Light and Dark Approaches in Agrobacterium-Mediated Transformation in 84K Poplar

High efficiency transformation and strong regeneration ability of plants are prerequisites to the establishment of good plant transgenic system. This paper describes two Agrobacterium-mediated transformation protocols for poplar, they are Light-induced method and dark-induced method by A. tumefaciens to introduce target genes into Populus alba X Populus glandulosa and comparatively analyzed two transgenetic systems. We summarized the advantages and disadvantages of the two methods. However, the dark-induced method had higher transformation efficiency and light-induced method has shorter period. So, a combination of the two methods can help maximize on efficiency and high levels of gene expression in transgenic plants. This study provides a useful foundation for further research toward the development of 84K Poplar.


Introduction
Since Parson et al. (1986) confirmed that the poplar can be genetically transformed in 1986, the genetic engineering of trees with poplar as the recipient has made great progress. With the extensive applications and in-depth studies of poplar tissue culture technology, plant cell dedifferentiation and re-differentiation, tissue and organ morphogenesis, various metabolic regulation mechanism, gene expression and regulation have been served in the production practice from multiple sources (Benyó et al., 2016).
Genetic engineering is a powerful method to change the characteristics of wood, and enhances the biotechnological use of biomass, but the challenge is how to establish a suitable transgenic method. As previously reported, although several transgenic methods for poplar have been developed, these procedures are time-consuming, unstable and the transformation efficiencies are low (Fungaro et al., 1995;Sandhu et al., 2001). Moreover, transgenetic integration may cause unintended alterations of the genome by deletions, insertions, or rearrangement, which is responsible for the pleio-tropic effects (Kuiper et al., 2001;Cellini et al., 2004;Garcí a-Cañas et al., 2010). Agrobacterium tumefaciens-mediated transformation (ATMT) is an efficient transgenetic method developed in recent 20 years (Michielse et al., 2005;Moon et al., 2008;Li et al., 2013). Because ATMT has many advantages, such as simple manipulations, high transformation efficiency and high single-copy rates, it has been employed as a routine procedure for a large number of plant species (Li et al., 2017) including Poplar (Maheshwari and Kovalchuk, 2016). Moreover, the leaf disc transgentic method (Nanjareddy et al., 2016) has been widely used in genetic engineering of plants, for it is simple, efficient and can be applied to plant species that are susceptible to A. tumqfaciens infection and can be regenerated from leaf explants.
The establishment of Populus alba X Populus glandulosa (84K Populus) can not only study many basic theories such as developmental physiology, cell differentiation and morphogenesis but also help improve poplar varieties and speed up the breeding process to promote the rapid propagation and reproduction of seedlings. Here, we report two robust transgentic protocols for Poplar using A. tumefaciens. Compared with other transgentic techniques, the ATMT method has been proved to increase transformation efficiencies and show a greater degree of transgenetic stability. Moreover, the study has shown that combined two transformation methods will perform better than one method.
The establishment of Populus alba X Populus glandulosa (84K Populus) can not only study many basic theories such as developmental physiology, cell differentiation and morphogenesis but also help improve poplar varieties and speed up the breeding process to promote the rapid propagation and reproduction of seedlings. Here, we report two robust transgentic protocols for Poplar using A. tumefaciens. Compared with other transgentic techniques, the ATMT method has been proved to increase transformation efficiencies and show a greater degree of transgenetic stability. Moreover, the study has shown that combined two transformation methods will perform better than one method.

Callus regeneration system
To explore the effects of different concentrations of hormones in the culture medium on the differentiation of callus, in this experiment, we added different concentrations of hormones and different ratio hormones to induce callus. It was shown that in the culture medium supplemented with different concentrations of hormones had a great influence on callus differentiation. Under different induction conditions, morphology and physical properties of callus had certain changes. Some callus were loose and vulnerable, milky white, transparent surface like water with long differentiation time of adventitious bud, low efficiency, and slow growth; some callus were compact , yellow green and white green. Adventitious bud induction was normal. The differentiation time shortened and the effects were same with 3 times of ordinary light. Even if the callus could continue differentiation, the differentiated seedlings were not normal, and some seedlings stopped differentiation and turn red until death. This paper summarizes these laws and provides theoretical guidance for regulating the growth of callus at different concentrations of hormones in the future.
According to the preliminary experiments, the optimal hormone concentrations were in dark-induced method. In the dark culture, after 20±2 days, the edges of the blade wounds gradually folded, and petiole began to expand and turned red. Callus appeared in about 25±2 days and continued to expand. Petiole, leaf tip and leaf back close to the culture medium appeared grain size of yellow callus. The structure was hard at the beginning with most of color being yellow and a few soft white. They then slowly turned green. After replacing callus induction medium with adventitious bud induction medium, adventitious buds started to induce from the callus.
By this protocol, we can produce thousands of transgenic plants. This transformation system further reduces the labor requirements and improves the transformation efficiency, which provides an easier, faster, and more convenient platform for production of transgenic Poplar and functional analysis of key poplar genes.

Direct differentiation and genetic transformation systems
According to the preliminary experiments, the whole process of light induced method was divided into four stages:(a) The shoot-induction selection: The leaf explants were cultured on selection medium with 50 mg/L kanamycin, and 300 mg/L cephalosporin for 3 to 4 weeks after co-cultivation. (b) After 5 weeks, most of the untransformed leaf explants died, and kana-resistant leaf explants were further cultured in the induction medium of adventitious bud. Buds start appearing in clusters.(c) the adventitious buds were thereafter, transferred to the elongation of adventitious buds medium.(d) In 3-4 weeks, roots appear. Let the shoots stay in the 1/2 MS medium for 1-2 more weeks for root system development, and then proceed to acclimatization.

Agrobacterium-mediated comparison of callus pathway and direct differentiation pathway
For establishing a high-throughput poplar transformation system, this paper systematically establishes two Agrobacterium-mediated transgenic poplar systems, more directly differentiated genetic systems and ones with the combination of dedifferentiation and redifferentiation (Figure 1).

Discussion
Although there has been great progress in transgenic system of poplar, especially ATMT, there are still many shortcomings such as low stability and transformation efficiency (Wendt et al., 2011).The objective of the work was to develop an efficient and applicable regeneration and genetic transformation system for Poplar by ATMT. In this paper, we successfully developed two ATMT method of Poplar.
In dark-induced method, the dedifferentiation and differentiation of the cells were separated by different culture conditions. The cells of the explants were returned to the level of meristematic cells after complete dedifferentiation, which easily accepted foreign genes to produce higher transformation efficiency and more transformants. The callus obtained by this method and the proportion of adventitious buds differentiated on this basis was higher. Light-induced method refers to the process of direct differentiation of explants from the adventitious buds without callus induction stage. Because of the short process of inducing callus, the experiment cycle was short and the operation was simple. And since the plants that have not been dedifferentiated directly differentiate into buds, the genetic transformation was relatively stable, but the transformation efficiency was very low. The results in the present study showed that the transformants in dark-induced method showed higher transformation efficiencies while protein expression levels of PCK in light-induced method were higher than that in dark-induced method.
In particular, there is a large difference between the transformation efficiency of the same transformation process in different laboratories. Even in the same laboratory, poor stability of the transformation efficiency limits further development of the poplar large-scale transgenic technology system. The results in the present study showed that the simultaneous use of the two transformation methods, callus transformation system and direct differentiation system can ensure the transformation efficiencies and exogenerous gene expression. This work makes a base for future Poplar improvement by gene manipulation and molecular biological studies In conclusion, dark-induced methods showed high transformation efficiency and regeneration, but time-consuming while light-induced method has higher rate of exogenerous gene expression.

Media preparation
Different concentrations of kinetin (KT) (0.05-1.0 mg/L), 2, 4-dichlorophenoxyacetic acid (2,4-D) (0.2-1.0 mg/L) and Thidiazuron (TDZ) (0.1-2.0 mg/L) used in the dark-induced transgenetic method during the period of callus induction. And different concentrations of 1-Naphthylacetic acid (NAA) and 6-benzylaminopurine (6BA) used in the light-induced transgenetic method during the period of adventitious buds induction. The prepared media were consisted of 30 g/L sucrose and 7 g/L gelrite agar and the pH was adjusted to be within 5.6-5.8 by using 1 mol/L HCl or NaOH after the addition of plant hormones. The media were autoclaved at 121°C under the pressure of 1.06 kg/cm 2 for 15 min prior to use.

Explant sterilization and propagation
The seedlings were initially washed under tap water for 1 h to remove dirt on the surface. Afterwards, surface sterilization procedures for seedlings were carried out by soaking the explants in filtered distilled water followed by 70% (v/v) ethanol for 3 min in a sterile beaker. After that, the explants were soaked in 8% sodium hypochlorite solution added with two drops of Tween 80 (Sigma, USA) for 30 min. The explants were then thoroughly rinsed with sterile distilled water to remove any traces of remaining detergents. The sterilized 84K Poplar were germinated on MS medium supplemented with 3% sucrose and solidified with 0.8% agar. The pH of the medium was adjusted to 5.8 before the addition of agar and the medium was autoclaved at 1 bar for 20 min prior to use.

Agrobacterium preparation and infection
A single colony from Agrobacterium strain was inoculated separately into 10 mL YEB broth amended with 50 mg mg/L kanamycin and 10 mg/L rifampicin. The cultures were incubated for 18 h in an orbital shaker with 180 rpm at 28°C . The Agrobacterium cultures were multiplied by sub-culturing 0.1 volume of bacterial culture into 200 mL YEB broth containing the above-mentioned antibiotics and incubated at 28°C in an orbital shaker set at 180 rpm until the bacterial broth reached an optical density (OD600) of 0.8. The bacterial cells were harvested by centrifuging at 5000 rpm for 10 min. The resulted pellets were suspended in 500 ml of 1/3 MS medium containing 3% sucrose supplemented with acetosyringone (200 μM) and the OD 600 was adjusted to 0.6.

Agrobacterium-mediated callus regeneration system
The callus induction system is also known as the "dark induced method" and "two steps to budding method". The explants were induced to form callus of about 1~2 cm in diameter under dark conditions (dedifferentiation) for about 1 to 2 months and then transferred into the light for a period of 1 to 2 months to obtain regenerated plants. This method can be used in a wide range of explants, and it has been proved by experiments that most plant tissues and organs can produce callus, which can be regenerated by in vitro culture. The Agrobacterium-mediated dark induction method was used to induce the callus after the explants were infected by A. tumefaciens for 1 ~ 2 months then move to light to induce callus formation. Bud induction was divided into 5 stages: co-culture; callus induction under darkness; Light induced adventitious bud of callus; adventitious buds, and rooting (Nahampun et al., 2016).

Agrobacterium-mediated direct differentiation
The direct differentiation system is also known as "light induced method" or "one step budding method". This refers to generation of adventitious buds of explants without callus formation (Pang et al., 2013). A. tumefaciens-mediated light induction was a transgenetic method for the differentiation of adventitious buds directly after exposure to light. This genetic transformation was divided into four stages: co-culture; adventitious bud induction; adventitious buds and rooting.
Authors' contributions LNW conceived the experiments; LNW and KW performed the experiments; LNW wrote the manuscript.