GRF-GIF chimeric proteins enhance in vitro regeneration and Agrobacterium-mediated transformation efficiencies of lettuce (Lactuca spp.)

Abstract

GRF-GIF chimeric proteins from multiple source species enhance in vitro regeneration in both wild and cultivated lettuce. In addition, they enhance regeneration in multiple types of lettuce including butterheads, romaines, and crispheads. The ability of plants to regenerate in vitro has been exploited for use in tissue culture systems for plant propagation, plant transformation, and genome editing. The success of in vitro regeneration is often genotype dependent and continues to be a bottleneck for Agrobacterium-mediated transformation and its deployment for improvement of some crop species. Manipulation of transcription factors that play key roles in plant development such as BABY BOOM, WUSCHEL, and GROWTH-REGULATING FACTORs (GRFs) has improved regeneration and transformation efficiencies in several plant species. Here, we compare the efficacy of GRF-GIF gene fusions from multiple species to boost regeneration efficiency and shooting frequency in four genotypes of wild and cultivated lettuce (Lactuca spp. L.). In addition, we show that GRF-GIFs with mutated miRNA 396 binding sites increase regeneration efficiency and shooting frequency when compared to controls. We also present a co-transformation strategy for increased transformation efficiency and recovery of transgenic plants harboring a gene of interest. This strategy will enhance the recovery of transgenic plants of other lettuce genotypes and likely other crops in the Compositae family.

Keywords: GIF; GRF; GROWTH-REGULATING FACTOR; Lettuce; Regeneration.

Fig. 1

Regeneration rates of Cobham Green and Armenian 999 after transformation with GRF–GIF fusions from tomato, pepper, citrus, and grape. a, b Box plots representing regeneration efficiency (a) and shooting frequency (b) of Cobham Green after 35 days in culture. c Tissue cultures of Cobham Green after 24 days on induction medium. d, e Box plots representing regeneration efficiency (d) and shooting frequency (e) of Armenian 999 after 45 days in culture. f Tissue cultures of Armenian 999 after 35 days on induction medium. Letters above each boxplot represent pairwise significance differences (Tukey HSD, α = 0.05) and p values were calculated using a one-way ANOVA (α = 0.05) (color figure online)

Fig. 2

Regeneration rates of Cobham Green and Armenian 999 after introduction of the wild-type and miR396 resistant tomato GRF–GIF fusions. a, b Box plots representing regeneration efficiency (a) and shooting frequency (b) of Cobham Green after 35 days in culture. c Tissue cultures of Cobham Green after 21 days on induction medium. d, e Box plots representing regeneration efficiency (d) of and shooting frequency (e) of Armenian 999 after 45 days in culture. f Tissue cultures of Armenian 999 after 30 days on induction medium. Letters above each boxplot represent pairwise significance differences (TukeyHSD, α = 0.05) and p values were calculated using a one-way ANOVA (α = 0.05) (color figure online)

Fig. 3

Regeneration rates of different lettuce genotypes after transformation with the grape rGRF4–GIF1 or an empty vector control. a–d Boxplots represent regeneration efficiency (RE) and shooting frequencies for each transformation of Cobham Green (a), Armenian 999 (b), Salinas (c), Valmaine (d) after 40 days on induction medium. p values were calculated using a Welch’s t test. e Forty-day-old tissue cultures of each genotype after introduction of an empty vector control (left) and the grape rGRF4–GIF1 fusion (right) (color figure online)

Fig. 4

Summary of the co-transformation experiment in Cobham Green using pLsUBI:dsRED:tLsUBI + Grape rGRF4–GIF1 selected on either kanamycin (Kan), BASTA^™ (BASTA) or kanamycin + BASTA^™ (Kan + BASTA) and pLsUBI:dsRED:tLSUBI + empty vector control (pTB005) selected on kanamycin (control). a–d Box plots representing regeneration efficiency (a), transformation efficiency (b), shooting frequency (c), and dsRED expression frequency (d) of each co-transformation. Letters above each boxplot represent pairwise significance differences (TukeyHSD, α = 0.05) and p values were calculated using a one-way ANOVA (α = 0.05) (a, c, d) or Welch’s t test (b). e Differences of regeneration in tissue culture between control (top) and Kan (bottom) co-transformations after 30 days in culture (color figure online)

Fig. 5

Summary of the co-transformation experiment in Armenian 999 using pLsUBI:dsRED:tLsUBI + Grape rGRF4–GIF1 selected on either kanamycin (Kan), BASTA^™ (BASTA) or kanamycin + BASTA^™ (Kan + BASTA) and pLsUBI:dsRED:tLSUBI + empty vector control (pTB005) selected on kanamycin (control). a–d Box plots representing regeneration efficiency (a), transformation efficiency (b), shooting frequency (c), and dsRED expression frequency (d) of each co-transformation. Numbers below the x-axis (d) indicated the total number of shoot regenerated and phenotyped for dsRED expression over all the replications. Letters above each boxplot represent pairwise significance differences (TukeyHSD, α = 0.05) and p values were calculated using a one-way ANOVA (α = 0.05) (a, c, d) or Welch’s t test (b). e Differences of regeneration in tissue culture between control (top) and Kan (bottom) co-transformations after 35 days in culture

Fig. 6

Frequency of regenerated Cobham Green and Armenian 999 shoots from the coTF Kan treatment containing each transgene after co-transformation with the grape rGRF–GIF and a gene of interest reporter, pLsUBI–dsRED–tLsUBI. The number of Cobham Green (left) or Armenian 999 (right) shoots that were PCR positive for selectable marker (kanamycin [nptII] and/or BASTA [bar]) and transgene (dsRED and/or rGRF–GIF). Each color represents the proportion of shoots that showed amplification of each specific gene target/s. The black numbers refer to the number of shoots PCR positive for each condition (color figure online)