VcFT is driven by a constitutive Cauliflower mosaic virus (CaMV) 35S promoter (Fig. 1A) (Song et al. 2013). One representative transgenic, hereafter named VcFT-Aurora, was used in this study.
Genetic background of VcFT-Aurora, Mu-Legacy, and their cross-progeny seedlings. (A) Transfer DNA (T-DNA) region in transgenic VcFT-Aurora. (B) T-DNA region in transgenic Mu-Legacy, showing VcDDF1 as the intended transgene and VcRR2 as an unintended activation due to insertion. (A and B) Polymerase chain reaction primer positions are marked. (C) Comparison of 8-month-old VcFT-Aurora and ‘Aurora’. (D) Comparison of 4-year-old ‘Legacy’ and Mu-Legacy. (E) Hybridization with VcFT-Aurora made on Mu-Legacy. (F) Hybridization with Mu-Legacy made on VcFT-Aurora. (G) In vitro germination of seeds from the hybridizations. LB = left border; RB = right border; GUS = β-glucuronidase. Primer names are shown in purple.
Citation: J. Amer. Soc. Hort. Sci. 150, 1; 10.21273/JASHS05447-24
‘Legacy’ is a midseason southern highbush blueberry (V. corymbosum L.) variety known for its top-ranked flavor. Mu-Legacy is a mutated ‘Legacy’ line developed through genetic transformation with the blueberry DWARF AND DELAYED FLOWERING 1 (VcDDF1) gene for increasing freezing tolerance (Song and Gao 2017; Walworth and Song 2018; Walworth et al. 2012). At the insertion site, the blueberry RESPONSE REGULATOR 2-like gene (VcRR2) was activated and promoted flowering (Fig. 1B) (Lin et al. 2019b; Song and Walworth 2018; Walworth et al. 2012). This Mu-Legacy was identified in a screening of 50 independent VcDDF1 transgenic lines (Walworth et al. 2012). Mu-Legacy is able to flower under nonchilling conditions and serves as a valuable material for studying chilling-mediated flowering mechanisms (Song and Walworth 2018; Surridge 2019).
Both ‘Aurora’ and ‘Legacy’ require a minimum of 800 chilling hours to break flower bud dormancy. All T0 transgenic and nontransgenic materials were maintained in vitro and transplanted to pots to obtain plants for this study.
Hybridization.
Three-year-old VcFT-Aurora and Mu-Legacy plants were exposed to ≈1000 chilling hours in a cold growth chamber. Afterward, the plants were transferred to a greenhouse to induce flowering. Emasculated flowers of VcFT-Aurora were pollinated with pollen from Mu-Legacy, and vice versa. The pollinated flowers were labeled and their mature fruit was harvested. Seeds were extracted from fruit stored in a freezer for a minimum of 3 months before it was used for germination.
For surface sterilization, seeds were soaked with agitation at 200 rpm in sodium hypochlorite (1%) for 10 min, then rinsed three times in sterile distilled water. The seeds were germinated on half-strength Murashige & Skoog (MS) medium (Murashige and Skoog 1962) in petri dishes at 25 °C at a 16-h photoperiod. Germinated seeds were transferred to half-strength MS medium in CultureJar™ G9 (PhytoTech Laboratories, Lenexa KS, USA). Elongated seedlings, 4 to 6 cm in length, were inserted into sphagnum moss in six-packs and grown in a plant culture room for 6 weeks before being transplanted into 4-inch pots and moved to a greenhouse in Jan 2023 (Song 2015). Ten months later, the plants were transplanted into 2-gal pots and placed in an unheated hoop house for the winter in East Lansing, MI, USA. All plants were moved out of the hoop house on 2 May 2024 and grown under open-air conditions. The plants were fertilized weekly with acid fertilizer and watered as needed.
Genotyping and phenotyping of the seedlings.
Polymerase chain reaction (PCR) was conducted to confirm the presence of the transgenes. Genomic DNA was extracted from young leaves using the cetyltrimethylammonium bromide method (Doyle and Doyle 1987). To detect the NEOMYCIN PHOSPHOTRANSFERASE II gene (nptII), the primers used were nptII-F (5′-GAGGCTATTCGGCTATGACTG-3′) and nptII-R (5′-ATCGGGAGCGGCGATACCGTA-3′) (Fig. 1A and B). To detect the transformed VcFT gene, primers targeting a segment of the CaMV 35S promoter and the VcFT gene were used: 35S-F (5′-TGACGCACAATCCCACTATC-3′) and VcFT-R (5′-GAGCTCTCAGCGTCGTCGTCCT-3′) (Fig. 1A). For detecting transformed genes in Mu-Legacy, the 35S-F primer was paired with a primer specific to the VcDDF1 gene, VcDDF1-R (5′-GAGTCTGACCCGATTGGAAA-3′) (Fig. 1B) (Walworth et al. 2012). PCR products were then run on a 1% agarose gel to verify the presence or absence of the target bands.
For phenotyping, the plants were monitored regularly, with photographs taken and flowering times recorded.
Results
Phenotypic characteristics of T0 VcFT-Aurora and T0 Mu-Legacy.
The VcFT-Aurora plantlets, after they were transplanted from in vitro culture into soil, flowered within 1 year without previous exposure to chilling hours. In contrast, the nontransgenic ‘Aurora’ plants did not flower within 3 years. Under normal growing conditions, VcFT-Aurora plants were initially shorter and had fewer branches during the first 2 years compared with the nontransgenic ‘Aurora’ plants (Fig. 1C). However, by the fifth year, VcFT-Aurora plants became taller than the nontransgenic plants (Supplemental Fig. 1). This is consistent with previous reports that CX-VcFT promotes flowering through complex gene interaction networks, suggesting that CX-VcFT is a significant flowering enhancer (Gao et al. 2016; Lin et al. 2019a; Song et al. 2013; Walworth et al. 2016).
Mu-Legacy, a transgenic southern highbush blueberry, is an early-flowering mutant that has been well studied in both T0 and self-pollinated T1 plants (Lin et al. 2019b; Song and Walworth 2018). In the presence of the transgenes, both T0 plants and T1 seedlings flowered after 1 year, whereas nontransgenic ‘Legacy’ plants and nontransgenic T1 seedlings did not flower until after 3 years. This suggests that the transgenes in Mu-Legacy are able to shorten the juvenile period of the seedlings. In addition, Mu-Legacy plants were shorter and produced more fruit than nontransgenic ‘Legacy’ plants (Fig. 1D), indicating a greater yield potential. Unlike VcFT-Aurora plants, Mu-Legacy plants did not show a reduction in the number of new branches.
Genotyping of the T1 seedlings.
Crosses between VcFT-Aurora and Mu-Legacy resulted in fruit and seed production (Fig. 1E–G). T1 seeds from these cross-pollinated fruit took up to 8 weeks to germinate, with germination rates of 53% for VcFT-Aurora and 63% for Mu-Legacy (Fig. 1G). Genotyping by PCR for the presence of the transgenes identified four genotype groups: nontransgenic, transgenic with the VcFT construct (VcFT+), transgenic with the VcDDF1 construct (VcRR2+), and transgenic with both constructs (VcFT+ and VcRR2+) (Table 1). A χ2 test confirmed that the segregation of these groups followed a 1:1:1:1 ratio (Table 1). Because Mu-Legacy carries a single copy of the transgenes, the transgenes in VcFT-Aurora must have segregated at a 1:1 ratio (transgenic vs. nontransgenic).
In vitro germination of seeds from the hybridizations and segregation of the transgenes in the germinated seeds.
Phenotyping of the T1 seedlings.
In the first year, seedlings grown in 4-inch pots in the greenhouse, before any chilling treatment, exhibited varying flowering times based on their genotype (Fig. 1A). Seedlings positive for both VcFT+ and VcRR2+ flowered in ≈2 months, whereas those positive for VcFT+ alone flowered 3 months after planting. Seedlings positive for VcRR2+ alone required 6 months to bloom. In addition, plants with the VcFT+ gene, whether alone or in combination with VcRR2+, flowered twice (Fig. 2B), whereas VcRR2+ plants flowered only once. None of the nontransgenic seedlings flowered in the first year.
Phenotyping of T1 seedlings in the first and second year. VcFT– and VcRR2–, nontransgenic seedlings; VcFT+, presence of the FLOWERING LOCUS T (VcFT) construct; VcRR2+, presence of the blueberry DWARF AND DELAYED FLOWERING 1 (VcDDF1) vector; VcRR2+ and VcFT+, presence of both VcDDF1 and VcFT constructs. (A and B) Seedlings growing in the greenhouse for 10 months. (C–I) Growth and flowering of 20-month-old seedlings under open-air conditions. Arrows show an additional growth on the petals.
Citation: J. Amer. Soc. Hort. Sci. 150, 1; 10.21273/JASHS05447-24
After the first round of flowering, 10 out of 11 plants positive for both VcFT+ and VcRR2+ produced more fruit than the 12 VcFT+ plants, which in turn bore more fruit clusters, except one seedling, than the VcRR2+ plants. In terms of plant architecture, the nontransgenic seedlings looked larger than the VcRR2+ plants, which were similar in size to seedlings positive for both VcFT+ and VcRR2+, but larger than the VcFT+ seedlings (Fig. 2A).
In the second year, fully chilled seedlings were grown in 2-gal pots under open-air conditions until the end of September. All transgenic seedlings flowered in the spring with normal-looking flowers, whereas the nontransgenic seedlings did not flower. Flowers from transgenic plants were similar morphologically to those of the nontransgenic cultivars Aurora and Legacy, and set fruit normally. In addition, all transgenic seedlings exhibited a second round of flowering on newly formed shoots as early as mid-July, whereas none of the nontransgenic seedlings flowered (Fig. 2C). This second round of continuous flowering lasted for ≈2.5 months under open-air conditions (Fig. 2D–2F). During this period, three distinct types of transgenic seedlings were visually discernible. Seedlings positive for VcFT+, either alone or in combination with VcRR2+, produced flowers not only at the shoot tips, but also along the middle sections of the branches. In contrast, the VcRR2+ transgenic lines developed flowers located primarily at the tips of shoots and branches (Fig. 2F). In addition, flowers from the VcFT+ genotype had unusual shapes characterized by a small, additional growth on the petals (Fig. 2G). This unusual flower morphology was also seen in the first year in VcFT+-positive plants (Fig. 2B), as well as in VcFT-Aurora plants flowering under nonchilling conditions. A key difference between the VcFT+-only plants and those positive for both VcFT+ and VcRR2+ was that the latter produced more new branches and had a greater percentage of normal flowers (Fig. 2D, E, G, and H).
At the end of September, we transferred the seedlings from open-air conditions to a heated greenhouse with a 14-h photoperiod and temperatures ranging from 21 to 28 °C to minimize seasonal effects on our study of blueberry flowering and fruit development. By the end of October, continuous flowering persisted in all transgenic seedlings, and fruit on each plant continued to develop (Fig. 3). This continuous flowering and fruiting suggest that transgenic seedlings containing either the VcFT+ or VcRR2+ genes not only facilitate the FasTrack breeding of blueberries, but also hold the potential for yield enhancement.
Continuous flowering of T1 transgenic seedlings during the second flowering cycle in the second year, which began in mid-July. Images were taken on 2 Nov 2024.
Citation: J. Amer. Soc. Hort. Sci. 150, 1; 10.21273/JASHS05447-24
Two years of phenotyping data on T1 seedlings have shown that both VcFT+ from VcFT-Aurora and VcRR2+ from Mu-Legacy promote early flowering by shortening the juvenile period of seedlings. However, VcFT+ proved more effective than VcRR2+. Consequently, VcFT-Aurora and Mu-Legacy are valuable resources for blueberry FasTrack breeding.
Discussion
FasTrack breeding materials, typically elite varieties or genotypes, are genetically engineered with specific genes, such as floral activators and repressors, to shorten the juvenile phase in plants. Hybridization, a fundamental plant breeding technique, enables the exchange of multiple genes between parent plants, including the introduced genetic modifications. Progenies from these crosses are expected to inherit the engineered genes and, consequently, display a reduced juvenile period. To date, FasTrack breeding materials have been developed and have shown promising results in subsequent generations of apple, citrus, and plum (Callahan et al. 2015; Endo et al. 2020; Patocchi et al. 2021; Schlathölter et al. 2018; Weigl et al. 2015). In our study, we demonstrate that VcFT-Aurora and Mu-Legacy are both excellent candidates for FasTrack breeding in blueberry.
Mu-Legacy has proved to be a reliable material for blueberry FasTrack breeding. Its seedlings from self-pollinated seeds flowered within 1 year under nonchilling conditions (Lin et al. 2019b; Song and Walworth 2018). Likewise, in the presence of Mu-Legacy–derived VcRR2+, seedlings from cross-pollinated seeds exhibited a shortened juvenile period and also flowered within 1 year. In our study, VcFT+ from VcFT-Aurora was highly effective in promoting flowering, highlighting VcFT-Aurora as an invaluable material for FasTrack breeding. Notably, seedlings with both VcFT+ and VcRR2+ showed even greater promise, demonstrating early flowering and high yield potential.
Although Mu-Legacy, VcFT-Aurora, and their transgenic seedlings carry genes beneficial for FasTrack breeding, they also serve as valuable materials for uncovering the mechanisms of blueberry flowering. Transcriptomic analyses have shown that the early flowering mechanism in VcFT-Aurora functions primarily through flowering pathway genes, whereas in Mu-Legacy it is driven by hormone-related pathways with few major flowering pathway genes involved (Lin et al. 2019b; Surridge 2019; Walworth et al. 2016). The transgenic seedlings obtained in this study offer new materials for studying the blueberry flowering mechanism and gene network.
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