Rapid Acquirement of Transgenic Rice Plants Derived from Callus of Mature Embryos Transformed by Agrobacterium Mediation  

Gang Guo1 , Jing Yu2 , Degang Zhao1,3
1. Guizhou Key Laboratory of Agro-Bioengineering, College of Life Sciences, Guizhou University, Guiyang, 550025, P.R. China
2. Guizhou Tongren flue-cured tobacco limited liability company, Tongren, 554300, P.R. China;
3. Guizhou Key Laboratory of Green Pesticide and Agricultural Biological Engineering, Ministry of Education, Guiyang, 550025, P.R. China
Author    Correspondence author
Molecular Plant Breeding, 2011, Vol. 2, No. 2   doi: 10.5376/mpb.2011.02.0002
Received: 13 Jul., 2010    Accepted: 23 Nov., 2010    Published: 05 Jan., 2011
© 2011 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Guo et al., 2011, Rapid acquirement of transgenic rice plants derived from callus of mature embryos transformed by agrobacterium mediation, Molecular Plant Breeding, Vol.2 No.2 (doi: 10.5376/mpb.2011.02.0002)


In this research we transformed calli of two elite germplasms, Taipei 309 and Nipponbare, derived from mature embryos using the construct of pCAMBIA0390 mediated by Agrobacterium tumefaciens strain EHA105. The expression plasmid pCAMBIA0390 was constructed with expressing elements of ubi:bar-Gus and ACT:Bt. Meanwhile We studied the effects of the factors on the regeneration rates of callus tissues. The results showed that the regeneration frequencies of Taipei 309 and Nipponbare increased up to 39% and 44% respectively, under the procedures as following: Callus was cultured 21days in the conditions of, 28℃, 2000 Lx, and 13 h/d illumination period. Infected callus air-dried 20 minutes and then co-cultured callus air-dried 20 minutes, finally, the callus desiccated using sterile filter paper of callus 8 hours. It shows that transformation cycle can shortened from 105 days to 60 days in absences of subculture stage. The GUS expression and PCR analysis indicated that the exogenous Bt gene has been integrated into the rice genome. 

Rice;Mature embryos;Rapid regeneration;Transformation mediated by Agrobacterium

Rice (Oryza Sativa L.) is one of the most important cereal crops. China is the biggest producer in the world. On November 27, 2009, Ministry of Agriculture of China (MOA), aproved and issued bio-safety certificate for two varieties of transgenic Bt rice, Restorer line, Hua Hui 1 and hybrid line, ShanYou-63, that indicated genetically modified rice is going from the lab to the field in China (Clive, 2010).

The early report on the Agrobacterium-mediated transformation with callus of japonica rice cultivar was documented in 1994 and done by Hiei et al. In 2006, Seiichi Toki et al reported that transgenic rice plants were acquired in around 30 days with the procedures of infection of mature embryso of Nipponbare using Agrobacterium after cultured mature embryo of Nipponbare one day in induction medium. And Su Yi et al. (2008) in China was confirmed that T0 rice plants were regenerated in 40 days using the same methods as Seiichi Toki. These researches proved that it is possible and feasible for rapid regeneration of rice mediated by Agrobacterium transformation.

Excellent rice callus was recognized as an important key factors that affects the efficiency of genetic transformation in the procedures of the Agrobacterium-mediated genetic transformation of rice. To improve callus quality, researchers studied lots of effece of the factors on callus regeneration rates focusing on endosperm content, illumination culture period, and drying methods etc. In this studies we developed the transformation procedures with combination of some results of previous studies such as mature embryo soaking, distilled water and removal of endosperm (Lin et al., 1996), light culture (Duan et al., 2005), drying method (Masayoshi et al., 1992), waiving subculture off (Seiichi Toki, 2006), to improve rates of induction and differentiation of rice mature embryo callus transformed by Agrobacterium mediation, and eventually to acquired the transgenic rice plants within the short period.

1 Results and analysis
1.1 Effect of different light culture conditions on the rate of callus differentiation
There were significantly different callus differentiation rates existed in the two tested varieties under light culture and non-light culture (<0.01) (Figure 1), which indicated that light culture has promotive effects on callus growth. We observed that callus of two varieties after three weeks non-light culture were getting dark, few granules appearing, callus surface drying and low rate of callus differentiation (18% and 21%, respectively), whereas under light culture 3 weeks, callus showed health growth in size and color.

Figure 1 Effect of different light culture coditions on the rate of callus differentiation

1.2 Effect of drying process on the rate of callus differentiation
It was reported that japonica rice callus with properly dehydrating treatment could promote rice regeneration (Masayoshi, 1992). In this research, Effects of dehydration treatment following infection and co-culture on rates of callus differentiation were analysed with the fixed dry time. The results were shown in Figure 2. It was significant that the rates of callus differentiation between Taipei 309 and Nipponbare were difference among four treatment groups based on multiple comparesions, A higher rates of callus differentiation came out for dehydration treatments both following post-infection and co-culture(figure 3).

Figure 2 Effect of dehydration treatment on the rate of callus differentiation

Figure 3 Selection of phosphinothricin -resistant callus and plant regeneration

1.3 Transgenic plants detected by histochemical staining and PCR test
GUS histochemical staining of the resistant rice plants were carried out in this research (Figure 4). It is easy to see the blue spots in transgenic leaf and root, whereas nothing in the control. Meanwhile we selected one transformed rice plant from 370 resistant plants for pcr test, a 875 bp length band were amplified from the selected plant, which was consistent with the target band size, whereas no bands appeared in the non-transgenic control plants (Figure 5).

Figure 4 GUS active transgenic plant by histochemical staining

Figure 5 The transgenic plant identified by PCR

2 Discussions
Definitely, the ideal material for rice transformation is immature embryos, but immature embryo is quite limited with harvest season, geographical limit, time-consuming for operations and specific facility etc. The advantages of mature embryos used for transformation are limitless material sources and simple operation procedures. Therefore, the use of mature embryos in rice transformation of rice become more common.

Regarding on issues of distilled water immersion and removal of endosperm processing, Lin et al. (1996) considered that regulation system for dormant and buffer action may exist in the mature rice seed. Therefore, induction expression is low when faced with external high-hormone environment. Removal of the endosperm part of the rice mature seed can broke the system stability of buffering capacity, resulting in absorbing much more growth hormone in the culture medium to promote cellular dedifferentiation into callus. Using single-distilled water immersion might promote cells of the mature seed to transform from the original hibernation status to the active differentiation stage. In addition, the authors believe that the explant sterilized by mercuric mercury method could be certain poisoned to affect the callus induction of explants in the later stage, while single-distilled water soaking and removal of endosperm treatment could alleviate explant toxicity to increase callus induction rate.

Light culture prior to differentiation is one of the methods to improve the rate of callus differentiation (Duan et al., 2001). In this study, subculture stage was removed during genetic transformation due to the reports on the rate of callus differentiation reduction following subculture. Our results confirmed the transformed plants can be acquired in short period even removal of subculture stage. Selecting embryonic callus with bright-yellow color, granular and dry surface for infection and callus culture with green spots will be easy to be differentiation.

Dehydration processes such as starvation and drying treatment will facilitate to callus differentiation (Lu et al., 2008; Masayoshi et al., 1992). The process for dehydration may lead to a callus cell physiological and biochemical changes, which might promote the absorption of the exogenous hormone. Dehydration by using natural drying on the clean bench may be suitable time period within the 30 miinutes based on the judgement of the status of callus, because callus dehydrated very quickly in environment of clean benches. If the drying time be longer, the vast part of the callus will be getting to die. In addition, there are some reports that indicated hypertonic environment in the internal of medium can be effective on rice callus dehydration, for example, increasing the agarose concentration in the medium, or adding a certain concentration of mannitol, sorbitol and so on.

In this study, the selectable marker Bar gene from soil Streptomyces (Streptomyces hygroscopieus), encoding phosphine chloramphenicol acetyltransferase (Phosp-hoinothricin), acetyltransferase was known to inhibit the activity of Gln synthetase in nitrogen metabolism of plant and to destruct photosynthesis of plant, which eventually the plant died dur to toxic ammonia accumulation (Zhao, 1999). while glutamine (Gln) inhibits action mode of acetyltransferase Therefore, glutamine should be removed in screening medium. Screening shoud be initiated in the phase of most of the callus with green spot appearing.

3 Materials and Methods
3.1 Plant Materials and culture medium
Experimental materials, Taipei 309 and Nipponbare, were planted in greenhouse of transgenic plant experimental station in the Guizhou University.

Plant medium were partly modified according to the literature (Lin et al., 2008; Xiang et al., 2004; Yu et al., 2008). Added 36 g/L glucose into the re-suspension, sterilization methods: high pressure and temperature (121℃, 15 minutes) (table 1).

Table 1 The composition of culture medium used in this study

3.2 Agrobacterium strain and plasmid
Monocot expression vector pCAMBIA0390 harboring the T-DNA region containing screening report fusion gene Bar::GUS drived by ubiquitin promoter Ubi1, Bt genes drived by rice actin promoter Actin1 and the terminator sequence nos. L and F were recognition sites loxp and frt of Cre and Flp recombinase, respectively, the sites can be recognised by FLP and the foreign gene canbe deleted on site (Zhao et al., 2008,; Luo et al., 2005). The structure of T-DNA region shown in figure 6.

Figure 6 The T-DNA Region of plasmid pCAMBIA0390

3.3 Explants and acquirement and Callus induction
In the experiment, we employed the mature embryo of Taipei 309 and Nipponbare as callus induction explant. Removed the seed-coat of the mature seeds, and select the seeds with normal color to be sterilized by using 75% alcohol 1min, and following by 0.1% HgCl2 10 min, washing 5 to 6 times by distilled water.

Sterilized explants soaked 8-12 hours in sterile distilled water, then excise the endosperm. the embryoes was planted on callus induction medium. Distilled water and immersed explants volume ratio is 4:1 (50 explants of rice each treatment, a total of 250 treatments, to remove contaminated callus, 200 rice explants were used for callus induction rate statistics analysis, callus induction rate(%)=no. of embryos with callus/no. of inoculated immature embryos×100%).

3.4 Light culture conditions
After buds grown in 28℃ non-light culture for 3-4 days, excise buds completely with a scalpel and inoculate mature embryos on induction medium by using the following procedures: (1)28℃, under 21 days light culture, with 2000 lux, 13 h/d. (2)28℃, 21 days non-light culture (50 explants of rice for each treatment, a total of 250 treatment, to remove contaminated callus, 200 rice explants were used for callus induction rate statistics analysis, callus differentiation rate(%)=callus with bud/total number of callus×100%).

3.5 Starvation and infection treatment of callus
Starvation treatment of callus were done before infection according to the Lu’s method(Lu et al., 2008), after precessing, placed the callus in Agrobacterium suspension (OD600≈0.4) to infect for 15min, then poure infected callus and absorb the excess bacteria using the dry sterile paper.

3.6 Dehydration treatment after infection and co-culture
Callus after infection were treated as following groups: A group: without drying, directly co-cultured, and then differentiation culture. B group: dried after infection. Dried procedure: callus were placed on absorbent paper within the clean benches and dried 20 minutes. Callus were cultured in media in petri-plates with two filter-papers following the method of Hamid Rashid et al., 1996 at 28℃ non-light culture for 3 days and using mannitol solution rinse 4-5 times after co-cultured (mannitol 0.1 mol/L, containing cefotaxime Cef 300 mg/L).

Calluses were treated as follows after co-cultured: C group: drying the callus which without drying after infection. D group: dried the callus with drying after the infection. Drying methods: air-dried for 20 minutes, then placed callus on petri-plates with two filter papers, dried 8 hours under light conditions (50 explants of rice each treatment, a total of 250 treatment, remove contaminated callus, 200 rice explants were used in callus induction rate statistics analysis, callus differentiation rate(%)=callus with bud/total number of callus×100%).

3.7 Callus differentiation and acquisition of resistant plants
The callus after co-culture were transferred on differentiation medium to culture about 7 days differentiation with the light culture condition as 2000 lux, 13 h/d and 26℃. Then start to differentiate approximately 14 days, the resistant seedlings were transferred into the rooting medium. After additional about 14 days, the regeneration rice plants with well-developed root refined and transplanted.

3.8 GUS histochemical detection of Transgenic Rice
GUS histochemical staining was followed with the procedure of Jefferson’s method (1987).

3.9 PCR Detection
Genomic DNA was extracted from the phosphinothricin-resistant rice plant following the guide of TIANGEN's plant genome extraction kit. According UbiI promoter sequence a pair of primers was designed and synthesized, that the upstream primer is sequence: 5'-CATCTCTGTCGCTGCCTCTG-3', and downstream primer sequence is: 3'-CTGAAGTCCAGCTGCCAGAA-5'. PCR reaction volume was 25 μl with upstream and downstream primers each 1.5 μl, 2 μl template DNA, 2.5 μl 10×EX Taq Buffer, 4 μl dNTPs (2.5 mM, Mg2+ plus), 0.15 μl TaKaRa EX Taq DNA polymerase, 11.35 μl ddH2O. PCR amplification program was followed as: 94℃ denaturation for 2 minutes, 94℃ 30s, 55℃ 40s, 72℃ 40s, 35 cycles; 72℃ extension 3 minutes, 4℃ preservation. PCR products was detacted with 1% agarose gel electrophoresis.

3.10 Statistical Analysis
Experimental data were analyzed using statistical software SPSS 13.0 for significance of difference analysis based on LSD method for multiple comparisons.

This Project was jointly supported by Cooperation Projects of Ministry of Science and Technology of China (2007DFA31260), National Science & Technology Pillar Program of Ministry of Science and Technology of China (2007BAD59B06) and Transgenic Special Projects of Guizhou Science and Technology department, China (2004NZO04).

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