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Abstract With the rapid development of molecular biology technique in recent years， significant progress also has been made in genetic engineering of tomato. We reviewed the research progress in disease resistance， insect resistance， stress， quality improvement and genetic resources utilization， respectively. The enormous application of transgenic tomato was also summarized in increasing resistance and improving quality. It was expected to provide references for the research and improvement of transgenic tomato.
Key words Tomato； Genetic engineering； Genetic transformation； Screening
Received： October 5， 2017 Accepted： January 11， 2018
Sponsored by Qing Lan Project of Colleges and Universities in Jiangsu Province in 2016 （young and middleaged academic leader）； Surface Project of National Natural Science Foundation of China （31672141）.
Yan AO （1974-）， female， PhD， associate professor， devoted to research about biostatistics and quantitative genetics， Email： 350685056@qq.com.
*Corresponding author. Email： wuqi@issas.ac.cn
Tomato is an annual or perennial plant in Lycopersicon of Solanaceae family， as well as a kind of vegetable crop with very high nutritive value. In recent years， with the continuous updating and development of molecular cloning techniques， researchers have made a great progress in research on characteristic improvement of tomato varieties， and good tomato varieties with strong resistance to virus， diseases and pests and stress and good quality have been obtained. With the deepening of the research， the lack of tomato germplasm resources has become an important inhibiting factor for the breeding of new tomato varieties. In 1990， Professor Rick from University of California set up a tomato gene resource center as a communication platform， which provides an effective way for the improvement of tomato varieties all over the world.
In this paper， the research progress in disease resistance， insect resistance， stress， quality improvement and genetic resources utilization were reviewed， so as to provide references for the research on transgenic tomato and variety improvement.
Research about Transgenic Antiviral Tomato
Tomato virus disease is spread by aphids and field operation， and the virus overwinters through plant residues in soil or seeds and other perennial plants. Currently， the most common viruses harming the growth of tomato include tobacco mosaic virus （TMV）， cucumbermosaic virus （CMV）， tobacco leaf curl virus （TLCV） and alfalfa mosaic virus （AMV）. In the research about transgenic antiviral tomato， Parrella et al.[1] found that there was a gene in Am family located on chromosome 6 of tomato， which could serve as a growth inhibitor which inhibits the overexpression of AMV protein in the early virus life cycle， and therefore， transforming the gene into tobedetected body of tomato could well play a role in resisting AMV. However， Hu et al.[2] screened a resistant gene Tm22 of TMV from closely related species in Solanaceae， and then cloned the resistant gene through qRTPCR technology and transformed it into vector pBIN19， and meanwhile， the vector pBIN19 was expressed by promoter CaMV 35S controlling gene. They concluded that plants transformed with Tm22 had better resistance than wild plants. Pratap et al.[3] directed CP gene of CMV into tomato by conventional Agrobacteriummediated method， and through inoculation and observation experiments， it was found that the transgenic progenies carrying CP gene of IB subgroup of CMV had higher resistance against CMV. Liao et al.[4] did not started from exogenous resistant gene， but promoted the massive expression of mRNA by silencing microRNAs gene through the insertion of the cloned fragment of CMV RNA3， thereby constructing a good CMVresistant vector for the improvement of the resistance to CMV. Anbinder et al.[5] screened a breeding line ‘TY172’ highly resistant to TLCV， and then mapped the genetic loci controlling TLCV resistance applying polymorphic DNA molecular marker technique and QTLs mapping. The results showed that in ‘TY172’， the resistance was controlled by one unknown major QTL and 4 minor QTLs， among which the major QTL was located on chromosome 4 and had a contribution rate of 39.7%-46.6%， and the minor QTLs were located on chromosomes 1， 7， 9 and 11， respectively， and exhibited the contribution rate of about 12%. With the continuous breeding of TLCVresistant plants， the virus also produces various subtypes itself， such as tomato leaf curl Taiwan virus （TOLCTWV） and Thailand tomato yellow leaf curl virus （TYLCV）. Chen et al.[6] constructed an expression vector by dualvirus RNA interference technology， and could obtain plants resistant to both above viruses.
In 1998， CMVresistant tomato PKTM8805R was developed by Beijing University for the first time. This variety was directed with exogenous CMVCP gene by Agrobacteriummediated method， and once produced a good economic benefit during its commercial planting in Xiamen City， Fujian Province. Research about Trangenic Pestresistant Tomato
Breeding for pest resistance is also an important means for the improvement of tomato yield. Since the 1980s， the research about breeding for pest resistance was started in China， pestresistant tomato germplasm resources were screened， and many excellent pestresistant genes were explored， laying a foundation for the research about further transgenic pestresistant tomato[7]. At present， the genes found and used for the improvement of pest resistance are mainly Bt toxin protein gene from Bacillus thuringiensis and cowpca trypsin inhibitor （CpTI）[8]. The Bt gene is a kind of specific insecticidal gene， which mainly kills Lepidoptera insects. Saker et al.[9] induced the overexperssion of Bt （Cry 2Ab） gene in transgenic gene by Agrobacteriummediated transformation， and the results showed that the Bt tomato expressing Cry 2Ab gene was resistant to Lepidoptera insects including America cotton bollworm and potato stem moth. CpTI gene is kind of gene with widespectrum resistance， and its albumin has insecticidal ability. In 2001， Ghoshal et al.[10] reported the transformation of CpTI gene into plants by direct PEG mediation， and found that under regulation of promoter CaMV 35s， CpTI protein had efficient catalytic activity. Hereafter， CpTI gene is widely used model crop including rice[11]， playing an important role to the improvement of plant pest resistance and the increase of yield.
With the deepening of the research， previous studies also found that the dualgene integrated answering effect is always better than the resistance effect controlled by single gene. Abdeen et al.[12] found that in homozygous tomato lines， the overexpression of PIII protein and carboxypeptidase inhibitor （PCI） gene resulted in higher resistance to larvae of cotton bollworm and Liriomyza trifolii， indicating that the interaction of two kinds of protease inhibitors could better control pests. Chan et al.[13] transformed plant cysteine protease inhibitor （CeCPI） gene and mycosin （PjCHI1） gene into tomato plants by skillfully use of dualgene overexpression system， and induced gene expression under the regulation of promoter pMSPOA， thereby inhibiting the growth of Meloidogyne incognita. Latest studies showed that βglucosidase and digesting enzyme extracted from tomato plants also could inhibit the growth and development of insects[14].
Since 1998， America approved the environmental safety and food safety evaluation of transgenic pestresistant tomato ‘5345’ by Monsanto company. In 2000， ‘5345’ was approved in Canada for eating. The transgenic pestresistant tomato ‘5345’ was bred through the transformation of insectidical Bt proteinCry1Ac into tomato cultivar ‘UC82B’ by Agrobacteriummediated method. It is also a successful case of the application of DNA recombination technology in crop improvement. When Cry1Ac is expressed in transgenic tomato ‘5345’ as endotoxin， it kills Lepidoptera pests with high selectivity， thereby exhibiting specific pestresistant character. Research about Transgenic Stressresistant Tomato
Research about transgenic saltresistant tomato
In recent years， saline and alkaline land in China shows an increasing trend， and plus other reasons such as inappropriate fertilizer and water management， most soil suffers from serious secondary salinization. These reasons lead to the decrease of tomato yield， and seriously restrict the commercialized production of tomato in China. Therefore， the research about the mechanism related to salt resistance in tomato becomes very important. Li et al.[15] found through gel hybridization experiments that after salt stress or osmotic stress induced by polyethylene glycol， monodehydroascorbate reductase （MDHAR） in chloroplast of wild tomato increased remarkably， and photoinhibition in PSII was alleviated through the improvement of ascorbic acid level， so as to induce enhancement of salt resistance in plant. According to this principle， MDHAR gene could be directed to tomato， to improve salt resistance in tomato， but no related studies have been reported. Recently， a type of prolinerich protein was found in plant， which could response to biotic or abiotic stress， especially high salt damage. The transformation of such protein gene into tomato plant could significantly improve the resistance to salt stress[16]. Of course， deeper studies have already been done on dualgene integrated regulation on slat resistance in tomato.
Viveros[17] reported that the overexpression of glyoxalase I （Gly I） and glyoxalase II （Gly II） in tomato plants， and through the detection of the stable expression in transgenic tomato after salt stress， it was demonstrated that GlyI and GlyII genes could enhance the resistance to salt stress through the reduction of oxidative stress capability in plant. However， above experiments did not verify whether the interaction between genes has a synergistic effect in improving salt resistance in tomato. In 2016， CAI et al.[18] reported that in tomato， SlDof gene could be combined at the promoter region of SlSOS1 gene， to regulate the resistance in plant to salt damage together.
Except the transformation of exogenous saltresistant gene， plants also could avoid or alleviate the effect of salt damage to plants through selfregulatory mechanism. Wang et al.[19] expressed a sequence of 3omega fatty acid desaturase gene regulated by CMV35S in a large quantity， then performed Western and Northern blot analysis based on reverse transcriptionPCR quantitative technique， and finally found by electron microscopy observation that LeFAD3 gene could maintain the integrity of cytomembrane， thereby improving the resistance in tomato to salt stress at seedling stage. Research about Transgenic Antifreezing Tomato
Tomato is also a kind of vegetable susceptible to cold injury， which is mainly reflected by the condition that liquid in injured parts produces ice crystals at first at the nursery stage， and the ice crystals grow bigger and cause the death of cells by extruding cell wall. Currently， there are two viewpoints which explain the antifreezing mechanism in plant： one is to transform antifreezing gene to improve tomato resistance， and the other is to add some regulation genes to induce the expression of antifreezing gene.
Kumar et al.[20] transformed the gene coding antifreezing protein （AFP） into tomato plants by Agrobacteriummediated method under the regulation by CaMV 35S， and proved the expression of AFP gene by Southern blot analysis and RTPCR technique. The results showed that the expression of antifreezing gene in carrot could improve the freezing resistance in tomato and improve yield in a large area. Subraman et al.[21] showed that the tomato inoculated with 1aminocyclopropane1carboxylic acid deaminase had higher resistance under cold injury， and freezing induction genes LeCBF1， LeCBF3 and ethylene response transcription factor ETF13 all could promote the expression of antifreezing gene. Latest studies showed that in polyamine transgenic tomato， pathogenyrelated protein PR1b1 could be upregulated under chilling condition to improve plant resistance[22]. In recent years， researchers also found a kind of antifreezing protein known as Maxi， which has no that much water molecules in its core as imagined， but achieves the stressresistant effect by the mechanism of interior folding and dynamic characteristics， which overturns the prediction of conventional proteins by scientists， which deems that the core of ordinary proteins is full of water molecules. The finding of such protein also added new interpretation to antifreezing mechanisms of plant[23].
Except that the expression of resistant genes is changed or regulated by other genes， plant hormones[24-25] also could respond to cold injury， thereby inducing a serious of molecular response mechanisms in plants. Zhu et al.[24] found through different concentrations of GA treatments that GA treatment could effectively alleviate cold injury， but genes involved in gibberellin synthesis pathway were remarkably downregulated. Meanwhile， gibberellin also could affect the response of plant under stress by inducing the biosynthesis of salicylic acid. Of course， in tomato plants， the expression of one gene is always not only a response mechanism of living organism to one kind of stress， but also a kind of comprehensive expression of the integrated effect of various environmental effects to multiple stress systems.
Zhang et al.[26] induced the specific DNA cleavage site of a selected marker gene using Cre/loxP DNA recombination system， thereby allowing the expression of AtIpk2b gene in tomato in a large quantity， and finally， the plant obtained higher resistance to drought， freezing and oxidation. Lyu et al.[27] extracted from Escherichia coli， a kind of trehalose6phosphate synthase/phosphatase fusion gene， which was transformed to tomato under the induction by CaMV 35S promoter. The results showed that compared with wild tomato， transgenic tomato has stronger photosynthesis tolerance under drought and salt stresses. Sun et al.[28] also demonstrated that SlWRKY39 regulates the adaption of tomato to biotic or abiotic stress by activating the expression of genes related to plant pathogenesis and stress. Also， in genetic breeding of crops， Saleem et al.[29] illuminated that the hybridization of different tomato lines （combination of different genotypes） has a remarkable superiority in resisting stress and improving yield in detail.
Several main conditions limiting normal growth of tomato were introduced in detail. In order to well introduce the mutual relation between various stresses during tomato growth and provide reference for tomato breeding， genes responding to stress were depicted in Fig. 1.
Fig. 1 Induced expression of related genes in tomato under stress
Yan AO et al. Research Progress on Transgenic Tomato
Research about Transgenic Quality
In recent years， with the development of economic and the improvement of living standard， people put forward higher requirements for tomato quality. Tomato having good quality often takes good fruit shape， no split， no bite， proper maturation and moderate sweet and sour taste as main indexes. Therefore， studies on the sweetness and sourness of tomato were reviewed below.
Transgenic research about improvement of sweetness of tomato
The improvement of sweetness of transgenic tomato was mainly conducted from two aspects： one is to transform sweetness genes into tomato by the method of directing exogenous genes to improve sweetness； and the other is to regulate the physiological and biochemical pathways such as glycometabolism of plants to change the concentration of sugar. Firsov et al.[30] found thaumatin II gene in Crassocephalum crepidioides， then inserted the cDNA fragment of thaumatin II gene into pBi121 vector under the regulation of CaMV 35S， and finally found the expression of thaumatin II gene in a large quantity by enzyme linked immunosorbent assay. The results also showed that transgenic tomato has better taste quality.
Reddy et al.[31] studied a kind of special sweet protein monellin in Dovyalis hebecarpa， transformed the monellin gene coding the sweet protein into tomato plants under the cocontrol of T7 promoter in E. coli and specific promoter of fruit maturation， and found the expression of monellin protein in a large quantity in plants by enzyme linked immunosorbent assay after normal culture in greenhouse gases. Compared with wild tomato， the sugar content in the transgenic tomato increased remarkably， which plays a positive role in the improvement of tomato flavor. Meanwhile， other genes regulating fruit sweetness are also found in succession.
One latest study shows that two types of transcription factors SlbZI1 and SlbZIP2 in tomato plants have a positive effect on the expression of sugarcoding genes， and further analysis shows that SlbZI1 could activate the activities of asparagine synthetase and proline dehydrogenase， thereby improving the contents of sucrose， glucose and fructose in transgenic plants[32].
Beauvoit et al.[33] studied the relation between enzymes related to glycometabolism process in tomato and vacuole expansion degree. The results showed that during cell division， the activities of fructokinase and phosphofructokinase were improved， and during vacuole expansion， the activities of sucrose synthetase， glucose6phosphate isomerase and ADPglucose pyrophosphorylase were also enhanced. Therefore， during vacuole expansion in cells， soluble sugar in vacuoles accumulates in a large quantity. These studies lay a theoretical basis for research about genes coding related synthetases in glycometabolism process and research about transsugar synthetase gene in tomato. However， at present， there were few reports about such research.
It is generally known that glycolytic pathway in plant cells plays a very important role in the regulation of plant growth and development， and the deficiency of phosphoenolpyruvate carboxykinase （PEPCK） activity has a profound influence on normal physiological metabolism of plant.
Huang et al.[34] initiated the degradation mechanism of mRNA sequence specificity using RNA interference technique， and affected the normal transcription of SlPEPCK gene， thereby repressing the synthesis process of PEPCK. Compared with wild tomato， the sugar content in the RNAinterfered tomato mutant had a remarkable decreasing trend， and the results showed that the deficiency of PEPCK would affect the accumulation of sugar content in tomato plants. In recent years， with the development of molecular biology study， the mechanisms of other synthetases regulating sugar synthesis in tomato plants during related glycolysis process are also known gradually. Meanwhile， with the coming of big data era， bioinformatics are widely applied to plant field. Ikeda et al.[35] revealed the significant changes of carbohydrate content and amino acid metabolism 20 after flowering of tomato， and further found that expression quantities of cell wallbound invertase， sucrose synthetase and glutamate synthase were also higher than wild varieties. They finally concluded that cell wallbound invertase and sucrose synthetase could affect the accumulation of glutamate synthase by regulating sugar concentration in carbon metabolism， thereby improving the sweetness of fruit. Transgenic research about improvement of sourness
Research shows that the sourness of tomato are very important to the color， taste and nutrition of tomato， and therefore， the physiological mechanisms of sourness to plant growth and development is attracting more and more attention. Sun et al.[36] repressed the expression of SINCED1 gene of 9cisepoxycarotenoid dioxygenases （NCED）， thereby affecting the biosynthesis of abscisic acid （ABA）. Experiments showed ABA could promote the decomposition of cell wall by regulating the expression levels of main tissue dissolution genes （SlPG， SlPME， SlXET， SlCels， SlExp）， which layed a theoretical basis for the exploration， cloning and transformation of SlNCED1. Latest studies show that the sugar content and sourness in some tomato mutants are in a dynamic changing process， and under stress， plants could realize the stability in plants through the sugarsourness interconversion mechanism， thereby improving the taste quality of plant. Therefore， it is urgent to explore major genes controlling interconversion reaction[37]. Also， there are studies showing that the mutual antagonism regulation by plant hormones gibberellin GA and abscisic acid ABA is beneficial to the improvement of tomato sourness. Consequently， the exploration of the key genes in the GA and ABA synthesis process becomes the focus of the public[38].
Tomato fruit has a short preservation time and rots rapidly after maturation， thereby losing commodity value. Therefore， in order to prolong the shelf life of tomato and bring convenience to market circulation， the research and development of shelfstable tomato has been conducted since the 1990s by transgenic technology. This work is mainly performed by transforming the antisense gene of tomato polygalacturonic acid （PG）， the antisense gene of ethylene synthetase， the Cry1Ac gene of Bacillus thuringiensis and the CMV coat protein （CP） gene into cultivated gene by Agrobacteriummediated method. In 1992， United States Department of Agriculture and Food and Drug Administration authorized the environmental release of storageresistant tomato "Flavr Savr"， and gave out foodsafety certification in 1994 for food and fodder. The variety is first case of transgenic crop approved from the market. Furthermore， Institute of Microbiology， Chinese Academy of Sciences directed the antisense gene of tomato ethylene synthetase into cultivated tomato by Agrobacteriummediated method， obtaining transgenic maturationdelayed tomato "Dadong 9". In 2000， "Dadong 9" was approved for commercial production in Beijing. Some other common transgenic maturationdelayed tomato varieties include ‘13454’， ‘351N’， ‘8338’， ‘B’， ‘Da’ and ‘F’. Conclusions
In recent 30 years， the gene transformation of tomato is also changed from the directional change of single character to the change of multiple characters， and finally developed towards the direction of medical treatment. Tomato transformed with multiple genes resistant to TMV， CMV， TLCV and AMV have been cloned in China[39]. As early as 1994， transgenic tomato variety "Flavr Savr" entered the market in America， and in China， in 1990， Huazhong Agricultural University started the research and development of transgenic storagetolerant tomato， which was approved by GMO Biosafety Committee， Ministry of Agriculture in 1996， and became the first agricultural biogenetic engineering product approved for commercial production in China， which was designated as "Huafan No. 1"[40]. The gene directed into "Huafan No. 1" is only to delay the maturation of tomato and inhibit the synthesis of partial special protein in tomato， so as to alleviate the degradation of cell wall and softening of fruit. However， with the development of science and technology， breeders obtained nontransgenic maturationdelayed tomato， the superiority of transgenic tomato in storage not only exists， and its low yield becomes a big problem. Plus thick peel and poor taste， transgenic tomato was directly weeded out in the market. Comprehensively， people still doubt whether transgenic technology is safe， the safety of transgenic crop needs to be verified by a long term of experiment as well. Therefore， the commercialization of transgenic tomato is facing bottleneck in Chinese market. With the finding of more and more marker genes， the development of a new generation of molecular marker technology also promoted the exploration， mapping and cloning of new genes， which provides technical support for the finding of more exogenous genes with practical value. Of course， with the rapid advance of sequencing technique， people have been able to find desired target genes at genomewide level， transcriptional level and translational level， which greatly enriches the diversity of germplasm resources of transgenic tomato， laying a foundation for genetic breeding work in future.
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（Continued on page 19）
Key words Tomato； Genetic engineering； Genetic transformation； Screening
Received： October 5， 2017 Accepted： January 11， 2018
Sponsored by Qing Lan Project of Colleges and Universities in Jiangsu Province in 2016 （young and middleaged academic leader）； Surface Project of National Natural Science Foundation of China （31672141）.
Yan AO （1974-）， female， PhD， associate professor， devoted to research about biostatistics and quantitative genetics， Email： 350685056@qq.com.
*Corresponding author. Email： wuqi@issas.ac.cn
Tomato is an annual or perennial plant in Lycopersicon of Solanaceae family， as well as a kind of vegetable crop with very high nutritive value. In recent years， with the continuous updating and development of molecular cloning techniques， researchers have made a great progress in research on characteristic improvement of tomato varieties， and good tomato varieties with strong resistance to virus， diseases and pests and stress and good quality have been obtained. With the deepening of the research， the lack of tomato germplasm resources has become an important inhibiting factor for the breeding of new tomato varieties. In 1990， Professor Rick from University of California set up a tomato gene resource center as a communication platform， which provides an effective way for the improvement of tomato varieties all over the world.
In this paper， the research progress in disease resistance， insect resistance， stress， quality improvement and genetic resources utilization were reviewed， so as to provide references for the research on transgenic tomato and variety improvement.
Research about Transgenic Antiviral Tomato
Tomato virus disease is spread by aphids and field operation， and the virus overwinters through plant residues in soil or seeds and other perennial plants. Currently， the most common viruses harming the growth of tomato include tobacco mosaic virus （TMV）， cucumbermosaic virus （CMV）， tobacco leaf curl virus （TLCV） and alfalfa mosaic virus （AMV）. In the research about transgenic antiviral tomato， Parrella et al.[1] found that there was a gene in Am family located on chromosome 6 of tomato， which could serve as a growth inhibitor which inhibits the overexpression of AMV protein in the early virus life cycle， and therefore， transforming the gene into tobedetected body of tomato could well play a role in resisting AMV. However， Hu et al.[2] screened a resistant gene Tm22 of TMV from closely related species in Solanaceae， and then cloned the resistant gene through qRTPCR technology and transformed it into vector pBIN19， and meanwhile， the vector pBIN19 was expressed by promoter CaMV 35S controlling gene. They concluded that plants transformed with Tm22 had better resistance than wild plants. Pratap et al.[3] directed CP gene of CMV into tomato by conventional Agrobacteriummediated method， and through inoculation and observation experiments， it was found that the transgenic progenies carrying CP gene of IB subgroup of CMV had higher resistance against CMV. Liao et al.[4] did not started from exogenous resistant gene， but promoted the massive expression of mRNA by silencing microRNAs gene through the insertion of the cloned fragment of CMV RNA3， thereby constructing a good CMVresistant vector for the improvement of the resistance to CMV. Anbinder et al.[5] screened a breeding line ‘TY172’ highly resistant to TLCV， and then mapped the genetic loci controlling TLCV resistance applying polymorphic DNA molecular marker technique and QTLs mapping. The results showed that in ‘TY172’， the resistance was controlled by one unknown major QTL and 4 minor QTLs， among which the major QTL was located on chromosome 4 and had a contribution rate of 39.7%-46.6%， and the minor QTLs were located on chromosomes 1， 7， 9 and 11， respectively， and exhibited the contribution rate of about 12%. With the continuous breeding of TLCVresistant plants， the virus also produces various subtypes itself， such as tomato leaf curl Taiwan virus （TOLCTWV） and Thailand tomato yellow leaf curl virus （TYLCV）. Chen et al.[6] constructed an expression vector by dualvirus RNA interference technology， and could obtain plants resistant to both above viruses.
In 1998， CMVresistant tomato PKTM8805R was developed by Beijing University for the first time. This variety was directed with exogenous CMVCP gene by Agrobacteriummediated method， and once produced a good economic benefit during its commercial planting in Xiamen City， Fujian Province. Research about Trangenic Pestresistant Tomato
Breeding for pest resistance is also an important means for the improvement of tomato yield. Since the 1980s， the research about breeding for pest resistance was started in China， pestresistant tomato germplasm resources were screened， and many excellent pestresistant genes were explored， laying a foundation for the research about further transgenic pestresistant tomato[7]. At present， the genes found and used for the improvement of pest resistance are mainly Bt toxin protein gene from Bacillus thuringiensis and cowpca trypsin inhibitor （CpTI）[8]. The Bt gene is a kind of specific insecticidal gene， which mainly kills Lepidoptera insects. Saker et al.[9] induced the overexperssion of Bt （Cry 2Ab） gene in transgenic gene by Agrobacteriummediated transformation， and the results showed that the Bt tomato expressing Cry 2Ab gene was resistant to Lepidoptera insects including America cotton bollworm and potato stem moth. CpTI gene is kind of gene with widespectrum resistance， and its albumin has insecticidal ability. In 2001， Ghoshal et al.[10] reported the transformation of CpTI gene into plants by direct PEG mediation， and found that under regulation of promoter CaMV 35s， CpTI protein had efficient catalytic activity. Hereafter， CpTI gene is widely used model crop including rice[11]， playing an important role to the improvement of plant pest resistance and the increase of yield.
With the deepening of the research， previous studies also found that the dualgene integrated answering effect is always better than the resistance effect controlled by single gene. Abdeen et al.[12] found that in homozygous tomato lines， the overexpression of PIII protein and carboxypeptidase inhibitor （PCI） gene resulted in higher resistance to larvae of cotton bollworm and Liriomyza trifolii， indicating that the interaction of two kinds of protease inhibitors could better control pests. Chan et al.[13] transformed plant cysteine protease inhibitor （CeCPI） gene and mycosin （PjCHI1） gene into tomato plants by skillfully use of dualgene overexpression system， and induced gene expression under the regulation of promoter pMSPOA， thereby inhibiting the growth of Meloidogyne incognita. Latest studies showed that βglucosidase and digesting enzyme extracted from tomato plants also could inhibit the growth and development of insects[14].
Since 1998， America approved the environmental safety and food safety evaluation of transgenic pestresistant tomato ‘5345’ by Monsanto company. In 2000， ‘5345’ was approved in Canada for eating. The transgenic pestresistant tomato ‘5345’ was bred through the transformation of insectidical Bt proteinCry1Ac into tomato cultivar ‘UC82B’ by Agrobacteriummediated method. It is also a successful case of the application of DNA recombination technology in crop improvement. When Cry1Ac is expressed in transgenic tomato ‘5345’ as endotoxin， it kills Lepidoptera pests with high selectivity， thereby exhibiting specific pestresistant character. Research about Transgenic Stressresistant Tomato
Research about transgenic saltresistant tomato
In recent years， saline and alkaline land in China shows an increasing trend， and plus other reasons such as inappropriate fertilizer and water management， most soil suffers from serious secondary salinization. These reasons lead to the decrease of tomato yield， and seriously restrict the commercialized production of tomato in China. Therefore， the research about the mechanism related to salt resistance in tomato becomes very important. Li et al.[15] found through gel hybridization experiments that after salt stress or osmotic stress induced by polyethylene glycol， monodehydroascorbate reductase （MDHAR） in chloroplast of wild tomato increased remarkably， and photoinhibition in PSII was alleviated through the improvement of ascorbic acid level， so as to induce enhancement of salt resistance in plant. According to this principle， MDHAR gene could be directed to tomato， to improve salt resistance in tomato， but no related studies have been reported. Recently， a type of prolinerich protein was found in plant， which could response to biotic or abiotic stress， especially high salt damage. The transformation of such protein gene into tomato plant could significantly improve the resistance to salt stress[16]. Of course， deeper studies have already been done on dualgene integrated regulation on slat resistance in tomato.
Viveros[17] reported that the overexpression of glyoxalase I （Gly I） and glyoxalase II （Gly II） in tomato plants， and through the detection of the stable expression in transgenic tomato after salt stress， it was demonstrated that GlyI and GlyII genes could enhance the resistance to salt stress through the reduction of oxidative stress capability in plant. However， above experiments did not verify whether the interaction between genes has a synergistic effect in improving salt resistance in tomato. In 2016， CAI et al.[18] reported that in tomato， SlDof gene could be combined at the promoter region of SlSOS1 gene， to regulate the resistance in plant to salt damage together.
Except the transformation of exogenous saltresistant gene， plants also could avoid or alleviate the effect of salt damage to plants through selfregulatory mechanism. Wang et al.[19] expressed a sequence of 3omega fatty acid desaturase gene regulated by CMV35S in a large quantity， then performed Western and Northern blot analysis based on reverse transcriptionPCR quantitative technique， and finally found by electron microscopy observation that LeFAD3 gene could maintain the integrity of cytomembrane， thereby improving the resistance in tomato to salt stress at seedling stage. Research about Transgenic Antifreezing Tomato
Tomato is also a kind of vegetable susceptible to cold injury， which is mainly reflected by the condition that liquid in injured parts produces ice crystals at first at the nursery stage， and the ice crystals grow bigger and cause the death of cells by extruding cell wall. Currently， there are two viewpoints which explain the antifreezing mechanism in plant： one is to transform antifreezing gene to improve tomato resistance， and the other is to add some regulation genes to induce the expression of antifreezing gene.
Kumar et al.[20] transformed the gene coding antifreezing protein （AFP） into tomato plants by Agrobacteriummediated method under the regulation by CaMV 35S， and proved the expression of AFP gene by Southern blot analysis and RTPCR technique. The results showed that the expression of antifreezing gene in carrot could improve the freezing resistance in tomato and improve yield in a large area. Subraman et al.[21] showed that the tomato inoculated with 1aminocyclopropane1carboxylic acid deaminase had higher resistance under cold injury， and freezing induction genes LeCBF1， LeCBF3 and ethylene response transcription factor ETF13 all could promote the expression of antifreezing gene. Latest studies showed that in polyamine transgenic tomato， pathogenyrelated protein PR1b1 could be upregulated under chilling condition to improve plant resistance[22]. In recent years， researchers also found a kind of antifreezing protein known as Maxi， which has no that much water molecules in its core as imagined， but achieves the stressresistant effect by the mechanism of interior folding and dynamic characteristics， which overturns the prediction of conventional proteins by scientists， which deems that the core of ordinary proteins is full of water molecules. The finding of such protein also added new interpretation to antifreezing mechanisms of plant[23].
Except that the expression of resistant genes is changed or regulated by other genes， plant hormones[24-25] also could respond to cold injury， thereby inducing a serious of molecular response mechanisms in plants. Zhu et al.[24] found through different concentrations of GA treatments that GA treatment could effectively alleviate cold injury， but genes involved in gibberellin synthesis pathway were remarkably downregulated. Meanwhile， gibberellin also could affect the response of plant under stress by inducing the biosynthesis of salicylic acid. Of course， in tomato plants， the expression of one gene is always not only a response mechanism of living organism to one kind of stress， but also a kind of comprehensive expression of the integrated effect of various environmental effects to multiple stress systems.
Zhang et al.[26] induced the specific DNA cleavage site of a selected marker gene using Cre/loxP DNA recombination system， thereby allowing the expression of AtIpk2b gene in tomato in a large quantity， and finally， the plant obtained higher resistance to drought， freezing and oxidation. Lyu et al.[27] extracted from Escherichia coli， a kind of trehalose6phosphate synthase/phosphatase fusion gene， which was transformed to tomato under the induction by CaMV 35S promoter. The results showed that compared with wild tomato， transgenic tomato has stronger photosynthesis tolerance under drought and salt stresses. Sun et al.[28] also demonstrated that SlWRKY39 regulates the adaption of tomato to biotic or abiotic stress by activating the expression of genes related to plant pathogenesis and stress. Also， in genetic breeding of crops， Saleem et al.[29] illuminated that the hybridization of different tomato lines （combination of different genotypes） has a remarkable superiority in resisting stress and improving yield in detail.
Several main conditions limiting normal growth of tomato were introduced in detail. In order to well introduce the mutual relation between various stresses during tomato growth and provide reference for tomato breeding， genes responding to stress were depicted in Fig. 1.
Fig. 1 Induced expression of related genes in tomato under stress
Yan AO et al. Research Progress on Transgenic Tomato
Research about Transgenic Quality
In recent years， with the development of economic and the improvement of living standard， people put forward higher requirements for tomato quality. Tomato having good quality often takes good fruit shape， no split， no bite， proper maturation and moderate sweet and sour taste as main indexes. Therefore， studies on the sweetness and sourness of tomato were reviewed below.
Transgenic research about improvement of sweetness of tomato
The improvement of sweetness of transgenic tomato was mainly conducted from two aspects： one is to transform sweetness genes into tomato by the method of directing exogenous genes to improve sweetness； and the other is to regulate the physiological and biochemical pathways such as glycometabolism of plants to change the concentration of sugar. Firsov et al.[30] found thaumatin II gene in Crassocephalum crepidioides， then inserted the cDNA fragment of thaumatin II gene into pBi121 vector under the regulation of CaMV 35S， and finally found the expression of thaumatin II gene in a large quantity by enzyme linked immunosorbent assay. The results also showed that transgenic tomato has better taste quality.
Reddy et al.[31] studied a kind of special sweet protein monellin in Dovyalis hebecarpa， transformed the monellin gene coding the sweet protein into tomato plants under the cocontrol of T7 promoter in E. coli and specific promoter of fruit maturation， and found the expression of monellin protein in a large quantity in plants by enzyme linked immunosorbent assay after normal culture in greenhouse gases. Compared with wild tomato， the sugar content in the transgenic tomato increased remarkably， which plays a positive role in the improvement of tomato flavor. Meanwhile， other genes regulating fruit sweetness are also found in succession.
One latest study shows that two types of transcription factors SlbZI1 and SlbZIP2 in tomato plants have a positive effect on the expression of sugarcoding genes， and further analysis shows that SlbZI1 could activate the activities of asparagine synthetase and proline dehydrogenase， thereby improving the contents of sucrose， glucose and fructose in transgenic plants[32].
Beauvoit et al.[33] studied the relation between enzymes related to glycometabolism process in tomato and vacuole expansion degree. The results showed that during cell division， the activities of fructokinase and phosphofructokinase were improved， and during vacuole expansion， the activities of sucrose synthetase， glucose6phosphate isomerase and ADPglucose pyrophosphorylase were also enhanced. Therefore， during vacuole expansion in cells， soluble sugar in vacuoles accumulates in a large quantity. These studies lay a theoretical basis for research about genes coding related synthetases in glycometabolism process and research about transsugar synthetase gene in tomato. However， at present， there were few reports about such research.
It is generally known that glycolytic pathway in plant cells plays a very important role in the regulation of plant growth and development， and the deficiency of phosphoenolpyruvate carboxykinase （PEPCK） activity has a profound influence on normal physiological metabolism of plant.
Huang et al.[34] initiated the degradation mechanism of mRNA sequence specificity using RNA interference technique， and affected the normal transcription of SlPEPCK gene， thereby repressing the synthesis process of PEPCK. Compared with wild tomato， the sugar content in the RNAinterfered tomato mutant had a remarkable decreasing trend， and the results showed that the deficiency of PEPCK would affect the accumulation of sugar content in tomato plants. In recent years， with the development of molecular biology study， the mechanisms of other synthetases regulating sugar synthesis in tomato plants during related glycolysis process are also known gradually. Meanwhile， with the coming of big data era， bioinformatics are widely applied to plant field. Ikeda et al.[35] revealed the significant changes of carbohydrate content and amino acid metabolism 20 after flowering of tomato， and further found that expression quantities of cell wallbound invertase， sucrose synthetase and glutamate synthase were also higher than wild varieties. They finally concluded that cell wallbound invertase and sucrose synthetase could affect the accumulation of glutamate synthase by regulating sugar concentration in carbon metabolism， thereby improving the sweetness of fruit. Transgenic research about improvement of sourness
Research shows that the sourness of tomato are very important to the color， taste and nutrition of tomato， and therefore， the physiological mechanisms of sourness to plant growth and development is attracting more and more attention. Sun et al.[36] repressed the expression of SINCED1 gene of 9cisepoxycarotenoid dioxygenases （NCED）， thereby affecting the biosynthesis of abscisic acid （ABA）. Experiments showed ABA could promote the decomposition of cell wall by regulating the expression levels of main tissue dissolution genes （SlPG， SlPME， SlXET， SlCels， SlExp）， which layed a theoretical basis for the exploration， cloning and transformation of SlNCED1. Latest studies show that the sugar content and sourness in some tomato mutants are in a dynamic changing process， and under stress， plants could realize the stability in plants through the sugarsourness interconversion mechanism， thereby improving the taste quality of plant. Therefore， it is urgent to explore major genes controlling interconversion reaction[37]. Also， there are studies showing that the mutual antagonism regulation by plant hormones gibberellin GA and abscisic acid ABA is beneficial to the improvement of tomato sourness. Consequently， the exploration of the key genes in the GA and ABA synthesis process becomes the focus of the public[38].
Tomato fruit has a short preservation time and rots rapidly after maturation， thereby losing commodity value. Therefore， in order to prolong the shelf life of tomato and bring convenience to market circulation， the research and development of shelfstable tomato has been conducted since the 1990s by transgenic technology. This work is mainly performed by transforming the antisense gene of tomato polygalacturonic acid （PG）， the antisense gene of ethylene synthetase， the Cry1Ac gene of Bacillus thuringiensis and the CMV coat protein （CP） gene into cultivated gene by Agrobacteriummediated method. In 1992， United States Department of Agriculture and Food and Drug Administration authorized the environmental release of storageresistant tomato "Flavr Savr"， and gave out foodsafety certification in 1994 for food and fodder. The variety is first case of transgenic crop approved from the market. Furthermore， Institute of Microbiology， Chinese Academy of Sciences directed the antisense gene of tomato ethylene synthetase into cultivated tomato by Agrobacteriummediated method， obtaining transgenic maturationdelayed tomato "Dadong 9". In 2000， "Dadong 9" was approved for commercial production in Beijing. Some other common transgenic maturationdelayed tomato varieties include ‘13454’， ‘351N’， ‘8338’， ‘B’， ‘Da’ and ‘F’. Conclusions
In recent 30 years， the gene transformation of tomato is also changed from the directional change of single character to the change of multiple characters， and finally developed towards the direction of medical treatment. Tomato transformed with multiple genes resistant to TMV， CMV， TLCV and AMV have been cloned in China[39]. As early as 1994， transgenic tomato variety "Flavr Savr" entered the market in America， and in China， in 1990， Huazhong Agricultural University started the research and development of transgenic storagetolerant tomato， which was approved by GMO Biosafety Committee， Ministry of Agriculture in 1996， and became the first agricultural biogenetic engineering product approved for commercial production in China， which was designated as "Huafan No. 1"[40]. The gene directed into "Huafan No. 1" is only to delay the maturation of tomato and inhibit the synthesis of partial special protein in tomato， so as to alleviate the degradation of cell wall and softening of fruit. However， with the development of science and technology， breeders obtained nontransgenic maturationdelayed tomato， the superiority of transgenic tomato in storage not only exists， and its low yield becomes a big problem. Plus thick peel and poor taste， transgenic tomato was directly weeded out in the market. Comprehensively， people still doubt whether transgenic technology is safe， the safety of transgenic crop needs to be verified by a long term of experiment as well. Therefore， the commercialization of transgenic tomato is facing bottleneck in Chinese market. With the finding of more and more marker genes， the development of a new generation of molecular marker technology also promoted the exploration， mapping and cloning of new genes， which provides technical support for the finding of more exogenous genes with practical value. Of course， with the rapid advance of sequencing technique， people have been able to find desired target genes at genomewide level， transcriptional level and translational level， which greatly enriches the diversity of germplasm resources of transgenic tomato， laying a foundation for genetic breeding work in future.
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