An Efficient Plant Regeneration and Transformation System of Ma Bamboo (Dendrocalamus latiflorus Munro) Started from Young Shoot as Explant

Abstract

Genetic engineering technology has been successfully used in many plant species, but is limited in woody plants, especially in bamboos. Ma bamboo (Dendrocalamus latiflorus Munro) is one of the most important bamboo species in Asia, and its genetic improvement was largely restricted by the lack of an efficient regeneration and transformation method. Here we reported a plantlet regeneration and Agrobacterium-mediated transformation protocol by using Ma bamboo young shoots as explants. Under our optimized conditions, embryogenic calluses were successfully induced from the excised young shoots on callus induction medium and rapidly grew on callus multiplication medium. Shoots and roots were regenerated on shoot induction medium and root induction medium, respectively, with high efficiency. An Agrobacterium-mediated genetic transformation protocol of Ma bamboo was established, verified by PCR and GUS staining. Furthermore, the maize Lc gene under the control of the ubiquitin promoter was successfully introduced into Ma bamboo genome and generated an anthocyanin over-accumulation phenotype. Our methods established here will facilitate the basic research as well as genetic breeding of this important bamboo species. Key achievements: A stable and high efficiency regeneration and Agrobacterium-mediated transformation protocol for Ma bamboo from vegetative organ is established.

Keywords: Ma bamboo; regeneration; shoots; transformation.

FIGURE 1

Regeneration of plantlet from young shoots of Ma bamboo. (A) Callus was induced from the shoots of Ma bamboo. (B) The callus grown on the callus multiplication medium. (C) Callus turns green on shoot induction medium. (D) The induced shoots on shoot induction medium. (E) The adventurous shoots grow in cluster. (F) Root was induced on root induction medium.

FIGURE 2

Growth status of regenerated plants after transplanted into soil. (A) The growth status of the regenerated plantlet 1-month after being transferred to the soil and grown in greenhouse. (B) The growth status of 3-month old plant. (C) One-year old plant grown in the green house. (D) Multiple new clusters of shoots were growing out of the soil. Bar = 2 cm.

FIGURE 3

Agrobacterium-mediated transformation of Ma bamboo. (A) Schematic representation of key elements of T-DNA in the binary vector pCAMBIA1301. LB, left border; T35S, terminator of CaMV35S; HPTII, hygromycin phosphotransferase gene; p35S, promoter of CaMV35S; MCS, multiple cloning sites; GUS, uiA β-glucuronidase gene; Tnos, NOS terminator; RB, right border. (B–D) Various factors that affect the frequencies of hygromycin-resistant shoot induction of Ma bamboo; including Agrobacterium strain B, the density of Agrobacterium C, and the duration of co-cultivation D. Data were collected around 7 months after infection. Results were presented as means and standard errors from at least three independent experiments. At least 100 calluses were used for each experiment. ^∗∗ indicates significant differences in comparison to control at p < 0.01 (Student’s t-test). (E) Key steps for Agrobacterium-mediated transformation of bamboo callus. (i) Callus status before Agrobacterium infection. (ii) New calli emerged from the transformed callus grown on the hygromycin containing medium. (iii) Morphology of 2-month old hygromycin-resistant calli. (iv) Status of the hygromycin-resistant calli that were used for shoot induction. (v) Green callus on shoot induction medium containing hygromycin. (vi) Regenerated shoots from hygromycin-resistant callus. (vii) The induced root on root induction medium. (viii) Regenerated plantlets grown in the soil. The enlarged pictures of represented callus were shown inside the panels. (F) PCR verification of the putative transgenic lines using 35S-forward and HPTII-reverse primers, which resulted in a 1044 bp product. (G) PCR verification of the putative transgenic lines using 35S-forward and GUS-reverse primers, which produced 1866 bp fragment. (H) GUS staining of the putative transformed callus (ii) and shoot (iv), with the uninfected callus or shoots (i,iii) as the negative control.

FIGURE 4

Overexpression of the maize Lc gene in Ma bamboo leads to the increased accumulation of anthocyanin. (A) Schematic representation of the putative inserted T-DNA region. The expression of maize Lc gene was controlled by ubiquitin promoter (pUBI), and HPTII gene driven by 35S promoter confers plant with hygromycin resistance. (B) Phenotypes of Lc-gene heterogonous expression lines during tissue culture. Purple pigment accumulation in the callus (left), regenerated shoot (middle and right panels) in the transgenic line (lower panel) compared with control (up panel). (C) Phenotype of Lc-gene heterogonous expression lines grown in the soil. Represented transgenic lines (ii) exhibit purple color compared with control (i). The enlarged picture of stem (iv), leaf blade (vi), and leaf (viii) and their corresponding controls (i,iii,v,vii, respectively) were shown. (D) PCR analysis of the transgenic lines. The expected 1044 bp product was detected with genomic DNA from putative transgenic bamboo as template. (E) qRT-PCR analysis of Lc-gene heterogonous expression lines. Total RNA was extracted from control and three represented transgenic lines (Lc-ox1, Lc-ox2, and Lc-ox3), and the qRT-PCR was performed using Lc gene specific primers. The Actin gene of Ma bamboo was used as internal control. The data shown represent mean values and standard errors obtained from at least two independent experiments with four technical repeats. ^∗∗ indicates significant differences in comparison to control at p < 0.01 (Student’s t-test). (F) Quantitative measurement of anthocyanins in 1-month-old seedlings of the control and three represented Lc-gene heterogonous expression lines. The data was shown as means and SD of three replicates. ^∗∗ indicates significant differences in comparison to control at p < 0.01 (Student’s t-test).