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Abstract [Objectives] The antifungal protein HAS1 newly obtained was evaluated for acute toxicity safety in KM mice according to relevant national regulations， so as to eliminate peoples concerns about the safety of transgenic plants.
[Methods] The acute toxicity of the purified protein HAS1 was observed by intragastric administration of mice， and the poisoning symptoms， poisoning degree， recovery and death were observed.
[Results] No abnormal toxicity symptoms were observed in the test group， the vehicle control group and the blank control group. The main tissues and organs were not abnormal in gross anatomy. The average body weight of each group showed an increasing trend compared with before administration at 1， 3， 7， 11 and 14 d after administration. It was found that after giving the purified protein HAS1 to KM mice at a cumulative dose of 64 mg/kg a day， no obvious toxicity was observed in the acute toxicity test， indicating that the test substance was nontoxic by oral administration.
[Conclusions] This study provides a basis for further use of the protein and its coding genes.
Key words Bacillus subtilis HAS; Purified protein HAS1; KM mice; Toxic reaction
Sugarcane （Saccharum officinarum L.） is the most important sugar crop in China. Chinas sugarcane planting area is about 1.2million hm2. Due to the lack of corresponding diseaseresistant varieties， sugarcane diseases have become increasingly serious， which has become one of the main factors restricting the sustainable development of sugarcane industry and sugar enterprises. Breeding diseaseresistant varieties is the most fundamental way to solve this problem， but conventional diseaseresistant breeding takes a long time and is far from meeting the needs of production[1-7]. Therefore， using plant genetic engineering technology to introduce disease resistance genes into good sugarcane varieties for effective expression and realization of disease resistance is the most effective way to cultivate sugarcane resistant varieties.
The HAS1 antifungal protein is a new antifungal protein isolated from the Bacillus subtilis HAS genome， which has a good inhibitory effect on various pathogenic fungi on sugarcane[8]. We intend to apply genetic engineering methods to introduce the HAS1 antifungal proteincoding gene into the main varieties of sugarcane production that are threatened by fungal diseases， thereby creating germplasm resources resistant to various fungal diseases. The safety of genetically modified organisms has always been the focus of attention， and we cant ignore this problem while carrying out genetic transformation of plants. Therefore， in this study， the acute toxicity safety of the obtained new antibacterial protein HAS1 in KM mice was evaluated in accordance with the relevant national regulations. This study provides experimental basis for further utilization of the protein and its coding gene， thereby eliminatingthe safety of transgenic plants.
Materials and Methods
Test drugs and animals
The purified protein HAS1 for expression was obtained by expression and purification in laboratory. The vehicle for the vehicle control group was prepared by the laboratory of the Safety Evaluation Center. The SPF grade KM mice were purchased from Changsha Tianqin Biotechnology Co.， Ltd. The SPF grade mouse feed was provided by Beijing Keao Xieli Feed Co.， Ltd.
Test animal grouping and administration method
Sixty KM mice that had a body weight meeting the standard and had undergone quarantine were selected and weighed in the range of 18-22 g. They were randomly divided into the test group， vehicle control group and blank control group， with 20 mice in each group， half male and half female. In each group， the female mice were not pregnant， and the differences between the body weights of KM mice did not exceed or were lower than 20% of the average body weight. In this test， the experiment was carried out by the "maximum administration dosage"， and the administration route was intragastric administration; and each group of the experiment was administered twice a day （one dose in the morning and in the afternoon）. The administration volume of the test group and the vehicle control group was 40 ml/kg. The mice were weighed on the day of administration， and the amount of administration per animal was calculated based on the body weight and the administration volume.
Index observation method
The observation was performed twice a day on the day of administration， and once a day from the second day to the 14thday of administration. Observations： Animal appearance， behavior， secretions and excretions were mainly observed; animal deaths （death time， presudden reaction， etc.） were observed; according to the characteristics of the test substance， the hypoglycemia reactions of the animals after administration （weakness of four limbs， reduced activity， coma， shock， death） were especially observed; weighing was performed on the day of grouping， 1st， 3rd， 7th， 11thand 14thd after administration using MP12001 or TP1200C electronic balance; and animals in the test group survived after the administration and the animals in the vehicle control group observed for 14 d after administration were subjected to cervical vertebrae dislocation and gross anatomy. The main tissues and organs such as heart， liver， spleen， lung， kidney， adrenal gland， thymus， bladder， uterus， ovary and intestine were observed by naked eyes. Data processing methods
All experiments were commissioned by the Hainan Provincial Drug Safety Evaluation Research Center. Animal body weight data were analyzed by ANOVA with SPSS 20.0 statistical software， and the differences in body weight between the test group and the vehicle control group were compared.
Results and Analysis
Signs and symptoms
The animals were closely observed for the reactions against the test substance within 4 h after the administration. The animals were observed twice for symptoms on the day of administration， and one symptom observation was performed from the 2ndd of administration to the 14thd after the administration. From the day of administration to the end of the experiment， no abnormal toxicity symptoms were observed in the KM mice of the test group， the vehicle control group and the blank control group， and the appearance signs， behavioral activities， respiration， fecal traits， water drinking and external reactivity were also observed. No obvious abnormalities were found.
Changes of body weight
Body weights were measured before administration and at 1， 3， 7， 11 and 14 d after administration. The results are shown in Fig. 1 and Table 1.
As can be seen from Table 1， the average body weights of the test group， the vehicle control group and the blank control group showed an increasing trend at 1， 3， 7， 11 and 14 d after administration compared with before administration. Using SPSS20.0 statistical analysis software， it was found that the weight of the blank control group was higher at 7 d after administration， with a extremely difference from the vehicle control group （P<0.01）， and there were no statistical differences in body weight between each group and the vehicle control group in other time periods. The body weight of the vehicle control group was similar to that of the test group at 7 d after administration， both were slightly lower than that of the blank control group， while on the day of administration， the vehicle control group was given ultrapure water， so the difference was caused by the individual difference， without biological significance.
Gross anatomy
At the end of the test period， the test group， vehicle control group and blank control group were sacrificed by cervical dislocation， and the animals were subjected to gross anatomy to examine the main living organs of KM mice such as heart， liver， spleen， lung， kidney， adrenal gland， thymus， bladder， uterus， ovary and intestine. No abnormalities were found. It indicated that the purified protein HAS1 was not significantly toxic to the internal organs of mice. Discussion and Conclusions
There have been many successful reports on the operation of transgenic technology for sugarcane， including resistance to viruses[14-17]， bacteria[18]， insects[19-20]， nematodes[21-22]， herbicides[23-25]， drought[26-27]and other aspects. The encoded gene of the purified protein in this study is the antigenic material for this engineering operation， so it is necessary to carry out preliminary research.
In addition， transgenic safety is one of the hot issues that people pay attention to. The safety evaluation of genetically modified organisms is usually the safety evaluation of genetically modified crops. In this study， toxicological research was conducted on the obtained genetically encoded protein prepared for transgenic technology operation， in order to provide experimental basis for subsequent work.
In this study， the collected purified HAS1 protein was administered to mice for observation of signs and visceral lesions， and the safety of the purified protein HAS1 was comprehensively evaluated. The mice were tested in the test group， vehicle control group and the blank control group by intragastric administration. It was found that after giving the purified protein HAS1 to KM mice at a cumulative dose of 64 mg/kg twice a day， no obvious toxicity was observed in the acute toxicity test， indicating that the test substance was less toxic by oral administration. These results provide a reference for further application of the purified protein HAS1 and its coding gene.
Guoru xiong et al. Study on Acute Toxicity of Purified Protein HAS1 Expression in KM Mice
References
[1] LU GD， LI CC， PAN CZ， et al. China sugarcane disease list[J]. Sugarcane， 1997， 4（4）： 19-23. （in Chinese）
[2] HUO XJ， LI CS， LU RS. Investigation and identification of sugarcane diseases in Guangxi[J]. Journal of Anhui Agricultural Sciences， 2013， 41（10）： 4368-4369. （in Chinese）
[3] HUANG YK， LI WF. Colored atlas of main diseases， insect pests and weeds of modern sugarcane[M]. Beijing： China Agriculture Press， 2011. （in Chinese）
[4] AN YX， GUAN CX. Atlas of sugarcane pests and diseases and control[M]. Guangzhou： Jinan University Press， 2009. （in Chinese）
[5] LI ZP， ZHANG SZ. Atlas of sugarcane pest and disease diagnosis in Hainan[M]. Beijing： China Agriculture Press， 2014. （in Chinese）
[6] XIONG GR， LI ZP， ZHAO TT， et al. Primary investigation to sugarcane on the diseases in Hainan Province[J]. Chinese Journal of Tropical Crops， 2010， 31（9）： 1-8. （in Chinese） [7] PENG SG. Sugarcane breeding[M]. Beijing： Agriculture Press， 1990. （in Chinese）
[8] LIU HQ， HUANG YT， DAI MY， et al. Identification of inintro antagonistic activity of HAS1 protein against Sporisorium scitanminea[J]. Agricultural Biotechnology， 2018， 7（06）： 9-11，15.
[9] China Food and Drug Administration. Drug registration management measures， 2007. （in Chinese）
[10] China Food and Drug Administration. Quality management regulations for nonclinical research， 2017. （in Chinese）
[11] China Food and Drug Administration. Drug Research Supervision and Management Measures （Trial）， 2005. （in Chinese）
[12] China Food and Drug Administration. Technical guidelines for singledose toxicity studies， 2014. （in Chinese）
[13] China Food and Drug Administration. Guidelines for nonclinical safety evaluation of therapeutic biological products， 2007. （in Chinese）
[14] SMITH GR， GAMBLEY RL. Progress in development of a sugarcane meristem transformation system and production of SCMVresistant transgenics[J]. Sugarcane， 1994， （6）： 22.
[15] SMITH RG， JOYCE PA， HANDLEY JA， et al. Genetically engineering resistance to sugarcane mosaic and Feiji disease viruses in sugarcane[C]//Sugar 2000 Symposium： Sugarcane： research towards efficient and sustainable production， 1996，12： 138-140.
[16] IVAN L INGELBRECHT， JAMES E IRVINE， T ERIK MIRKOV. Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homologydependent virus resistance in a monocot that has a complex polyploid genome[J]. Plant Physiology， 1999， 119： 1187-1198.
[17] YAO W， YU AL， XU JS， et al. Analysis and identification for transgenic sugarcane of ScMVCP gene[J]. Molecular Plant Breeding， 2004， 2（1）： 13-18. （in Chinese）
[18] ZHANG LH， XU JL， ROBERT G BIRCH. Engineered detoxification confers resistance against a pathogenic bacterium[J]. Nature Biotechnology， 1999， 17（10）： 1021-1024.
[19] ARIEL ARENCIBIA， ROBERTO I VAZQUEZ， DMITRI PRIETO， et al. Transgenic sugarcane plants resistant to stem borer attack[J]. Molecular Breeding， 1997， 3（4）： 247-255.
[20] ZHANGSUN DT， LUO SL， CHEN RK， et al. Improved agrobacteriummediated genetic transformation of GNA transgenic sugarcane[J]. Section Cellular and Molecular Biology， 2007，62（4）：386-393.
[21] JOANNE CH TAN， MICHAEL GK JONES， JOHN FOSUNYARKO. Gene silencing in root lesion nematodes （Pratylenchus spp.） significantly reduces reproduction in a plant host[J]. Experimental Parasitology， 2013， 133（2）：166-178. [22] CHEN PH， XU LP， CHEN RK. Construction of expression vector of nematoderesistant gene and transformation of sugarcane[J]. Chinese Journal of EcoAgriculture， 2004， 12（4）： 57-59. （in Chinese）
[23] GIL A， ENRIQUEZOBREGON， ROBERTO I VAZQUEZPADRON， DMITRI L PRIETOSAMSONOV， et al. Herbicideresistant sugarcane （Saccharum officinarum L.） plants by agrobacteriummediated transformation[J]. Planta， 1998， 206： 1367-1374.
[24] FALCO MC. TULMANN NETO A， ULIAN EC. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane[J]. Plant Cell Reports， 2000， 19 （12）： 1188-1194.
[25] MANICKAVASAGAM M， GANAPATHI A， ANBAZHAGAN VR， et al. Agrobacteriummediated genetic transformation and development of herbicideresistant sugarcane （Saccharum species hybrids） using axillary buds[J]. Plant Cell Rep， 2004， 23（3）： 134-43.
[26] ZHANG SZ， ZHENG XQ. Study on microprojectile bombardmentmediated transformation of sugarcane （Saccharum officinarum）[J]. Chinese Journal of Tropical Crops， 2001， 22（1）： 35-41. （in Chinese）
[27] ZHANG SZ， ZHENG XQ， LIN JF， et al. Cloning of trehalose synthase gene and transformation into sugarcane[J]. Chinese journal of agricultural biotechnology， 2000， 8（4）： 385-388. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
[Methods] The acute toxicity of the purified protein HAS1 was observed by intragastric administration of mice， and the poisoning symptoms， poisoning degree， recovery and death were observed.
[Results] No abnormal toxicity symptoms were observed in the test group， the vehicle control group and the blank control group. The main tissues and organs were not abnormal in gross anatomy. The average body weight of each group showed an increasing trend compared with before administration at 1， 3， 7， 11 and 14 d after administration. It was found that after giving the purified protein HAS1 to KM mice at a cumulative dose of 64 mg/kg a day， no obvious toxicity was observed in the acute toxicity test， indicating that the test substance was nontoxic by oral administration.
[Conclusions] This study provides a basis for further use of the protein and its coding genes.
Key words Bacillus subtilis HAS; Purified protein HAS1; KM mice; Toxic reaction
Sugarcane （Saccharum officinarum L.） is the most important sugar crop in China. Chinas sugarcane planting area is about 1.2million hm2. Due to the lack of corresponding diseaseresistant varieties， sugarcane diseases have become increasingly serious， which has become one of the main factors restricting the sustainable development of sugarcane industry and sugar enterprises. Breeding diseaseresistant varieties is the most fundamental way to solve this problem， but conventional diseaseresistant breeding takes a long time and is far from meeting the needs of production[1-7]. Therefore， using plant genetic engineering technology to introduce disease resistance genes into good sugarcane varieties for effective expression and realization of disease resistance is the most effective way to cultivate sugarcane resistant varieties.
The HAS1 antifungal protein is a new antifungal protein isolated from the Bacillus subtilis HAS genome， which has a good inhibitory effect on various pathogenic fungi on sugarcane[8]. We intend to apply genetic engineering methods to introduce the HAS1 antifungal proteincoding gene into the main varieties of sugarcane production that are threatened by fungal diseases， thereby creating germplasm resources resistant to various fungal diseases. The safety of genetically modified organisms has always been the focus of attention， and we cant ignore this problem while carrying out genetic transformation of plants. Therefore， in this study， the acute toxicity safety of the obtained new antibacterial protein HAS1 in KM mice was evaluated in accordance with the relevant national regulations. This study provides experimental basis for further utilization of the protein and its coding gene， thereby eliminatingthe safety of transgenic plants.
Materials and Methods
Test drugs and animals
The purified protein HAS1 for expression was obtained by expression and purification in laboratory. The vehicle for the vehicle control group was prepared by the laboratory of the Safety Evaluation Center. The SPF grade KM mice were purchased from Changsha Tianqin Biotechnology Co.， Ltd. The SPF grade mouse feed was provided by Beijing Keao Xieli Feed Co.， Ltd.
Test animal grouping and administration method
Sixty KM mice that had a body weight meeting the standard and had undergone quarantine were selected and weighed in the range of 18-22 g. They were randomly divided into the test group， vehicle control group and blank control group， with 20 mice in each group， half male and half female. In each group， the female mice were not pregnant， and the differences between the body weights of KM mice did not exceed or were lower than 20% of the average body weight. In this test， the experiment was carried out by the "maximum administration dosage"， and the administration route was intragastric administration; and each group of the experiment was administered twice a day （one dose in the morning and in the afternoon）. The administration volume of the test group and the vehicle control group was 40 ml/kg. The mice were weighed on the day of administration， and the amount of administration per animal was calculated based on the body weight and the administration volume.
Index observation method
The observation was performed twice a day on the day of administration， and once a day from the second day to the 14thday of administration. Observations： Animal appearance， behavior， secretions and excretions were mainly observed; animal deaths （death time， presudden reaction， etc.） were observed; according to the characteristics of the test substance， the hypoglycemia reactions of the animals after administration （weakness of four limbs， reduced activity， coma， shock， death） were especially observed; weighing was performed on the day of grouping， 1st， 3rd， 7th， 11thand 14thd after administration using MP12001 or TP1200C electronic balance; and animals in the test group survived after the administration and the animals in the vehicle control group observed for 14 d after administration were subjected to cervical vertebrae dislocation and gross anatomy. The main tissues and organs such as heart， liver， spleen， lung， kidney， adrenal gland， thymus， bladder， uterus， ovary and intestine were observed by naked eyes. Data processing methods
All experiments were commissioned by the Hainan Provincial Drug Safety Evaluation Research Center. Animal body weight data were analyzed by ANOVA with SPSS 20.0 statistical software， and the differences in body weight between the test group and the vehicle control group were compared.
Results and Analysis
Signs and symptoms
The animals were closely observed for the reactions against the test substance within 4 h after the administration. The animals were observed twice for symptoms on the day of administration， and one symptom observation was performed from the 2ndd of administration to the 14thd after the administration. From the day of administration to the end of the experiment， no abnormal toxicity symptoms were observed in the KM mice of the test group， the vehicle control group and the blank control group， and the appearance signs， behavioral activities， respiration， fecal traits， water drinking and external reactivity were also observed. No obvious abnormalities were found.
Changes of body weight
Body weights were measured before administration and at 1， 3， 7， 11 and 14 d after administration. The results are shown in Fig. 1 and Table 1.
As can be seen from Table 1， the average body weights of the test group， the vehicle control group and the blank control group showed an increasing trend at 1， 3， 7， 11 and 14 d after administration compared with before administration. Using SPSS20.0 statistical analysis software， it was found that the weight of the blank control group was higher at 7 d after administration， with a extremely difference from the vehicle control group （P<0.01）， and there were no statistical differences in body weight between each group and the vehicle control group in other time periods. The body weight of the vehicle control group was similar to that of the test group at 7 d after administration， both were slightly lower than that of the blank control group， while on the day of administration， the vehicle control group was given ultrapure water， so the difference was caused by the individual difference， without biological significance.
Gross anatomy
At the end of the test period， the test group， vehicle control group and blank control group were sacrificed by cervical dislocation， and the animals were subjected to gross anatomy to examine the main living organs of KM mice such as heart， liver， spleen， lung， kidney， adrenal gland， thymus， bladder， uterus， ovary and intestine. No abnormalities were found. It indicated that the purified protein HAS1 was not significantly toxic to the internal organs of mice. Discussion and Conclusions
There have been many successful reports on the operation of transgenic technology for sugarcane， including resistance to viruses[14-17]， bacteria[18]， insects[19-20]， nematodes[21-22]， herbicides[23-25]， drought[26-27]and other aspects. The encoded gene of the purified protein in this study is the antigenic material for this engineering operation， so it is necessary to carry out preliminary research.
In addition， transgenic safety is one of the hot issues that people pay attention to. The safety evaluation of genetically modified organisms is usually the safety evaluation of genetically modified crops. In this study， toxicological research was conducted on the obtained genetically encoded protein prepared for transgenic technology operation， in order to provide experimental basis for subsequent work.
In this study， the collected purified HAS1 protein was administered to mice for observation of signs and visceral lesions， and the safety of the purified protein HAS1 was comprehensively evaluated. The mice were tested in the test group， vehicle control group and the blank control group by intragastric administration. It was found that after giving the purified protein HAS1 to KM mice at a cumulative dose of 64 mg/kg twice a day， no obvious toxicity was observed in the acute toxicity test， indicating that the test substance was less toxic by oral administration. These results provide a reference for further application of the purified protein HAS1 and its coding gene.
Guoru xiong et al. Study on Acute Toxicity of Purified Protein HAS1 Expression in KM Mice
References
[1] LU GD， LI CC， PAN CZ， et al. China sugarcane disease list[J]. Sugarcane， 1997， 4（4）： 19-23. （in Chinese）
[2] HUO XJ， LI CS， LU RS. Investigation and identification of sugarcane diseases in Guangxi[J]. Journal of Anhui Agricultural Sciences， 2013， 41（10）： 4368-4369. （in Chinese）
[3] HUANG YK， LI WF. Colored atlas of main diseases， insect pests and weeds of modern sugarcane[M]. Beijing： China Agriculture Press， 2011. （in Chinese）
[4] AN YX， GUAN CX. Atlas of sugarcane pests and diseases and control[M]. Guangzhou： Jinan University Press， 2009. （in Chinese）
[5] LI ZP， ZHANG SZ. Atlas of sugarcane pest and disease diagnosis in Hainan[M]. Beijing： China Agriculture Press， 2014. （in Chinese）
[6] XIONG GR， LI ZP， ZHAO TT， et al. Primary investigation to sugarcane on the diseases in Hainan Province[J]. Chinese Journal of Tropical Crops， 2010， 31（9）： 1-8. （in Chinese） [7] PENG SG. Sugarcane breeding[M]. Beijing： Agriculture Press， 1990. （in Chinese）
[8] LIU HQ， HUANG YT， DAI MY， et al. Identification of inintro antagonistic activity of HAS1 protein against Sporisorium scitanminea[J]. Agricultural Biotechnology， 2018， 7（06）： 9-11，15.
[9] China Food and Drug Administration. Drug registration management measures， 2007. （in Chinese）
[10] China Food and Drug Administration. Quality management regulations for nonclinical research， 2017. （in Chinese）
[11] China Food and Drug Administration. Drug Research Supervision and Management Measures （Trial）， 2005. （in Chinese）
[12] China Food and Drug Administration. Technical guidelines for singledose toxicity studies， 2014. （in Chinese）
[13] China Food and Drug Administration. Guidelines for nonclinical safety evaluation of therapeutic biological products， 2007. （in Chinese）
[14] SMITH GR， GAMBLEY RL. Progress in development of a sugarcane meristem transformation system and production of SCMVresistant transgenics[J]. Sugarcane， 1994， （6）： 22.
[15] SMITH RG， JOYCE PA， HANDLEY JA， et al. Genetically engineering resistance to sugarcane mosaic and Feiji disease viruses in sugarcane[C]//Sugar 2000 Symposium： Sugarcane： research towards efficient and sustainable production， 1996，12： 138-140.
[16] IVAN L INGELBRECHT， JAMES E IRVINE， T ERIK MIRKOV. Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homologydependent virus resistance in a monocot that has a complex polyploid genome[J]. Plant Physiology， 1999， 119： 1187-1198.
[17] YAO W， YU AL， XU JS， et al. Analysis and identification for transgenic sugarcane of ScMVCP gene[J]. Molecular Plant Breeding， 2004， 2（1）： 13-18. （in Chinese）
[18] ZHANG LH， XU JL， ROBERT G BIRCH. Engineered detoxification confers resistance against a pathogenic bacterium[J]. Nature Biotechnology， 1999， 17（10）： 1021-1024.
[19] ARIEL ARENCIBIA， ROBERTO I VAZQUEZ， DMITRI PRIETO， et al. Transgenic sugarcane plants resistant to stem borer attack[J]. Molecular Breeding， 1997， 3（4）： 247-255.
[20] ZHANGSUN DT， LUO SL， CHEN RK， et al. Improved agrobacteriummediated genetic transformation of GNA transgenic sugarcane[J]. Section Cellular and Molecular Biology， 2007，62（4）：386-393.
[21] JOANNE CH TAN， MICHAEL GK JONES， JOHN FOSUNYARKO. Gene silencing in root lesion nematodes （Pratylenchus spp.） significantly reduces reproduction in a plant host[J]. Experimental Parasitology， 2013， 133（2）：166-178. [22] CHEN PH， XU LP， CHEN RK. Construction of expression vector of nematoderesistant gene and transformation of sugarcane[J]. Chinese Journal of EcoAgriculture， 2004， 12（4）： 57-59. （in Chinese）
[23] GIL A， ENRIQUEZOBREGON， ROBERTO I VAZQUEZPADRON， DMITRI L PRIETOSAMSONOV， et al. Herbicideresistant sugarcane （Saccharum officinarum L.） plants by agrobacteriummediated transformation[J]. Planta， 1998， 206： 1367-1374.
[24] FALCO MC. TULMANN NETO A， ULIAN EC. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane[J]. Plant Cell Reports， 2000， 19 （12）： 1188-1194.
[25] MANICKAVASAGAM M， GANAPATHI A， ANBAZHAGAN VR， et al. Agrobacteriummediated genetic transformation and development of herbicideresistant sugarcane （Saccharum species hybrids） using axillary buds[J]. Plant Cell Rep， 2004， 23（3）： 134-43.
[26] ZHANG SZ， ZHENG XQ. Study on microprojectile bombardmentmediated transformation of sugarcane （Saccharum officinarum）[J]. Chinese Journal of Tropical Crops， 2001， 22（1）： 35-41. （in Chinese）
[27] ZHANG SZ， ZHENG XQ， LIN JF， et al. Cloning of trehalose synthase gene and transformation into sugarcane[J]. Chinese journal of agricultural biotechnology， 2000， 8（4）： 385-388. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU