Overexpression of the AtSHI gene in poinsettia, Euphorbia pulcherrima, results in compact plants

Abstract

Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI) gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21-52%) and internode lengths (31-49%) were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1). The indole-3-acetic acid (IAA) content appeared lower (11-31% reduction) in the transgenic lines compared to the wild type (WT) controls, with the lowest level (31% reduction) in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced.

Figure 1. Gene construct: pKanIntron-35S-SHI used for Agrobacterium-mediated transformation of poinsettia.

Figure 2. PCR analysis of poinsettia transformed with the AtSHI gene (genomic DNA was extracted from leaves).

Lane 1∶100 bp marker, lane 2: plasmid control, lane 3: blank, lanes 4–11: eight plants from independent transgenic lines 1 (TL1) (individuals 1–8), lane 12: TL2, lane 13: TL3 and lane 14: WT control line. The arrow indicates the 500 bp band.

Figure 3. Southern blot analysis of PCR positive transgenic poinsettia lines overexpressing the AtSHI gene.

HindIII-digested total genomic DNAs were hybridized with a AtSHI probe (the 500 bp PCR product). Lane 1: positive control, the 500 bp PCR product; lane 2–4: TL1–3, TL1–4, TL1–6 of the transgenic line one (TL1); lane 5: TL2; lane 6: TL3, lane 7: WT control.

Figure 4. Quantitative real-time PCR analysis of AtSHI transgene in different transgenic lines of poinsettia.

Two microgram total RNAs from transgenic poinsettia lines and the endogenous control, α-tubulin gene were used for synthesize cDNAs prior the real-time qPCR analysis. Values are means of three technical replications except TL2 (n = 2). Data were analyzed using the 2^−△△C _T method and represented as log₁₀ values. Vertical bars represent the ± SE (standard error).

Figure 5. Height comparison among the different transgenic lines (TL) of AtSHI overexpressing poinsettia and untransformed control plants grown under short day (10 h) (A) and long day conditions (16 h) (B).

Vertical bars represent the ± SE (standard error), n = 11–12 and 6–12 in A and B, respectively.

Figure 6. Transgenic AtSHI overexpressing poinsettia plants (A), petioles (B) and leaves (C).

In each figure: from the left different transgenic lines; TL1, TL2, TL3 and non-transformed control plants are shown. The plants were grown at 21±2°C, a 10 h photoperiod at an irradiance of 100±20 µmol m⁻² s⁻¹.

Figure 7. Effects of AtSHI overexpression on internode length (A), total internode number (sum of bracts and leaves) (B), bract number (C) and bract area (D) of different transgenic lines and control plants of poinsettia.

Plants were grown under short day conditions of a 10 h photoperiod, n = 11–12. Mean values with different letters are significantly different based on ANOVA followed by a Tukey’s test at p≤0.05. Vertical bars represent the ± SE (standard error).