Plant Cell Tiss Organ Cult DOI 10.1007/s11240-014-0497-1
RESEARCH NOTE
Test pigments in media for tissue culture and transformation Ying Liu • Yang Li • Jianfeng Liu • Xia Li Lifang Sun • Jianhui Xiao • Chao Li • Ze Meng • Ying Ren • Xianggan Li
•
Received: 19 December 2013 / Accepted: 21 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Accurate uses of key components and right media during tissue cultures are vital for a particular process and repeatable outcome in plant transformation. If additional components, as indicators, can be added into media, it could avoid some of the issues, such as adding a wrong component or misrecognition of a right medium. We tested to add a specific pigment into a set of media sharing a key common element, such as a selection agent, so that all media containing the same selection agent appeared in a certain coloration to provide a convenient marker for all people involved in the process of transformation. The effect of four color pigments on tissue cultures of corn, rice, and soybean was evaluated in this study. The results show pigment brilliant blue, Pyla-Cert green, and mixture purple neither impacted soybean cotyledon growth nor callus formation and shoot regenerations of corn and rice. In soybean hair root transformation, addition of pigment blue, green, erythrosine red and mixture purple into the selection media did not reduce its transformation frequency and copy number. However, erythrosine red resulted in lower callus formation rates and low regeneration rates among rice and corn. Further, fresh weight and dry weight of soybean cultures were increased on the media containing erythrosine red. Therefore, we recommend using pigment brilliant blue, Pyla-Cert green and mixture purple as
Y. Liu Y. Li J. Liu X. Li L. Sun J. Xiao C. Li Z. Meng Y. Ren X. Li Syngenta Biotechnology China Co. Ltd., No. 25 Life Science Park Road, Changping District, Beijing 102206, People’s Republic of China X. Li (&) Syngenta Biotechnology Incorporated, 3054 E. Cornwallis Rd, Durham, NC 27709, USA e-mail:
[email protected]
indicators for selection agents in the transformation process of maize, soybean, and rice to prevent misuse of wrong components or wrong media. Keywords Pigments Media Tissue culture Transformation Abbreviations TF Transformation frequency GFP Green fluorescent protein IEs Immature embryos CIM Callus induction medium
Plant tissue culture techniques have become vitally important for pursuing a wide range of fundamental and applied research and development. It encompasses a variety of procedures used for specific purposes, such as artificial seeds production, in vitro plant regeneration, and genetic transformation. All of these procedures have depended on the use of suitable nutrient media (Li and Murashige 1977). Plant tissue culture media contain correct proportion of different components to satisfy nutritional as well as the physiological support of many plant cells (Gamborg et al. 1976). Accurate uses of key elements, such as selection agents like glyphosate and glufosinate during plant transformation process, are vital for a particular process and repeatable outcomes in plant genetic engineering. There are multiple choices of different selection agents for different selection markers with a crop. The combinations of selection agents for a set of crops make it challenging to track different media. If additional components, as indicators, can be added into media, it could avoid some of the issues that have occurred during medium
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preparations and plant transformations, such as adding a wrong component or misrecognition of a right medium. We propose to add one specific pigment into the stock solution of a selection agent so that all media containing the same selection agent, such as selection media, shoot regeneration media, or root regeneration media, will appear in a certain coloration to provide a convenient marker for all people involved in the process of transformation. Here, we report our effort to identify pigments or mixtures of pigments to code media with different pigments. Brilliant blue is a valuable nontoxic dye tracer with distinct visibility, and is relatively stable (Flury and Fluhler 1994). Although it is known that brilliant blue is the neutral or anionic property and has large water solubility, it was still suspected this dye may be absorbed onto substrate due to possibly formation of ion pairs with calcium ions (Flury and Fluhler 1995). In searching for a red dye to be used as a component marker, we decide to test erythrosine which is provided by PhytoTechnology as media component. The supplier reported that no negative impact was observed on tissue culture, although it is known erythrosine is a potent inhibitor of the plant plasma membrane Ca-ATPase (Williams et al. 1990). The inhibition is concentration dependent based on assay results from red beet plasma membrane vesicles. Furthermore, erythrosine alkalized growth medium during assaying modulation of plasma membrane H?ATPase activity (Schaller and Oecking 1999). Additionally, it was reported that erythrosine may be a cause of atopic disease (Uysal and Aral 1998), and carcinogenic nature. It also may lead to tumors, although it is a major food color additive with processed food (Tabara et al. 2011). Besides, erythrosine, phloxine, and rose bengal can be adsorbed to the surface of charred cellulose granules while blur brilliant blue cannot (Tabara et al. 2011). As one component of the purple pigment, amaranth is a wellknown azo dye, which is widely used for coloring textile materials, paper, wood, leather and foodstuff for several years. But for the last few years the carcinogenicity and other toxic effects of the dye compelled authorities towards its legal prohibition in many countries (Gupta et al. 2012). Food color green, as synonyms as Pyla-Cert Green MX415, contains FD&C Green #3 & FD&C Yellow #5 soluble in water with odourless. It has been tested with Boston fern and hosta to ensure there are no phytotoxic impurities that would cause morphological aberrations to plants (Seckingger 2012). With the conflicted information in mind, we decided to conduct a systematic verification of different pigments in our tissue culture system. Therefore, the aim of this study was to evaluate impacts of the four pigments: brilliant blue, Pyla-Cert green, erythrosine red, and mixture purple on tissue culture and transformation of soybean, maize, and rice so that we can decide which color pigment can be
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added as an indicator for a particular selection agent. Pigment brilliant blue, Pyla-Cert green, and erythrosine red were purchased from PhytoTechnology Laboratories with corresponding catalogue numbers as F322, F320, and F323. Mixture purple (a mixture of amaranth and FD&C blue #1) was ordered from a local company named as Teng Zhou Fei Yi biotechnology Co., Ltd. All pigments were dissolved into water. Then, these stock solutions were filter sterilized using 0.2 lm membrane filter and added into media after autoclaved and before pouring. Final concentrations were 4, 4, 4, and 2 mg/L for brilliant blue, PylaCert green, erythrosine red, and mixture purple respectively. These concentrations are high enough to trigger our visual detection. The test control (CK) was with the same medium without any pigment (pigment free). Actual views of five medium plates with various colorations are shown as Fig. 1a. It provides clearly visual differences among four pigments plus media. We observed that Pigment red faded during tissue culture process (Fig. 1b). We examined these pigments with the media associated with tissue cultures and transformations of soybean, maize, and rice. For in vitro regeneration and transformation process of soybean variety of Jack and Williams, these pigments were examined with the two important steps as the recovery step after Agrobacterium inoculation and the shoot regeneration prior to the shoot elongation (Khan 2004). First, cotyledons alone were tested on five different media in the experiment consisting of 600 explants with three replicates for Jack. These cotyledons were isolated and inserted into the recovery medium (Khan 2004). Then, cultures were maintained at 24–26 °C with 60–80 lEm-2 S-1 light intensity and 16/8 h light/dark photoperiod for 10 days. The fresh weight and dry weight of cotyledons cultured in the recovery medium containing pigment blue, green and purple were not altered in comparison to the pigment-free control (Table 1). Further, both fresh weight and dry weight of cotyledons with Pigment red were increased statistically significantly than that of other pigments and the control (Table 1) when Fisher’s least significant difference (LSD) was performed following Williams and Abdi (2010) with differences described as being significant where P B 0.05, and not significant where P [ 0.05. It was observed that cotyledons in contact with media containing Pigment red became dark, while other cotyledons did not appear such change in color (Fig. 1c). The second soybean experiment was to test the impact of pigments plus media on the regeneration of soybean cultivar Williams 82. Soybean meristems with one cotyledon and epicotyl were wounded, infected, and co-cultured for 5 days, and subsequently transferred to the recovery medium and the regeneration media for 21 days (Khan 2004). The fresh weight of cotyledons cultured in media containing red pigment significantly increased
Plant Cell Tiss Organ Cult
a
c
b2
b1
CK
Blue
Green
d1
Red
Fig. 1 Appearance of colored medium plates, cotyledons, and hairy roots with GFP. a Colorations of solid media plates with different pigments. Each plate from left to right containing soybean elongation media without pigment, with pigment blue, green, red, and purple respectively in Petri dishes (9 cm diameter); same sizes of petri dishes used throughout of this study; b soybean elongation plates with pigment red under darkness (b1) and light (b2) after 3 days,
d2
indicating red coloration faded under light. c Appearance of cotyledons cultured on recovery media without any pigment (CK), with pigment blue, green, red, and purple respectively; d hairy roots with GFP expression after transformation selection for 2 weeks, indicating there isn’t any interference between the GFP and pigment red (d1) and pigment purple (d2) as well as pigment green and pigment purple (photos not shown)
Table 1 Weight of cotyledons after cultured on the recovery medium
A B
Treatments
No. of cotyledons
Fresh weight (g)/10 cotyledons*
Dry weight (g)/10 cotyledons*
Recovery—CK
120
2.81a
0.945a
120
a
0.919a
a
Recovery—blue
2.93
C D
Recovery—green Recovery—red
120 120
2.72 4.47b
0.934a 1.08b
E
Recovery—purple
100
2.76a
0.928a
* Data represent means of three replicates with different superscript letters (a vs. b) within the same column indicating significant difference at a = 0.05 by using Fisher’s LSD test
Table 2 Weight of cotyledons after cultured on the regeneration medium
A
Treatments
No. of cotyledons
Fresh weight (g)/5 cotyledons*
Dry weight (g)/5 cotyledons*
Regeneration—CK
48
1.9a
0.17a
a
B
Regeneration—blue
54
2.1
0.21b
C
Regeneration—green
49
2.1a
0.21b
56
b
0.27c
a
0.21b
D E
Regeneration—red Regeneration-purple
43
2.4
2.0
* Data represent means of three replicates with different superscript letters (a vs. b or b vs. c) in the same column indicating significant difference at a = 0.05 by using Fisher’s LSD test
(Table 2, Fisher’s LSD). Other pigments did not generate such impact. Also, the dry weight of cotyledons with the four medium treatments was significantly higher than the control, especially the red pigment (Fisher’s LSD). In addition, no difference in fresh weight was observed with regenerated shoots (excluded cotyledons) among control and media treatments (data not shown).
Subsequently we tested four pigments on soybean hair root transformation. Seeds sterilization, seed germination, Agrobacterium streaking, and suspending followed the protocol published by our colleague (Khan 2004). Then the cotyledons were placed onto co-cultivation media (Cao et al. 2009; Gurel et al. 2009). After 4 days co-culture at 24–28 °C, explants were incubated for about 2 weeks at 24–28 °C. Subsequently,
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Plant Cell Tiss Organ Cult Table 3 Maize callus formation rates and shoot regeneration rates Treatments
A
Callus induction—CK
Callus induction stage
Shoot regeneration stage
No. of embryos
Callus formation rates (%)*
No. of calli
Regeneration rate (%)*
140
78.6a
104
78.9a
a
B
Callus induction—blue
140
76.4
104
86.5a
C
Callus induction—green
140
77.1a
96
81.3a
140
b
48
33.3b
a
76
84.2a
D E
Callus induction—red Callus induction—purple
140
40.8
70.0
* Data represent means of three replicates with different superscript letters (a vs. b) in the same column indicating significant difference at a = 0.05 by using Fisher’s LSD test
the induced roots were observed under fluorescence microscope. Number of transformed cotyledons with GFP positive hair roots (Kereszt et al. 2007) was counted to calculate transformation frequency percentage against the total number of cotyledons infected. Pigment green, blue, red and purple did not generate any negative impact on soybean hairy root tissue culture, transformation frequencies, and number of single copy events produced (data not shown). Also, additions of four pigments onto soybean hair root transformation media did not interfere with the observation of GFP intensity and fluorescent pattern (Fig. 1d). Since callus induction rates and shoot regeneration rates are crucial for maize transformation we investigated the effect of four pigment treatments on these two stages. Immature embryos (IEs) of an Syngenta inbreed corn line were isolated and placed on callus induction medium (CIM) and shoot regeneration media (Li et al. 2003) to compare whether callus induction rates and shoot regeneration rates were maintained with pigment plus media and pigment free medium. The IEs with sizes ranged between 0.7 and 1.2 mm were isolated from ears produced in greenhouse (Li et al. 2003). Then, IEs were placed on CIM containing different pigments to induce calli by following the same protocol published from our lab (Li et al. 2003). The number of IEs, which formed calli, was counted after 4 weeks at 28 °C under darkness. Then, good looking calli were selected and transferred to the regeneration media (Li et al. 2003), containing different pigments as well. The number of the callus lines which regenerated shoots was counted after 28 days of cultures, with 14 days under dark and 14 days under light. The control was the CIM without any pigment. The corn experiments were repeated three times with total 140 IEs for each treatment. No difference was observed in callus formation rates among the control, pigment green, blue and purple (Table 3). However, as showed in Fig. 2 and Table 3, pigment red produced smaller calli and showed significant lower callus formation rate (Fisher’s LSD). In addition, calli regenerated normal sizes of shoots on regeneration media containing pigment green, blue, and purple (Fig. 2). But the media containing
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pigment red produced less than half of shoots in comparison with the pigment free control (Table 3), indicating that the red pigment exerted a negative impact on both callus formation and shoot regeneration of maize. Both callus induction and regeneration are also crucial for rice transformation (Duan et al. 2012). We further investigated the pigments impact on the two stages of Indica rice transformation with variety IR58025B. Mature seeds embryos were separated and placed on CIM (Duan et al. 2012) with different pigments. The number of embryos that produced callus was counted after 25 days culture under darkness. Callus derived from mature seeds was then placed on regeneration media (Duan et al. 2012) with different pigments for 28 days under darkness. The number of calli that generated seedlings was then counted. The control was media without any pigment. Our rice experiment indicated pigment blue, green and purple have no impact on callus induction rates and regeneration rates (data not shown), which is very similar to that of maize. Whereas pigment red produced notable negative response both on callus induction and plant regeneration (Fig. 2, far right panel, Fig. 3). Therefore, we conclude that red pigment should not be used in tissue culture systems of either rice or maize for our purpose. According to the product information sheet provided by PhytoTechnology Laboratories, the red pigment was tested in tissue culture with Boston fern and tobacco callus. The green and blue pigments were tested with Boston fern and hosta. Test concentrations by the supplier were 100 mg/L. We used 2–4 mg/L pigments in media and did not test even higher concentrations since the lower concentration generated enough color difference to be recognizable. However, our study showed that the red pigment generated impact on tissue culture of all three crops (soybean, maize and rice) such as significantly increase in weight of soybean cotyledon and negative impact on maize and rice callus formation and shoot regeneration. Also, the attachment portion of corn and rice calli to the media turned red. Similarly, corn callus that touched blue pigment media turned blue as well.
Plant Cell Tiss Organ Cult
Fig. 2 Maize callus formation (a, b, c, d, e), maize shoot regeneration (f, g, h, i, j), and rice callus formation (k, l, m, n, o) on media without any pigments (top row, a, f, k), and with Pigment blue (the 2nd row, b, g, i), pigment green (the 3rd row, c, h, m), pigment red (the 4th row, d, i, n) and pigment purple (the 5th bottom row, e, j, o)
respectively. Petri dishes with 9 cm diameter were used. For maize callus formation and regeneration, sixteen immature embryos at 4 9 4 arrays plated per plate and, after callus induction, good callus lines transferred onto the shoot regeneration media from the corresponding CIM media
In addition, we found that the media with the red pigment (erythrosine) started fading after 2 days storage under light condition. An example of soybean elongation medium
with the red pigment is shown in Fig. 1b to indicate the color fading after 3 days of light exposure. Media with purple started fading after 2 weeks under light. Media with
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Plant Cell Tiss Organ Cult
a
b
c
d
e
Fig. 3 Rice shoot regenerations on the media containing different pigments. Five plates are shown in a row for the same treatment. Photos from top to bottom row represent the pigment free control (a), pigment blue (b), green (c), red (d), and purple (e). Petri dishes with 9 cm diameter used through this study
blue and green did not fade within 1 month. But, under dark storage, media with all the four pigments maintained as expected coloration. In a summary, pigment brilliant blue, Pyla-Cert green, and mixture purple neither impacted soybean cotyledon growth in the recovery stage and soybean hairy roots transformation nor callus formation and shoot regenerations of corn and rice. However, erythrosine red resulted in lower callus formation rates and low regeneration rates among both rice and corn. Further, fresh weight and dry weight of soybean cotyledons in both recovery stage and shoot regeneration stage were increased on the media containing erythrosine red. Therefore, we recommend to use pigment brilliant blue, Pyla-Cert green and mixture purple as indicators for selection agents in the transformation process of maize, soybean, and rice to prevent misuse of wrong components or wrong media. Acknowledgments We thank our colleagues from the Maize Transformation and Media teams in Research Triangle Park, North Carolina, USA, for sharing their information including usage of Pigment blue, concentration and photos with us. We are indebted to Shikui Song and Huaping Gui for their advice in experimental design.
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