论文部分内容阅读
Abstract [Objectives] This study was conducted to improve the genetic transformation rate of maize. [Methods] The seeds of maize "Zheng 58" as the experimental material were germinated and treated under freezing temperature of -18 ℃ for 30 and 35 min. The EGFP gene was transformed into the growth points of these seed buds by Agrobacterium tumefaciens EHA105. The transformation effect was determined by fluorescence protein detection on transformed buds. [Results] After a certain period of treatment at -18 ℃ following germination and transformation of maize bud growth points with A. tumefaciens， although the survival rate of maize buds was reduced， the percentage of transformed cells significantly increased. [Conclusions] Low temperature treatment can improve the transformation efficiency of A. tumefaciens to the growth point of maize bud.
Key words Maize buds； Low temperature treatment； Agrobacterium； Transgenic
Maize is a food crop distributed the widest in the world， as well as one of the most important crops in China[1]. The research on genetic transformation of maize is of great significance. At present， Agrobacterium-mediated method is the most popular genetic transformation method for plant with the characteristics of low cost， low exogenous gene copy number， and stable genetic expression of exogenous gene， which is more suitable for large-scale transgenic technical system[1]. Phenolic substances including AS and HO-AS as main inducers are mainly synthesized in plant cell wall of dicotyledon， and could not be found in monocotyledon usually， and therefore， ratification addition of AS during transformation process could promote A. tumefaciens to infect monocotyledon[2-3]. After taking these measures， satisfactory transformation result still could not be obtained when performing genetic transformation of monocotyledon plants such as maize through Agrobacterium. It indicates that except these factors， there are also unknown factors influencing transformation. The transformation of plant cells with Agrobacterium should be performed through cytomembrane which is a protective screen for cells， and opening this passage by a proper method is beneficial to the improvement of transformation efficiency. In this study， the cell membrane permeability at the growth points of maize buds was improved through low temperature treatment on maize buds， thereby promoting the infection of the growth points of maize buds with A. tumefaciens， and then the transformed buds were cultured under certain condition to allow recovery of the slightly-injured cell membrane to normal state and differentiation and budding of seeds to normal plants， so as to achieve the effect of improving transformation. Materials and Methods
Experimental materials
Receptor material： Mature seeds of maize selfing line "Zheng 58" were subjected to germination treatment and freezing treatment， and the growing points were used as the transformation receptor. The used plant transgenic vector PEGAD contained enhanced green fluorescent protein （EGFP）， and the Agrobacterium strain was EHA105.
Experimental method
Germination of maize seeds： Intact mature maize seeds free of mildewing were cleaned and sterilized with 0.1% HgCl2 for 12 min. The seeds were flushed with sterile water for five times， and the sterilized seeds were placed in a culture dish with a diameter of 9 cm subjected to high-temperature sterilization laid with two layers of filter. In each culture dish， 20 maize seeds were cultured in 8 ml of 210 μM acetosyringone water solution at 25 ℃ for 3 d under dark condition.
Preparation of Agrobacterium-mediated transformation liquid： Single colony was picked and inoculated to 50 ml of LB liquid medium containing 50 mg/L kanamycin sulfate +40 mg/L rifampin. The bacterial liquid was cultured at 28 ℃ under 210 r/min of oscillation until the OD600 of the liquid was 0.5. Centrifugation was performed at 4 000 r/min at 5 ℃ for 5 min， and the supernatant was discarded. Into the precipitate， 2 ml of hypertonic infection base liquid was added， obtaining the Agrobacterium-mediated transformation liquid after shaking. The hypertonic infection base liquid was salt of 1/10 MS medium + 68.5 g/L sucrose +30 g/L glucose +400 mg/L MES +100 μmol/L acetosyringone + 100 mg/L pluronic F68， with pH in the range of 5.4-5.6.
Transformation treatment and detection： When the maize seeds were cultured under dark condition for 3 d， germinated maize seeds were subjected to low temperature treatment at -18 ℃ for 30 and 35 min， respectively， as treatments 1 and 2， respectively， and the treatment free of low temperature treatment served as control （CK）. The obtained growing points of maize buds were air-dried for 30 min and then subjected to transformation treatment. After transformation， each culture dish was placed with 6-10 seeds， which were air-dried for 30 min again. Into each culture dish， 1 ml of sterile water was added， and the seeds were then cultured in the dark at 23-25 ℃ for 4 d. Then， 2 ml of water was added into each culture dish， and the seeds were cultured with illumination for 1 d. Finally， green fluorescent protein detection was performed to transformed buds with a fluorescent stereoscopic microscope， and the transformation effect was determined according to whether the green fluorescent protein is expressed as well as the expression strength of green fluorescent protein. Results and Analysis
Effect of low temperature treatment on rate of positive buds after transformation
The results of green fluorescent protein detection （Table 1） on transformed maize seeds showed that after 30 and 35 min of low temperature treatment at -18 ℃ and transformation with A. tumefaciens， the rates of positive buds were both lower than the CK. This is because that maize buds were injured after low temperature treatment， resulting in reduced emergence rate. After 35 min of low temperature treatment， only one bud survived， and the emergence rate after transformation was thus affected. Therefore， longer time of low temperature treatment reduced the rate of positive buds after transformation.
Effect of low temperature treatment on transformation degree
It could be seen from the expression condition of green fluorescent protein in transformed maize buds subjected to low temperature treatment that the expression level of green fluorescent protein after low temperature treatment was higher than that of the CK （Fig. 2）， and the green fluorescent protein was expressed in deeper cells of maize buds， while the CK only expressed the fluorescent protein on the surfaces of buds at a lower level. It indicates that low temperature treatment is more beneficial to transformation of more maize bud cells， and except surface cells， deeper cells could be also transformed， which is beneficial to the formation of transgenic chimeric plants with more positive cells， which could improve the probability of obtaining transgenic seeds.
Discussion
The maize plants obtained through Agrobacterium transformation using growing points of maize seed buds as transformation receptor and direct regeneration without tissue culture are always transgenic chimeras， the higher the proportion of transformed cells in chimeras， the more likely they will get transgenic offspring， and more transgenic seeds would be obtained afterwards. The results of this study showed that through low temperature treatment on maize seed buds as receptor， the growing points of maize seed buds were injured to certain degree， the permeability of cytomembrane was improved， which facilitated infection and transformation with A. tumefaciens， and more transformed cells were obtained. An optimal transformation effect could be achieved by finding the optimal balance point through control of time and temperature of the low temperature treatment. Under this condition， not only the growing points of maize seed buds are injured to a certain degree， resulting in improved cell membrane permeability， but also self-healing is allowed at the co-culture stage， i.e.， no fatal effect is caused to the growth of maize seeds. Therefore， the proportion of transformed cells is improved， which is beneficial to the acquisition of offspring materials， and the purpose of improving transformation efficiency is achieved. Maize was used as the experimental material in this study， if other crops are used as receptor materials， whether an ideal transformation effect could be obtained through similar treatment still needs further study.
References
[1] LIU YJ， JIA ZW， LIU Y， et al. Establishment and Application of Large-Scale Transformation Systems for Maize[J]. Scientia Agricultura Sinica， 2014， 47（21）： 4172-4182.
[2] LIU ZX， MA XQ， HE YY， et al. Improvement of the assistant method in Agrobacterium-mediated transformation[J]. Journal of Fudan University： Science Edition， 1999， 38（5）： 601-604.
[3] ZHANG LJ， CHEN LM， DU JZ， et al. Establishment and optimization of puna chicory genetic transformation system with Agrobacterium-mediated method[J]. Acta Agrectir Sinica， 2011， 19（6）： 1042-1050.
Key words Maize buds； Low temperature treatment； Agrobacterium； Transgenic
Maize is a food crop distributed the widest in the world， as well as one of the most important crops in China[1]. The research on genetic transformation of maize is of great significance. At present， Agrobacterium-mediated method is the most popular genetic transformation method for plant with the characteristics of low cost， low exogenous gene copy number， and stable genetic expression of exogenous gene， which is more suitable for large-scale transgenic technical system[1]. Phenolic substances including AS and HO-AS as main inducers are mainly synthesized in plant cell wall of dicotyledon， and could not be found in monocotyledon usually， and therefore， ratification addition of AS during transformation process could promote A. tumefaciens to infect monocotyledon[2-3]. After taking these measures， satisfactory transformation result still could not be obtained when performing genetic transformation of monocotyledon plants such as maize through Agrobacterium. It indicates that except these factors， there are also unknown factors influencing transformation. The transformation of plant cells with Agrobacterium should be performed through cytomembrane which is a protective screen for cells， and opening this passage by a proper method is beneficial to the improvement of transformation efficiency. In this study， the cell membrane permeability at the growth points of maize buds was improved through low temperature treatment on maize buds， thereby promoting the infection of the growth points of maize buds with A. tumefaciens， and then the transformed buds were cultured under certain condition to allow recovery of the slightly-injured cell membrane to normal state and differentiation and budding of seeds to normal plants， so as to achieve the effect of improving transformation. Materials and Methods
Experimental materials
Receptor material： Mature seeds of maize selfing line "Zheng 58" were subjected to germination treatment and freezing treatment， and the growing points were used as the transformation receptor. The used plant transgenic vector PEGAD contained enhanced green fluorescent protein （EGFP）， and the Agrobacterium strain was EHA105.
Experimental method
Germination of maize seeds： Intact mature maize seeds free of mildewing were cleaned and sterilized with 0.1% HgCl2 for 12 min. The seeds were flushed with sterile water for five times， and the sterilized seeds were placed in a culture dish with a diameter of 9 cm subjected to high-temperature sterilization laid with two layers of filter. In each culture dish， 20 maize seeds were cultured in 8 ml of 210 μM acetosyringone water solution at 25 ℃ for 3 d under dark condition.
Preparation of Agrobacterium-mediated transformation liquid： Single colony was picked and inoculated to 50 ml of LB liquid medium containing 50 mg/L kanamycin sulfate +40 mg/L rifampin. The bacterial liquid was cultured at 28 ℃ under 210 r/min of oscillation until the OD600 of the liquid was 0.5. Centrifugation was performed at 4 000 r/min at 5 ℃ for 5 min， and the supernatant was discarded. Into the precipitate， 2 ml of hypertonic infection base liquid was added， obtaining the Agrobacterium-mediated transformation liquid after shaking. The hypertonic infection base liquid was salt of 1/10 MS medium + 68.5 g/L sucrose +30 g/L glucose +400 mg/L MES +100 μmol/L acetosyringone + 100 mg/L pluronic F68， with pH in the range of 5.4-5.6.
Transformation treatment and detection： When the maize seeds were cultured under dark condition for 3 d， germinated maize seeds were subjected to low temperature treatment at -18 ℃ for 30 and 35 min， respectively， as treatments 1 and 2， respectively， and the treatment free of low temperature treatment served as control （CK）. The obtained growing points of maize buds were air-dried for 30 min and then subjected to transformation treatment. After transformation， each culture dish was placed with 6-10 seeds， which were air-dried for 30 min again. Into each culture dish， 1 ml of sterile water was added， and the seeds were then cultured in the dark at 23-25 ℃ for 4 d. Then， 2 ml of water was added into each culture dish， and the seeds were cultured with illumination for 1 d. Finally， green fluorescent protein detection was performed to transformed buds with a fluorescent stereoscopic microscope， and the transformation effect was determined according to whether the green fluorescent protein is expressed as well as the expression strength of green fluorescent protein. Results and Analysis
Effect of low temperature treatment on rate of positive buds after transformation
The results of green fluorescent protein detection （Table 1） on transformed maize seeds showed that after 30 and 35 min of low temperature treatment at -18 ℃ and transformation with A. tumefaciens， the rates of positive buds were both lower than the CK. This is because that maize buds were injured after low temperature treatment， resulting in reduced emergence rate. After 35 min of low temperature treatment， only one bud survived， and the emergence rate after transformation was thus affected. Therefore， longer time of low temperature treatment reduced the rate of positive buds after transformation.
Effect of low temperature treatment on transformation degree
It could be seen from the expression condition of green fluorescent protein in transformed maize buds subjected to low temperature treatment that the expression level of green fluorescent protein after low temperature treatment was higher than that of the CK （Fig. 2）， and the green fluorescent protein was expressed in deeper cells of maize buds， while the CK only expressed the fluorescent protein on the surfaces of buds at a lower level. It indicates that low temperature treatment is more beneficial to transformation of more maize bud cells， and except surface cells， deeper cells could be also transformed， which is beneficial to the formation of transgenic chimeric plants with more positive cells， which could improve the probability of obtaining transgenic seeds.
Discussion
The maize plants obtained through Agrobacterium transformation using growing points of maize seed buds as transformation receptor and direct regeneration without tissue culture are always transgenic chimeras， the higher the proportion of transformed cells in chimeras， the more likely they will get transgenic offspring， and more transgenic seeds would be obtained afterwards. The results of this study showed that through low temperature treatment on maize seed buds as receptor， the growing points of maize seed buds were injured to certain degree， the permeability of cytomembrane was improved， which facilitated infection and transformation with A. tumefaciens， and more transformed cells were obtained. An optimal transformation effect could be achieved by finding the optimal balance point through control of time and temperature of the low temperature treatment. Under this condition， not only the growing points of maize seed buds are injured to a certain degree， resulting in improved cell membrane permeability， but also self-healing is allowed at the co-culture stage， i.e.， no fatal effect is caused to the growth of maize seeds. Therefore， the proportion of transformed cells is improved， which is beneficial to the acquisition of offspring materials， and the purpose of improving transformation efficiency is achieved. Maize was used as the experimental material in this study， if other crops are used as receptor materials， whether an ideal transformation effect could be obtained through similar treatment still needs further study.
References
[1] LIU YJ， JIA ZW， LIU Y， et al. Establishment and Application of Large-Scale Transformation Systems for Maize[J]. Scientia Agricultura Sinica， 2014， 47（21）： 4172-4182.
[2] LIU ZX， MA XQ， HE YY， et al. Improvement of the assistant method in Agrobacterium-mediated transformation[J]. Journal of Fudan University： Science Edition， 1999， 38（5）： 601-604.
[3] ZHANG LJ， CHEN LM， DU JZ， et al. Establishment and optimization of puna chicory genetic transformation system with Agrobacterium-mediated method[J]. Acta Agrectir Sinica， 2011， 19（6）： 1042-1050.