耐寒相关基因转化华南木薯品种研究
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摘要
木薯(Manihot esculenta Crantz)是世界热带亚热带地区重要的粮食与经济作物之一。传统的育种方式为选育木薯良种方面作出了重要的贡献,但是由于木薯是高度杂合的特质加上种质资源的限制及常规育种方法的局限性,让木薯进一步的遗传改良陷入困境。随着分子生物学研究的深入,基因的鉴定、克隆、重组技术及转基因技术的日趋成熟,利用基因工程手段进行木薯遗传改良,增强木薯抗病虫和耐逆境能力,提高木薯块根产量,改善木薯淀粉品质及降低有害物质的含量等,已成为木薯育种的一种有效手段。木薯是一种抗逆性比较强的热带、亚热带作物,但是地域性的适应特性决定其不能忍受低温的环境。通过农杆菌法将抵抗逆境相关的基因引入木薯基因组,可以改善木薯的耐寒品质,使其能在较低温度的环境中种植,为木薯北移或特异性区域种植提供品种保障。
本研究以四个木薯优良品种华南5号、6号、7号和8号为实验材料,进行了以下方面的研究：建立了木薯无菌苗繁殖系统；木薯体胚的循环诱导及体胚子叶再生培养条件研究；消毒剂种类、取材时间及取材地点对木薯外植体消毒效果的影响；6-BA对木薯单芽茎段生长的影响；2,4-D和picloram对不同品种木薯体胚发生能力的影响；体胚成熟时间对子叶植株再生能力的影响；建立了木薯遗传转化受体体系；利用建立的木薯遗传转化受体系统,采用农杆菌(GV3101)介导对木薯体胚子叶进行遗传转化,并对共培养时间对遗传转化的影响进行研究；用六个植物表达载体对华南木薯优良品种华南5号、6号和8号进行转化,并获得再生植株共1279株；对再生植株进行抗性检测及分子检测；将转基因植株炼苗移栽至花盆,成功移栽了600多株苗；对转基因株系进行组培苗和盆栽苗的低温处理鉴定其低温胁迫下的适应能力。主要研究结果如下：
1.取材时间地点及消毒剂对木薯启动培养消毒影响效果不同,研究表明最佳的消毒方式是：取室内培养的木薯外植体材料,经过洗衣粉水浸泡10 min,流水冲洗20 min,75%酒精浸泡40s,0.1% HgCl2消毒10 min,可达到很好的消毒效果,无菌率在90%以上。
2.6-BA对抑制木薯顶端优势很明显,浓度越高抑制越强。它不仅抑制木薯芽的径向生长,也抑制根的径向生长。但是适宜的浓度可以促使木薯腋芽萌发产生丛生芽。对于需要木薯植株快速成苗的情况,以不添加任何激素的MS基本培养基培养效果最好。
3.两种激素2,4-D(8 mg/l)和picloram(12 mg/l)对四个木薯初级体胚的形成都有很强的促进作用,其效果相当。初级体胚继代后除华南7号木薯外都能达到100%的产胚率,而华南7号得木薯初级体胚却在继代后衰败。
4.CaCl2在木薯初级体胚的诱导过程中作用不明显,而继代后30d就有了明显的结果,实验设计范围内的高浓度CaCl2对继代后的体胚形成有促进作用。15 mmol／l的CaCl2可以保持体胚100d的活力。
5.成熟培养不同时间对木薯体胚子叶器官发生很有影响。成熟10-15 d的木薯体胚子叶器官发生率比较好,诱导频率在70%以上。
6.华南木薯良好的遗传转化体系：将培养10-15d的子叶胚尽量切小,用OD600在0.9-1.1之间的工程菌液进行侵染45 min,然后共培养3d后清洗去菌,转到含500mg/l羧苄青霉素和10mg/l潮霉素的低选择压器官发生培养基上进行初筛及高抗菌素脱菌1周后,再进行500mg/l羧苄青霉素和20mg/l潮霉素正常选择压器官发生培养基筛选2-3周。经过两次筛选的组织已有器官发生,选择有芽器官的组织转到茎器官伸长培养基上培养直到芽长至1-2cm。切下芽接种到无激素培养基上成苗培养。适量扩繁转化苗后,切取1-2cm顶端部分茎段进行10mg/l潮霉素选择压生根筛选。对于选择压下有根形成的转化株系再进行后续的分子检测。该体系适合华南5号,6号,8号;但转化效率略有差别。以华南8号得转化效率最好,华南5号和华南6号效果相当。
7.通过PCR检测的方法对筛选压上形成的转化植株进行了检测,共鉴定出：pVKH-35S-AtGolS2-pA转基因木薯198个PCR阳性株系,其中PCR为阳性且在筛选压上生根的株系有81个。pVKH-35S-AtGolS2-ipt-pA转基因木薯203个阳性株系,其中PCR为阳性且在筛选压上生根的株系有79个。pVKH-35S-CBF3-pA转基因木薯167个阳性株系,其中PCR为阳性且在筛选压上生根的株系有50个。pCA-CP-CBF3-pA转基因木薯123个阳性株系,其中PCR为阳性且在筛选压上生根的株系有78个。pVKH-35S-AtGolS3-pA转基因木薯309个阳性株系,其中PCR为阳性且在筛选压上生根的株系有93个。pCA-CP-CBF3-35S-AtGolS3-pA转基因木薯104个阳性株系,其中PCR为阳性且在筛选压上生根的株系有25个。
8.通过RT-PCR对PCR为阳性且筛选压上可以生根的株系进行对应的检测。结果得到pVKH-35S-AtGolS2-pA转基因木薯65个阳性株系;pVKH-35S-AtGolS2-ipt-pA转基因阳性株系48个；pVKH-35S-CBF3-pA转基因阳性株系39个;pCA-CP-CBF3-pA转基因57个阳性株系;pVKH-35S-AtGolS3-pA转基因木薯64个阳性株系;pCA-CP-CBF3-35S-AtGolS3-pA转基因木薯20个阳性株系。
9.对部分RT-PCR结果为阳性的植株进行了PCR-southern杂交结果也为阳性。这一系列的检测进一步证明了抗寒基因在实验的3个华南木薯品种中得以整合并表达。转基因植株的基因组酶切Southern杂交正在探索中。
10.本文通过对木薯组培苗移栽基质的选择、移栽季节的选择及苗的状态方面的研究,表明适合木薯移栽的经济方法是：采用海南红土+细沙(+腐殖土)=2：2(：1)作为栽培基质,选择在每年的2-3月份,将培养1月的组培苗进行水培闭口3 d,开口4 d炼苗后,移栽,移栽后浇足定根水。通过此法,本研究成功移栽600多株木薯转基因苗。本研究还发现,对越冬木薯组培移栽苗进行春季修剪,留下基本10 cm左右主茎,可以促进木薯苗的快速生长。
11.本研究对转基因木薯组培苗及盆栽苗进行了低温处理检测。通过对转基因木薯组培苗和非转基因组培苗进行低温胁迫观察表型变化,发现在相同的处理条件下,非转基因植株因不能适应低温的环境,完全枯死,而部分木薯转AtGolS2、AtGolS2-ipt、CBF3基因株系却表现出了很强的低温耐受性,植株顶部生长组织并没有被低温完全迫害,恢复室温后又能继续生长。通过对盆栽苗进行4℃低温处理及生理指标的检测发现,部分转AtGolS2、AtGolS2-ipt、CBF3等基因的木薯株系,SOD活性在低温胁迫后增幅非常大,甚至有的超过常温时的3倍,与之相对应的MDA含量比常温下的含量还要低。进一步证明了,基因的转化对改良木薯耐寒品质具有一定的效果。田间耐低温试验将是下一步必须的检测工作。
Cassava(Manihot esculenta Crantz) is one of the most important food and economic crops in tropic and subtropic areas of the world.The traditional breeding method has greatly improved cassava characteristics.As the higher heterozygosity trait and the limitation of cassava germplasm resource, it becomes much difficult to further improvement. With the development of molecular biology, and the mature technologies of gene manipulation, it becomes a useful method to increase cassava resistance to disease, insects and environment stress, or enhance cassava "storage roots" production, starch quality, or decrease harmful matter content in cassava. Cassava has a good ability of stress resistances, but it is short to cold tolerance. In this research, some of cold related genes have been transformed to three cassava cultivars through Agrobacterium to improve its cold resistance.
In this thesis, four cassava cultivars (SC5,SC6, SC7, SC8) were used for the following researches:establishment of cassava in vitro culture system; researches on cassava somatic embryo cycle induction culture and somatic embryo cotyledon organogenesis;the effects of sterilants, sample collecting time and conditions on the cassava sterilization; the effects of 6-BA on growth of cassava seedlings; the effects of 2,4-D and picloram on cassava somatic embryogenesis;the effects of CaCl2 on cassava somatic embryo activity; the effects of mature time of somatic embryo on cotyledon organogenesis; establishment of gene transformation systems for the three cassava cultivars; transformation of six vector into three cassava cultivars by Agrobacterium; research the effects of co-culture time on gene transformation; selection and detection the transgenic cassava plants;transplant the transgenic cassava into pots and detection their ability to low temperature in both in vitro plants and pot plants.The main results are as follows:
All factors such as sample collecting time, area and sterilants affected cassava sterilization. The best method for cassava sterilization was:cassava materials were collected from the room cultured plants;foam water to wash 10 min; fresh water to wash 20 min; 75% ethanol for 40 s;0.1% HgCl2 or 10 min. The sterilization ratio was up to more than 90%.
6-BA can obviously inhibit the growth of cassava seedling tips, which was more serious in higher concentration. The inhibition was on the growth of radial buds and roots. However, suitable content on medium could induce radial buds to form thickly growth buds.For seedling faster growth in cassava in vitro culture system,the MS media with no hormone was the best one.
Both 2,4-D and picloram could induce cassava primary somatic embryos, which was the similar to the 4 cassava cultivars. When the primary somatic embryos were continually subcultured, only three of them (SC5,SC6 and SC8)could obtain 100% secondary somatic embryos.The somatic embryos of SC7 became decline after subculture.
The effects of CaCl2 on cassava primary somatic embryo induction was not obviously clear, but after subculture for 30 days, high concentration of CaCl2(15 mmol/1) could improve somatic embryogenesis, and keep the activity till 100 days.
The mature time of somatic embryos affected the rate of the cotyledon regeneration.10～15 d mature somatic embryo cotyledon regenerated well and rate of the organogenesis was up to 70%.
The suitable gene transformation system for cassava cultivars (SC) was:little pieces of 10～15 d mature somatic embryo cotyledon were infected by Agrobacterium which OD600 was around 0.9～1.1,for 45 min, then were co-cultured on organogenesis media for 3 d and washed away the bacterium; the transferred plantlets were firstly cultured on the low stress selection organogenesis media for 1 week, then on the higher stress selection organogenesis media with 500 mg/1 carbenicillin and 20 mg/1 hygromycin for anyone 2-3 weeks. After selection, many resistant shoot clusters were induced. The resistant shoot clusters were placed on the shoot elongation media till the shoots grew to 1-2 cm in length, then cultured them on the media without any hormone to regenerate plants.For root selection,1-2 cm tip part of the plants were places on the selection media with 10 mg/1 hygromycin for root growth. Finally, the plants with roots were detected by molecular methods. This transformation system was suitable for SC5,SC6 and SC8,while the transgenic ratio was a little different. It was best for SC8,and the same for SC5 and SC6.
After detection of the putative transgenic plants by PCR,198 positive transformants of pVKH-35S-AtGolS2-pA were selected, in which 81 transformants developed roots on stress medium; 203 positive transformants of pVKH-35S-AtGol52-ipt-pA were selected, in which 79 transformants developed roots on stress medium; 167 positive transformants of pVKH-35S-CSF3-pA were selected, in which 50 transformants developed roots on stress medium; 123 positive transformants of pCA-CP-CSF3-pA were selected, in which 78 transformants developed roots on stress medium; 309 positive transformants of pVKH-35S-AtGolS3-pA were selected, in which,93 transformants developed roots on stress medium; 104 positive transformants of pCA-CP-CBF3-35S-AtGolS3-pA were selected, in which 25 transformants developed roots on stress medium.
The positive transformants,which developed roots on stress medium and identified by PCR, were detected by RT-PCR. The result:65 positive transformants from 81 of pVKH-35S-AtGolS2-pA were identified by RT-PCR; 48 positive transformants from 79 of pVKH-35S-AtGolS2-ipt-pA were identified by RT-PCR; 39 positive transformants from 50 of pVKH-35S-CBF3-pA were identified by RT-PCR; 57 positive transformants from 78 of pCA-CP-CBF3-pA were identified by RT-PCR; 64 positive transformants from 93 of pVKH-35S-AtGolS3-pA were identified by RT-PCR; 20 positive transformants from 25 of pCA-CP-CBF3-35S-AtGolS3-pA were identified by RT-PCR.
Parts of the positive transformants were detected by PCR-southern, All of them were showed positive results.After the serials of detection, the cold related genes have been transformed into the 3 cassava cultivar genome (SC5,SC6, SC8).
The most economic methods for cassava transplanting was:The ground substance:red soil+sand(+decay soil);Time:Feb.-Mar. in a year; Plants:1 month old in vitro plants were cultured in water 3 d under closed conditions, then for another 4 d under open conditions;finally, these plants were transferred into the pots with soil., By this method we have obtained 600 transgenic plants.In the spring, clip the transplants which transferred in the winter of last year to 10 cm of stem in length. They grew better.
The low temperature treatment on transgenic cassava in vitro plants and pot plants indicated that:after 4℃treatment for 10 days and recovery for 20 days, all control plants were died, while some of AtGolS2,AtGolS2-ipt、CBF3 transgenic plants were still alive. For the pots plants,after 4℃treatment for 36 h, SOD activity was higher than controls in some of the AtGolS2, AtGolS2-ipt, CBF3 transgenic plants, which the highest was 3 folds of the control plants;meanwhile MDA content was lower than controls in these transgenic plants at natural temperature. Thus, it indicates that the transgenic cassava plants will be better to low temperature.
本研究以四个木薯优良品种华南5号、6号、7号和8号为实验材料,进行了以下方面的研究：建立了木薯无菌苗繁殖系统；木薯体胚的循环诱导及体胚子叶再生培养条件研究；消毒剂种类、取材时间及取材地点对木薯外植体消毒效果的影响；6-BA对木薯单芽茎段生长的影响；2,4-D和picloram对不同品种木薯体胚发生能力的影响；体胚成熟时间对子叶植株再生能力的影响；建立了木薯遗传转化受体体系；利用建立的木薯遗传转化受体系统,采用农杆菌(GV3101)介导对木薯体胚子叶进行遗传转化,并对共培养时间对遗传转化的影响进行研究；用六个植物表达载体对华南木薯优良品种华南5号、6号和8号进行转化,并获得再生植株共1279株；对再生植株进行抗性检测及分子检测；将转基因植株炼苗移栽至花盆,成功移栽了600多株苗；对转基因株系进行组培苗和盆栽苗的低温处理鉴定其低温胁迫下的适应能力。主要研究结果如下：
1.取材时间地点及消毒剂对木薯启动培养消毒影响效果不同,研究表明最佳的消毒方式是：取室内培养的木薯外植体材料,经过洗衣粉水浸泡10 min,流水冲洗20 min,75%酒精浸泡40s,0.1% HgCl2消毒10 min,可达到很好的消毒效果,无菌率在90%以上。
2.6-BA对抑制木薯顶端优势很明显,浓度越高抑制越强。它不仅抑制木薯芽的径向生长,也抑制根的径向生长。但是适宜的浓度可以促使木薯腋芽萌发产生丛生芽。对于需要木薯植株快速成苗的情况,以不添加任何激素的MS基本培养基培养效果最好。
3.两种激素2,4-D(8 mg/l)和picloram(12 mg/l)对四个木薯初级体胚的形成都有很强的促进作用,其效果相当。初级体胚继代后除华南7号木薯外都能达到100%的产胚率,而华南7号得木薯初级体胚却在继代后衰败。
4.CaCl2在木薯初级体胚的诱导过程中作用不明显,而继代后30d就有了明显的结果,实验设计范围内的高浓度CaCl2对继代后的体胚形成有促进作用。15 mmol／l的CaCl2可以保持体胚100d的活力。
5.成熟培养不同时间对木薯体胚子叶器官发生很有影响。成熟10-15 d的木薯体胚子叶器官发生率比较好,诱导频率在70%以上。
6.华南木薯良好的遗传转化体系：将培养10-15d的子叶胚尽量切小,用OD600在0.9-1.1之间的工程菌液进行侵染45 min,然后共培养3d后清洗去菌,转到含500mg/l羧苄青霉素和10mg/l潮霉素的低选择压器官发生培养基上进行初筛及高抗菌素脱菌1周后,再进行500mg/l羧苄青霉素和20mg/l潮霉素正常选择压器官发生培养基筛选2-3周。经过两次筛选的组织已有器官发生,选择有芽器官的组织转到茎器官伸长培养基上培养直到芽长至1-2cm。切下芽接种到无激素培养基上成苗培养。适量扩繁转化苗后,切取1-2cm顶端部分茎段进行10mg/l潮霉素选择压生根筛选。对于选择压下有根形成的转化株系再进行后续的分子检测。该体系适合华南5号,6号,8号;但转化效率略有差别。以华南8号得转化效率最好,华南5号和华南6号效果相当。
7.通过PCR检测的方法对筛选压上形成的转化植株进行了检测,共鉴定出：pVKH-35S-AtGolS2-pA转基因木薯198个PCR阳性株系,其中PCR为阳性且在筛选压上生根的株系有81个。pVKH-35S-AtGolS2-ipt-pA转基因木薯203个阳性株系,其中PCR为阳性且在筛选压上生根的株系有79个。pVKH-35S-CBF3-pA转基因木薯167个阳性株系,其中PCR为阳性且在筛选压上生根的株系有50个。pCA-CP-CBF3-pA转基因木薯123个阳性株系,其中PCR为阳性且在筛选压上生根的株系有78个。pVKH-35S-AtGolS3-pA转基因木薯309个阳性株系,其中PCR为阳性且在筛选压上生根的株系有93个。pCA-CP-CBF3-35S-AtGolS3-pA转基因木薯104个阳性株系,其中PCR为阳性且在筛选压上生根的株系有25个。
8.通过RT-PCR对PCR为阳性且筛选压上可以生根的株系进行对应的检测。结果得到pVKH-35S-AtGolS2-pA转基因木薯65个阳性株系;pVKH-35S-AtGolS2-ipt-pA转基因阳性株系48个；pVKH-35S-CBF3-pA转基因阳性株系39个;pCA-CP-CBF3-pA转基因57个阳性株系;pVKH-35S-AtGolS3-pA转基因木薯64个阳性株系;pCA-CP-CBF3-35S-AtGolS3-pA转基因木薯20个阳性株系。
9.对部分RT-PCR结果为阳性的植株进行了PCR-southern杂交结果也为阳性。这一系列的检测进一步证明了抗寒基因在实验的3个华南木薯品种中得以整合并表达。转基因植株的基因组酶切Southern杂交正在探索中。
10.本文通过对木薯组培苗移栽基质的选择、移栽季节的选择及苗的状态方面的研究,表明适合木薯移栽的经济方法是：采用海南红土+细沙(+腐殖土)=2：2(：1)作为栽培基质,选择在每年的2-3月份,将培养1月的组培苗进行水培闭口3 d,开口4 d炼苗后,移栽,移栽后浇足定根水。通过此法,本研究成功移栽600多株木薯转基因苗。本研究还发现,对越冬木薯组培移栽苗进行春季修剪,留下基本10 cm左右主茎,可以促进木薯苗的快速生长。
11.本研究对转基因木薯组培苗及盆栽苗进行了低温处理检测。通过对转基因木薯组培苗和非转基因组培苗进行低温胁迫观察表型变化,发现在相同的处理条件下,非转基因植株因不能适应低温的环境,完全枯死,而部分木薯转AtGolS2、AtGolS2-ipt、CBF3基因株系却表现出了很强的低温耐受性,植株顶部生长组织并没有被低温完全迫害,恢复室温后又能继续生长。通过对盆栽苗进行4℃低温处理及生理指标的检测发现,部分转AtGolS2、AtGolS2-ipt、CBF3等基因的木薯株系,SOD活性在低温胁迫后增幅非常大,甚至有的超过常温时的3倍,与之相对应的MDA含量比常温下的含量还要低。进一步证明了,基因的转化对改良木薯耐寒品质具有一定的效果。田间耐低温试验将是下一步必须的检测工作。
Cassava(Manihot esculenta Crantz) is one of the most important food and economic crops in tropic and subtropic areas of the world.The traditional breeding method has greatly improved cassava characteristics.As the higher heterozygosity trait and the limitation of cassava germplasm resource, it becomes much difficult to further improvement. With the development of molecular biology, and the mature technologies of gene manipulation, it becomes a useful method to increase cassava resistance to disease, insects and environment stress, or enhance cassava "storage roots" production, starch quality, or decrease harmful matter content in cassava. Cassava has a good ability of stress resistances, but it is short to cold tolerance. In this research, some of cold related genes have been transformed to three cassava cultivars through Agrobacterium to improve its cold resistance.
In this thesis, four cassava cultivars (SC5,SC6, SC7, SC8) were used for the following researches:establishment of cassava in vitro culture system; researches on cassava somatic embryo cycle induction culture and somatic embryo cotyledon organogenesis;the effects of sterilants, sample collecting time and conditions on the cassava sterilization; the effects of 6-BA on growth of cassava seedlings; the effects of 2,4-D and picloram on cassava somatic embryogenesis;the effects of CaCl2 on cassava somatic embryo activity; the effects of mature time of somatic embryo on cotyledon organogenesis; establishment of gene transformation systems for the three cassava cultivars; transformation of six vector into three cassava cultivars by Agrobacterium; research the effects of co-culture time on gene transformation; selection and detection the transgenic cassava plants;transplant the transgenic cassava into pots and detection their ability to low temperature in both in vitro plants and pot plants.The main results are as follows:
All factors such as sample collecting time, area and sterilants affected cassava sterilization. The best method for cassava sterilization was:cassava materials were collected from the room cultured plants;foam water to wash 10 min; fresh water to wash 20 min; 75% ethanol for 40 s;0.1% HgCl2 or 10 min. The sterilization ratio was up to more than 90%.
6-BA can obviously inhibit the growth of cassava seedling tips, which was more serious in higher concentration. The inhibition was on the growth of radial buds and roots. However, suitable content on medium could induce radial buds to form thickly growth buds.For seedling faster growth in cassava in vitro culture system,the MS media with no hormone was the best one.
Both 2,4-D and picloram could induce cassava primary somatic embryos, which was the similar to the 4 cassava cultivars. When the primary somatic embryos were continually subcultured, only three of them (SC5,SC6 and SC8)could obtain 100% secondary somatic embryos.The somatic embryos of SC7 became decline after subculture.
The effects of CaCl2 on cassava primary somatic embryo induction was not obviously clear, but after subculture for 30 days, high concentration of CaCl2(15 mmol/1) could improve somatic embryogenesis, and keep the activity till 100 days.
The mature time of somatic embryos affected the rate of the cotyledon regeneration.10～15 d mature somatic embryo cotyledon regenerated well and rate of the organogenesis was up to 70%.
The suitable gene transformation system for cassava cultivars (SC) was:little pieces of 10～15 d mature somatic embryo cotyledon were infected by Agrobacterium which OD600 was around 0.9～1.1,for 45 min, then were co-cultured on organogenesis media for 3 d and washed away the bacterium; the transferred plantlets were firstly cultured on the low stress selection organogenesis media for 1 week, then on the higher stress selection organogenesis media with 500 mg/1 carbenicillin and 20 mg/1 hygromycin for anyone 2-3 weeks. After selection, many resistant shoot clusters were induced. The resistant shoot clusters were placed on the shoot elongation media till the shoots grew to 1-2 cm in length, then cultured them on the media without any hormone to regenerate plants.For root selection,1-2 cm tip part of the plants were places on the selection media with 10 mg/1 hygromycin for root growth. Finally, the plants with roots were detected by molecular methods. This transformation system was suitable for SC5,SC6 and SC8,while the transgenic ratio was a little different. It was best for SC8,and the same for SC5 and SC6.
After detection of the putative transgenic plants by PCR,198 positive transformants of pVKH-35S-AtGolS2-pA were selected, in which 81 transformants developed roots on stress medium; 203 positive transformants of pVKH-35S-AtGol52-ipt-pA were selected, in which 79 transformants developed roots on stress medium; 167 positive transformants of pVKH-35S-CSF3-pA were selected, in which 50 transformants developed roots on stress medium; 123 positive transformants of pCA-CP-CSF3-pA were selected, in which 78 transformants developed roots on stress medium; 309 positive transformants of pVKH-35S-AtGolS3-pA were selected, in which,93 transformants developed roots on stress medium; 104 positive transformants of pCA-CP-CBF3-35S-AtGolS3-pA were selected, in which 25 transformants developed roots on stress medium.
The positive transformants,which developed roots on stress medium and identified by PCR, were detected by RT-PCR. The result:65 positive transformants from 81 of pVKH-35S-AtGolS2-pA were identified by RT-PCR; 48 positive transformants from 79 of pVKH-35S-AtGolS2-ipt-pA were identified by RT-PCR; 39 positive transformants from 50 of pVKH-35S-CBF3-pA were identified by RT-PCR; 57 positive transformants from 78 of pCA-CP-CBF3-pA were identified by RT-PCR; 64 positive transformants from 93 of pVKH-35S-AtGolS3-pA were identified by RT-PCR; 20 positive transformants from 25 of pCA-CP-CBF3-35S-AtGolS3-pA were identified by RT-PCR.
Parts of the positive transformants were detected by PCR-southern, All of them were showed positive results.After the serials of detection, the cold related genes have been transformed into the 3 cassava cultivar genome (SC5,SC6, SC8).
The most economic methods for cassava transplanting was:The ground substance:red soil+sand(+decay soil);Time:Feb.-Mar. in a year; Plants:1 month old in vitro plants were cultured in water 3 d under closed conditions, then for another 4 d under open conditions;finally, these plants were transferred into the pots with soil., By this method we have obtained 600 transgenic plants.In the spring, clip the transplants which transferred in the winter of last year to 10 cm of stem in length. They grew better.
The low temperature treatment on transgenic cassava in vitro plants and pot plants indicated that:after 4℃treatment for 10 days and recovery for 20 days, all control plants were died, while some of AtGolS2,AtGolS2-ipt、CBF3 transgenic plants were still alive. For the pots plants,after 4℃treatment for 36 h, SOD activity was higher than controls in some of the AtGolS2, AtGolS2-ipt, CBF3 transgenic plants, which the highest was 3 folds of the control plants;meanwhile MDA content was lower than controls in these transgenic plants at natural temperature. Thus, it indicates that the transgenic cassava plants will be better to low temperature.
引文
1.Allen RD,1995,Dissection of oxidase stress tolerance using transgenic plants [J].Plant Physiol,107:1049～1054.
2. Alves AAC,2002, Cassava Botany and Physiology. In:Cassava Biology, Production and Utilization (eds.) Hillocks RJ, Thresh JM and Bellotti AC. CABI Publishing, Oxon, New York.
3.Anderson MD,Busk PK, Svendsen I et al.,2000, Cytochromes P450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin [J].JBiol Chem,275:1966～1975.
4. Angelov MN, Sun J, Byrd GT et al.,1993,Novel characteristics of cassava (Manihot esculenta Crantz),a C3-C4 intermediate photosynthesis species [J].Photosyn Res,38:61～72.
5.Apel K and Hirt H,2004, Reactive oxygen species:Metabolism, oxidative stress, and signal transduction[J].Annu Rev Plant Biol,(55):373～379.
6. Ariizumi T, Kishitani S,Inatsugi R et al.,2002, An increase in unsaturation of fatty acids in phosphatidyl glycerol from leaves improves the rates of photosynthesis and growth at low temperatures in transgenic rice seedlings [J].Plant Cell Physiol,43(7):751～758.
7.Arruda SCC, Souza GM, Almeida M et al,2004, Anatomical and biochemical characterization of the calcium effect on Eucalyptus urophylla callus morphogenesis in vitro [J].Plant Cell, Tissue and Organ Culture,63(2):142-154.
8.Bachmann M, Matile P, Keller F.,1994, Metabolism of the raffinose family oligo saccharides in leaves of Ajuga reptans L. cold acclimation translocation, sink to source transition: discovery of chain elongation enzyme [J].Plant Physical,(105):1335～1345.
9. Barrios EA, Bressani R,1967, Composicion quimica de la rainy de la hoja de algunas variedades de yucca Manihot [J].Turrialba,17:314～320.
10. Bastow R, Mylne JS, Lister C et al.,2004, Vernalization requires epigenetic silencing of FLC by histone methylation [J].Nature,427:164～167.
11.Bellotti AC,2002, Arthopod pests. In:Hillocks RJ and Thresh JM (Eds.)Cassava:Biology, production and Utilization, CABI, Oxon, pp:209-235.
12. Brenac P, Horbowicz M, Downer SM et al.,1997,Raffinose accumulation related to desiccation tolerance during maize (Zeamays L.) seed development and maturation [J].Plant Physiol,150:481-488.
13.Cerqueira YM,1989, Efeito da deficiencia de agua na anatomia foliarde cultivares de mandio ca (Manihot esculenta Crantz). MSc Thesis, UniversidadeFederal da Bahia, Cruz das Almas, Brazil.
14. Chafouleas JG, Bolton WE, Boyd III AE et al.,1982, Calmodulin and the cell cycle: involvement in regulation of cell-cycle progression [J].Cell,28,41～50.
15.Chafouleas JG, Lagace L, Bolton WE et al.,1984, Changes in calmodulin and its mRNA accompany reentry of quiescent cells into the cell cycle [J].Cell,36:73～81.
16. Cheng M, Fry JE, Pang S,1997,Genetic Transformation of wheat mediated by Agrobacterium tumefaciens [J]. Plant Physiol,115(3):971～980.
17.Cheung WY,1980, Calmodulin plays a pivotal role in cellular regulation [J].Science, 207:19-27.
18.Conde P, Sousa A, Costa A et al,2008,A protocol for Ulmus minor Mill. Micropropagation and acclimatization[J].Plant Cell Tiss Organ Cult,92:113～119.
19. Cooke RD, Coursey DG,1981,Cassava:a major cyanidecontaining food crop. In. Vennesland B,Conn EE and Knowles CJ (eds) Cyanide in Biology. Academic Press, New York.
20. Dey PM,Dixon RA,1985,Biochemistry of storage carbohydrates in green plants [M].New York:Academic Press.
21.Dhandapani M, Hong SB, Aswath CR et al.,2008,Regeneration of zoysia grass (Zoysia matrella L. Merr) cv. Konhee from young inflorescences and stem nodes [J].In Vitro Cell Dev Biol-Plant,44:8-13.
22. Dickau R, Ranere AJ, Cooke RG,2007,Starch grain evidence for the preceramic dispersals of maize and root crops into tropical dry and humid forests of Panama [J]. Proc Natl Acad Sci USA,104:3651-3656.
23.Edwards GE, Sheta E, Moore BD et al.,1990, Photosynthetic characteristics of cassava (Manihot esculenta)a C3 species with chloronchymatous bundle sheath cell [J].Plant Cell Physiol,31:1199～1206.
24. Eklund L, Eliasson L,1990, Effects of calcium ion concentration on cell wall synthesis [J]. Journal of Experimental Botany,41:863～7.
25.El-Sharkawy MA, Cock JH,1987,C3-C4 intermediate photosynthesis characteristics of cass ava (Manihot esculenta Crantz).I [J].Gas Exchange Photosyvethesis Research,12:219～236
26. El-Sharkawy MA and Cock JH,1990, Photosynthesis of cassava (Manihot esculeuta) [J].Exeprimental Agriculture,26:325～340.
27. Etienne H, Lartaud M, Carron MP et al.,1997, Use of calcium to optimize long term proliferation of friable embryogenic calluses and plant regeneration in Hevea brasiliensis [J]. JExp Bot,48:129～137.
28.Faiss M, Zalubilova J, Strnad M et al.,1997, Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants [J]. Plant J,12:401～415.
29. Fan Y, Liu B,Wang H et al.,2002, Cloning of an antifreeze protein gene from carrot and its influence on cold tolerance in transgenic tobacco plants [J].Plant Cell Rep,21:296～301.
30. Frischmuth T, Stanley J,1991,African cassava mosaic virus DI DNA interferes with the replication of both genomic components [J].Virology,183:539～544.
31.Georges F, Saleem M, Cutler AJ,1990, Design and cloning of a synthesis gene for the flounder antifreeze protein and its expression in plant cells [J].Gene,91(2):159～165.
32. Ghosh SP, Ramanujam T, Jos JS et al,1988, Tuber Crops. Oxford & TBH Publishing Co., New Dehli,pp:3～146.
33.Gilmour S, Sebolt AM,2000, Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation [J].Plant Physiol,124:1854～1865.
34.Gilmour SJ,Zarka DG, Stockinger EJ et al.,1998,Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptionalactivators as an early step in cold-induced COR gene expression [J].The Plant Journal,16(4):433-442.
35.Gleeson D, LeluWalter MA, Parkinson M.,2005,Over production of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frost [J].Molecular Breeding,15:21～29.
36. Griffth M and Ala P,1992, Antifreeze protein produced endogenously in winter rye leaves [J]. Plant Physiol,100:593～596.
37.Gupta AS, Webb RP, Holaday AS et al.,1993,over expression of superoxide dismutase protects plants from oxidature stress [J].Plant Physiol,103:1067～1073.
38.Haake V, Cook D, Riechmann JL et al.,2002, Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis [J].Plant Physiol,130(2):639～648.
39. Hayashi H and Alia ML,1997, Transformation of Arabidopsis thaliana with the codA gene for choline oxidase:accumulation of glycinebetaine and enhanced tolerance to salt and cold stress [J].Plant,12:133.
40. Heather MW, Louise VM, Olga S et al,2003, Functional characterization of two cytochrome b5 fusion desaturases from Anemone leveillei:the unexpected identification of a fatty acid A6 desaturase[J].Planta,217:983～992.
41.Hewelt A, Prinsen E, Schell J et al.,1994, Promoter tagging with a promoterless ipt gene leads to cytokinin-induced phenotypic variability in transgenic tobacco plants:implications of gene dosage effects [J].Plant J,6:879～891.
42. Highttower R, Baden C,1991,Expression of antifreeze proteins in transgenic plant [J].Plant Mol Biol,17:1013～1021.
43.Highttower R, Baden C,1991,Expression of antifreeze proteins in transgenic plant [J].Plant Mol Biol,17:1013～1021.
44.Hillocks RJ,2002, Cassava in Africa. In:Hillocks RJ, Thresh JM, Bellotti AC (eds) Cassava: biology, production and utilization. CABI Publishing, New York, pp:41-54.
45.Hinesley LE, Pharr DM, Snelling LK et al.,1992, Foliar raffinose and sucrose in four conifer species:relationship to seasonal temperature [J]. Am Soc Hortic Sci,(117):852～855.
46.Holmberg N,2001,Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco [J].Gene,275(1):115～124.
47. Hong Y and Stanley J,1996, Virus resistance in Nicotiana benthamiana conferred by African cassava mosaic virus replication associated protein (ACI)[J].Mol Plant Microbe Interact, 9:219～225.
48.Horsch RB,Fraley RT, Rogers SG et at.,1984, Inheritance of function foreign genes in plants [S].Science,223:496～498.
49. Howeler KH,Cadavid LF,1983,Accumulation and distribution of dry matter and nutrients during a 12 month growth cycle of cassava [J].Field Crop Research,7:123～139.
50. Hsieh TH, Lee JT, Yang PT et al.,2002, Heterology expression of the Arabidopsis C-repeat/dehydration response element binding Factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato [J].Plant Physiol,129:1086～1094.
51.Hu T, Metz S, Chay C et al.,2003,Agroobacteriuim-mediated large-scale transformation of wheat(Triticum aestivum L.) using glyphosate selection [J].Plant Cell Rep,21:1010～1019.
52.Huang J, Hirji R, Adam L et al.,2000, Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants:metabolic limitations [J].Plant Physiol, 122:747～756.
53. Huang T, Nicodemus J, Zarka DG et al.,2002, Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature [J].Plant Molecular Biology,50:333～344.
54. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG et al,1998,Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance [J].Science,280(5360):104～106.
55.Jansen MAK, Booij H, Schel JHN et al.,1990, Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures [J].Plant Cell Reports,9:221～223.
56. Jo UA, Murthy HN, Hahn EJ et al.,2008,Micropropagation of Alocasia amazonica using semisolid and liquid cultures [J].In Vitro Cell Dev Biol-Plant,44:26～32.
57.Johanson U, West J, Lister C et al,2000, Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time [J]. Science,290:344～347.
58.John D,Plant Genetic Transformation and Gene Expression A Laboratory Manual. Blackwell Scientific Publications Oxford London Edingburgh.
59. Kasuga M, Liu Q, Miura S et al,1999, Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress inducible transcription factor [J]. Nature Biotechnology,17: 287～291.
60. Katia H,Katia and A B,1993,Terence Enhance transformation of tomato co-cultivated with Agrobacterium tumefaciens C58el Rifr:pGSR.1161 in the presence of acetosyringone [J]. Plant cell report,12:422～425.
61.Keating BA, Evenson JP, Fukai S,1982b, Environmental effects on growth and development of cassava (Manihot esculenta crantz).Ⅲ.Assimilate distribution and storage organ yield. Field Crops Research,5:293～303.
62. Keating BA, Evenson JP, Fukai S,1982, Environmental effects on growth and development of cassava (Manihot esculenta Crantz) I. Crop development [J].Field Crops Research, 5:271～281.
63.Khodakovskaya M, McAvoy R, Peters J et al.,2006, Enhanced cold tolerance in transgenic tobacco expressing a chloroplast ω3 fatty acid desaturase gene under the control of a cold inducible promoter [J]. Planta,223:1090-1100.
64. Kim JH,Cetiner S, Jaynes JM,1992, Enhancing the nutritional quality of crop plants: design and expression of an artificial plant storage protein gene. In:Bhatnager D and Cleveland TE (Eds.) Molecular Approaches to Improving Food Quality and Safety, Avi Books,New York,1-36.
65.Kirsten J,Saren B,Peter K,2005,Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers.Distribution of cyanogenic glucosides, their site of synthesis and transport and blockage of the biosynthesis by RNA interference technology [J].Plant Physiol, 139:363～374.
66. Kochetov AV,2004, Suppressor of proline dehydrogenase Gene, Are characterized by higher proline content and cytoplasm osmotic pressure [J].Russian Journal of Genetics,2:216～218.
67.Kodama H,1995,Fatty acid desaturation during chilling acclimation is one of the factors involved in conferring low temperature tolerance to young tobacco leaves [J].Plant Physiol, 107(4):1177～1185.
68.Kumar S, Dhingra A, Danlel H,2004, Plastid expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confer enhanced salt tolerance [J].Plant Physiol,136:2843～2854.
69. Ladino J, Echeverry M, Mancilla L et al.,2002, Genetic transformation of cassava: confirmation of transgenesis in clone 60444 and analysis of CRY1Ab protein in transgenic lines. Preliminary data on transformation of farmer preferred cultivars SM12199 and CM33064. Annual Report, CIAT, Cali,Colombia.
70. Li Q, Robson PR, Bettany AJE et al.,2004, Modification of senescence in ryegrass transformed with ipt under the control of a monocot senescence-enhanced promoter [J].Plant Cell Rep,22:816～821.
71.Li Y, Hagen G, Guilfoyle TJ,1992, Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs [J].Dev Biol,153:386～395.
72.Li,HQ, Sautter C, Potrykus I,1996, Genetic transformation of cassava (Manihot esculenta Crantz)[J].Nature Biotech.14:736～740.
73.Lilius C,Holmberg N, Bulow L,1996, Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase [J].Biotechnology,14:177.
74. Liorente F, Lopezc RM,Catala R et al,2002, A novel cold inducible gene from Arabidopsis, RCI3,encodes a peroxidase that constitutes a component for stress tolerance [J].Plant J, 32:13～24.
75.Liu JJJ, Krenz DC, Galvez AF et al.,1998,Galactinol synthase increased enzyme activity and levels of mRNA due to cold and desiccation [J].Plant Sci,134:11～20.
76. Liu Q, Kasuga M, Sakuma Y et al.,1998,Two transcription factors:DREB1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in droughtand low temperature responsive gene expression, respectively, in Arabidopsis [J].Plant Cell,10:1391～1406.
77.Lorenzi JO,1978,Absorcao de micronutrients a acumulacao de material seca para duas cultivares do mandioca.MSc Thesis Universidade do Sao Paulo, Escola Supcrior de Agricultura Luis de Queiroz, Piracicaba,Brazil,92.
78.Ma J and Liu D,1996, Cloning of Spinach SAD gene, its construction and transformation to tobacco [J].Supplement to Plant Physiol,111(2):814.
79. Mahon JD, Lowe SB,Hunt LA et al.,1977,Envionmental effects on photosynthesis and transpiration in attached leaves of cassava (Manihot esculenta Crantz) [J].Photosnthetica, 11:121-130.
80. Makunga NP and Staden J,2008,An efficient system for the production of clonal plantlets of the medicinally important aromatic plant (Salvia africanalutea L.) [J]. Plant cell Tiss Organ Cult,(92):63～72.
81.Martinea UB,1995,Highsolids tomato shows promise in the field [J].Agbiotech News and Information,7(5):104～105.
82.Martineaub,1995,High-solid stomato shows promise in the field [J].Agbiotech News and Information,7(5):104-105.
83.MC Kersie BD,Chen Y, Beus M et al.,1993,Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.) [J].Plant Physiol,
103(4):1155～1163.
84. Mckersie BD, Chen Y, Beus M et al.,1993,Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.) [J].Plant Physiol,103(4):1155-1163.
85.McMahon JM, White WLB, Sayre RT,1995,Cyanogenesis in cassava (Mahihot esculenta Crantz) [J].Journal of Experimental Botany,46:731～741.
86. Medina J, Bargues M, Terol J et al.,1999, The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration [J].Plant Physiol,119:463～470.
87. Meyer K,1999, A leucinerich repeat protein of carrot that exhibits antifreeze activity [J]. FEBS Letters,447:171～178.
88.Miceli FAG, Estudillo AD, Archila MA et al.,2008,Optimization of Renealmia mexicana (Klotzsch ex. Petersen)cultivation in vitro[J].In Vitro Cell Dev Biol-Plant,44:32-39.
89. Mitten DH, Redenbaugh MK, Sovero M et al.,1992, Safety assessment and commerciali-zation of transgenic fresh tomato products and transgenic rape seed oil products [R].
90. Mkpong O, Yan H, Chism G et al.,1990, Purification, chatacterization and localization of linamarase in cassava [J].Plant Physiol,15:176～181.
91.Monger WA, Seal S, Cotton S,2001,Identification of different isolates of cassava brown streak virus and development of a diagnostic test [J].Plant Path,50:768～775.
92. Montoro P, Etienne H, Micbaux-Ferriere N et al.,1993,Callus friability and somatic embryogenesis in Hevea brasiliensis [J].Plant Cell Tissue and Organ Culture,33:331～338.
93.Morgan AJ, Cox PN, Turner DA et al.,1987, Transformation of tomato using an Riplasmi dvector [J].Plant science,49:37～49.
94. Motohashi T, Toda MI, Kondo K,2008,Adventitious embryo formation derived from zygotic embryos in Cycas revoluta [J].Plant biotechnology,25:589～591.
95.Munyikwa T, Raemakers C, Schreuder M et al.,1998,Pinpointing towards improved transformation and regeneration of cassava [J].Plant Sci,135:87～101.
96. Murata N,Ishizaki NO, Higashi S et al.,1992, Genetically engineered alteration in the chilling sensitivity of plants[J].Nature,356:710～713.
97.Nanjo T, Kobayashi M, Yoshiba Y et al.,1999, Antisense suppression of proline degradation ilmproves tolerance to freezing and salinity in Arabidopsis thaliana [J], FEBS Lett,461: 205～210.
98.Nilgun, Mkeith ET, Richard SN et al.,1987, Expression of alfalfa mosaic virus coat protein gene confers cross protection in transgenic tobacco and tomato plant [J]. The EM BO Journal, 6(5):181～188.
99. Nishizawa A, Yabuta Y, Shigeoka S,2008,Galactinol and raffinose constitute a novel function to protect plants from oxidative damage [J].Plant Physiol,147:1251～1263.
100. Nomura K, Komamine A,1986, Somatic embryogenesis in cultured carrot cells [J]. Development Growth and Differentiation,28:511～517.
101.Nweke FI, Spencer D, Lynam JK,2002, Cassava Transformation:Africa's Best Kept Secret East Langsing. Michigan Sate University Press.
102.Obrien G, Taylor A, Poulter N,'1991,Improved enzymatic assay for cyanogens in fresh and processed cassava [J].JSci Food Agric,56:277～289.
103.Oelsligle D,1975, Accumulation of dry matter, nitrogen, phosphorus and potassium in cassava(Manihot esculenta Crantz)[J].Turrialba,25:85～87.
104. Oh SJ, Song SI, Kim YS et al,2005, Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth [J].Plant Physiol, 138:341～351.
105.Olsen KM,Schaal BA,1999, Evidence on the origin of cassava:phylogeography of Manihot esculenta [J].Proc Natl Acad Sci USA,96:5586～5591.
106.Panikulangara TJ, Eggers-Schumacher G, Wunderlich M et al.,2004, Galactinol synthasel:a novel heat shock factor target gene responsible for heatinduced synthesis of raffinose family oligosaccharides in Arabidopsis [J].Plant Physiol,136:3148～3158.
107. Parvanova D,Popova A, Zaharieva I et al.,2004, Low temperature tolerance of tobacco plants transformed to accumulate proline, fructans or glycine betaine.Variable chlorophyll fluorescence evidence [J].Photosynthetica,42(2):179～185.
108.Pierrobon D,Virgilio FO, Pozzea T,1990, Structural and functional aspects of calcium homeostasis in eukaryotic cells [J].European Journal of Biochemistry,193:559～622.
109. Pilonsmits EAH, Ebakamp MJM, Jenken MJW et al.,1995,Improved performance of transgenic fructan accumulating tobacco under drought stress [J].Plant Physiology, 107:125-130.
110. Pita JS,Fondong VN,Sangare A,2001,Recombination, pseudore combination and synergism of geminiviruses are determinant keys to the epidemic of severe cassava mosaic disease in Uganda [J].J Gen Virol,82:655～665.
111.Plumbley RA, Rickard JE,1991,Postharvest deterioration of cassava [J].Tropical Sci, 31:295～303.
112. Polashock J, Lipson D,1993,Expression of yeast Δ9 fatty acid desaturase activity enhances chilling resistance in tobacco [J].Supplement to Plant Physiol,102:160.
113.Poovaiah BW, Reddy ASN,1987, Calcium messenger system in plants [J]. CRC in Plant Science 6:47～103.
114. Raemakers CJJM,1993,Primary and cyclic embryogenesis in cassava.PhD thesis, Wageningen Agricultural University, Wageningen, The Netherlands.
115.Raemakers CJJM, Jacobsen E, Visser RGF,1997, Micropropagation of Manihot esculenta (Crantz) cassava. In:Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 39. High tech and micropropagation. Springer, Berlin Heidelberg New York, pp 77～102.
116. Raemakers C, Schreuder M, Pereira I et al,2003,Production of amylose-free cassava plants by genetic modification. In:Fauquet CM, Taylor NJ (eds.) Cassava:An Ancient Crop for Modern Times, Proceedings of the 5th International Meeting of the Cassava Biotechnology Network.
117. Reilly K, Gomez Vasquez R, Buschmann H et al,2003,Oxidative stress responses during cassava postharvest physiological deterioration [J].Plant Mol Biol,53:669～685.
118.Rogers DJ,1965,Some botanical and ethnological considerations of Manihot esculenta [J]. Econ Bot,194:769～773.
119. Roosens NH, Al Bitar F, Loenders K et al.,2002, Over expression of ornithine-δ-aminotrans-ferse increases proline biosynthesis and confers osmotolerance in transgenic plants [J].Mol Breed,9:73～80.
120. Rupp HM, Frank M, Werner T et al,1999, Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem [J].Plant J,18:557～563.
121.Samis K, Bowley S, Mckersie BD,2002, Pyramiding Mrisuperoxide dismutase transgenes to improve persistence and biomass production in alfalfa [J].Jexpbot Oxford:Oxford University Press,53(372):1343～1350.
122. Saravitz DM,Pharr DM, Carrer TE,1987, Galactinol synthase activity and soluble sugarsin developing seeds of four soybean gene types [J].Plant Physical, (83):185～189.
123. Sarria R, Torres E, Angel F et al,2000, Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation [J].Plant Cel Rep,19:339～344.
124. Sato Y, Murkami T, Funatsuki H et al,2001,Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings [J].J Expe Bot,52:145～151.
125.Schopke C, Taylor N, Carcamo R et al,1996,Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures [J]. Nature Biotech,14:731～735.
126. Shinwari ZK, Nakashima K, Miura S et al,1998,An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low temperature responsive gene expression [J]. Biochem Biophys Res Commun,250:161～170.
127.Silva P, Ricardo CPP,1992, β-Fructosidases and in vitro dedifferentiation-redifferentia-tion of carrot cells[J].Phytochemistry,31:1507～1511.
128.Siritunga D, AriasGarzon D,White W et al,2004, Overexpression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification [J].Plant Biotech J, 2:37-43.
129. Siritunga D, Sayre RT,2003,Generation of cyanogens-free transgenic cassava [J].Planta, 217:367～373.
130.Smigocki A, Neal JWJ,McCanna I et al,1993,Cytokinin-mediated insect resistance in Nicotiana plants transformed with the ipt gene [J].Plant Mol Biol,23:325～335.
131.Smigocki AC,1991,Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene [J].Plant Mol Biol,16:105～115.
132.Stamp JA, Henshaw GG,1982, Somatic embryogenesis in cassava [J].Z P glanzen physiol, 105:183～187.
133.Stocking EJ, Gilmour SJ, Thomashow MF,1997,Arabidopsis thaliana CBF1 encodes an AP2 domaincontaining transcriptional activator that binds to Crepeat/DRE, a cisacting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proc Nat Acad Sci USA,94:1035～1040.
134. Su J, Hirji R, Zhang L et al.,2006, Evaluation of the stressinducible production of choline oxidase in transgenic rice as a strategy for producing the stress protectant glycine betaine [J]. J Expe Bot,57(5):1129-1135.
135.Taji T, Ohsumi C, Hchi S et al.,2002, Important roles of drought and cold inducible genes for galactinol syntheses in stress tolerance in Arabidopsis thaliana [J].Plant Jour, 29(4):417-426.
136.Tavora FJAT, Melo FIO, Pinho JLN et al,1995,Yield crop growth rate and assimilatory characteristics of cassava at the coastal area of Ceara [J].Revista Brasileira de Fisiologoa hegetal,7:81～88.
137.Thomashow M F,1998,Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance [J].Science,280:104～106.
138.Thomashow MF,1999, Plant cold acclimation:freezing tolerance genes and regulatory mechanisms[J].Annu Rev Plant Physiol Plant Mol Biol,50:571～599.
139. Thresh JM, Fargette D,1994, Effects of African cassava mosaic geminivirus on the yield of c assava [J].Trop Sci,34:26～42.
140. Tian HQ, Yuan T,2000, Calcium function in fertilization process in angiosperms [J].Acta Phytophysiol Sin,26:369～380.
141.Timmers ACJ, Schel JHN(1991).Localization of cytosolic Ca2+ during carrot somatic embryogenesis using confocal scanning laser microscopy. In:Karssen CM, van Loon LC, Vreugdenhil D (eds).Progress in plant growth regulation. Dordrecht:Kluwer Academic Publishers,347～353.
142.Touchell DH, Chiang VL, Tsai CJ,2002, Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification [J].Plant Cell Rep,21:118～224.
143.Ueno 0 and Agarie S,1997,The intercellular distribution of glycine decarboxylasre in leaves of cassava in relation to the photosynthetic mode and leaf anatomy [J].Japanese Journal of crop Science,66:268-278.
144. Ugent D, Pozorski S, Pozorski T,1986, Archaeological manioc (Manihot) from coastal Peru [J].Econ Bot,40:78～102.
145.Urrutia ME, Duman JG, Knight CA,1992, Plant themal hysteresis proteins [J].Biochim Biophy Acta,1121(1):199～206.
146. Vargas Bonilla HL, Bolivar LR, Arias V et al.,2002, Nataima31 variedad de yucca (Manihot esculenta Crantz) resistant a mosca blanca (Aleurotrachelus socialias Bondar) para el valle calido del Alto Magdalena. Publication CORPOICA Regional Seis.Codigo 28306322002. A. Postal 064, El Espinal, Tolima, Colombia.
147. Verdus MC, Dubois T, Dubois J et al.,1993,Ultra structural changes in leaves of Cichorium during somatic embryogenesis [J].Annals of Botany,11:375～383.
148.Vladinir S,Larry G, Prince A et al.,2006, Agrobacteriummediated transformation of seeding derived maize callus [J].Plant Cell Reports,25:320～328.
149.Wada H, Gombos Z, Murata N,1990, Enhancement of chilling tolerance of facyano bacterium genetic manipulation of fatty acid desaturation [J].Nature,347:13,200.
150. Wallis JG, Wang H, Guuerra DJ et al.,1997, Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures [J].Plant Molecular Biology, 35(3):323～330.
151.Wallis JG, Wang HY, Daniel J,1997, Structure, function and evolution of antifreeze proteins [J].Plant Molecular Biology,35:323～330.
152. Wheatley CC, Chuzel G,1993,Cassava:the nature of the tuber and use as a raw material. In: Macrae R, Robison RK and Saddler MJ (Eds.) Encyclopedia of Food science, Food technology and Nutrition. Academic Press, San Diego, CA. pp:734～743.
153.Wheatley CC, Orrego JI, Sanchez T et al.,1993,Quality evaluation of cassava core collection at CIAT. In:Roca WM, Thro AM (eds). Proceedings of the Girst International Scientific Meeting of the Cassava Biotechnology Net, work. CIAT, Cartegena, Colombia. pp:255-264.
154. White W, AriasGarzon D, Mahon JM et al.,1998,Cyanogenesis in cassava:the role of hydroxynitrile lyase in root cyanide production [J].Plant Physiol,116:1219～1225.
155. Worrall D, Elias L, Ashford D et al.,1998, A carrot leucine rich repeat protein that inhibits ice recry stallization [J]. Science,282(2):115～117.
156. Yamaguchi-Shinozaki K,Shinozak K,2006, Transcriptional regulatory networks in cellular re sponses and tolerance to dehydration and cold stresses[J].Annu Rev Plant Biol,57:781～803.
157.Yang HY,1999, The role of calcium in the fertilization process in flowering plants [J].Acta Bot Sin,41:1027～1035.
158.Yokio S, Higashi SL, Kishitani S,1998, Introduction of the cDNA for Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acid and chilling tolerance of photosynthesis on rice [J].Mol Breeding,4:269～275.
159.Zhang P, Gruissem W,2004, Production of Transgenic cassava (Manihot esculenta Crantz). In:Cuitis IS.(ed.),Transgenic crops of the world-essential protocols,301～319.
160. Zhang P, Jaynes J, Potrykus I et al,2003,Transfer and expression of an artificial storage protein(ASP1)gene in cassava (Manihot esculenta Crantz) [J].Transgenic Res,12:243～250.
161.Zhang P, Vanderschuren H, Futterer J et al.,2005,Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes [J].Plant Biotechnol J,385～391.
162.Zhao J, Ren W, Zhi D et al,2007,Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress [J].Plant Cell Rep,26(9):1521～1528.
163.陈儒钢,巩振辉,逯明辉等,2008,植物抗寒基因工程研究进展[J].西北植物学报,28(6)：1274-1280.
164.陈旭微,杨玲,章艺,2004,类脂对植物生长和发育的作用[J].植物生理学通讯,40(3)：373-378.
165.陈永波,赵清华,2005,魔芋试管苗批量生产过程中外植体消毒灭菌技术研究[J].氨基酸和生物资源，27(3)：27-29.
166.德庆措姆,布穷,2000,银白杨组织培养中外植体消毒时间初探[J].辽宁林业科技，4：29,37.
167.段永波,赵德刚,赵丰兰等,2007,低温胁迫下ipt基因水稻某些生理特性研究[J],山地农业生物学报,26(2)：95-98.
168.樊军锋,2002,mtlD/gutD双价耐盐基因转化杨树、猕猴桃的研究[T].西北农林科技大学博士学位论文.
169.冯金玲,陈辉,杨志坚等,2006,锥栗组织培养外植体消毒和选抒[J].福建林学院学报，26(1)：22-26.
170.付永彩,刘新仿,曹守云等,1999,水稻中抑制衰老的嵌合基因的基因枪转化和表达分 析[J].农业生物技术学报，(1)：17-22.
171.付永彩,孙传清,李自超等,2000,农杆菌介导的抑制衰老的嵌合基因转化籼稻的研究[J].农业生物技术学报，8(1)：28,32.
172.耿飒,麻密,李国凤等,2000,ipt基因定位表达对转基因烟草育性的影响[J].植物学报，42(2)：217-220.
173.黄洁,李开绵,叶剑秋等,2008,我国的木薯优势区域概述[J].广西农业科学，39(1)：104-108.
174.黄永芬,汪清胤,付桂荣等,1997,美洲拟鲽抗冻蛋白基因(afp)导入番茄的研究[J].生物化学杂志，13(4)：418-42.
175.贾庚祥,2002,BADH基因转化番茄提高耐盐性的研究[T].中国科学院研究生院植物研究所博士学位论文.
176.焦芳婵,2001,转基因改良植物的胁迫耐性[J].生物技术通讯，12(2)：137.
177.金建凤,高强,陈勇等,2005,转移拟南芥CBF1基因引起水稻植株脯氨酸含量提高[J].细胞生物学杂志，27(1)：73-76.
178.金万梅,董静,尹淑萍等,2007,转移拟南芥CBF1基因引起水稻植株脯氨酸含量提高[J].西北植物学报，27(2)：223-227.
179.赖玉洁,路丙社,陈书明等,2007,青榨槭外植体消毒方法初步研究[J].河北林果研究，22(2)：143-146.
180.李开绵,林雄,黄洁,2001,国内外木薯科研发展概况[J].热带农业科学，(1)：56-60.
181.林茂,闫海霞,眭顺照等,2008,植物CBF转录因子及其在基因工程中的应用[J].广西农业科学，39(1)：21-25.
182.林雄,李开绵,1992,海南岛木薯种质资源考察报告[A].华南热带作物科学研究院,中国农业科学院作物品种资源研究所.海南岛作物(植物)种质资源考察文集[C].北京：农业出版社.
183.刘雪红,刘俊华,张孝霖,2009,冬枣组织培养的消毒方法[J].北方园艺，2：97-98.
184.罗小英,崔衍波,邓伟等,2004,超量表达苹果酸脱氢酶基因提高苜蓿对铝毒的耐受性[J].分子植物育种，2(5)：621-626.
185.马国华,许秋生,羡蕴兰,1998,从木薯嫩叶直接诱导初生体细胞胚胎发生和芽的形成[J].植物学报，40(6)：503-507.
186.马华升,孙玉强,童建新等,2008,千层塔组织培养中外植体消毒灭菌研究初报[J].杭州农业科技，3：15-18.
187.毛自朝,于秋菊,甄伟等,2002,果实专一性启动子驱动ipt基因在番茄中的表达及其对番茄果实发育的影响[J].科学通报，47(6)：444-448.
188.酶、超氧歧化酶活性测定[J].哈尔滨师范大学自然科学学报，14(4)：188-193.
189.孟凤,郁松林,郑强卿等,2008,甜菜碱与植物抗逆性关系之研究进展[J].中国农学通 报，24(4)：225-228.
190.沈漫,王明麻,黄敏仁,1997,植物抗寒机理研究进展[J].植物学通报，14(2)：1-8.
191.汤绍虎,孙敏,周启贵等,2004,采用正交设计快速获得梨无菌外植体的研究[J].西南师范大学学报(自然科学版),29(2)：282-284.
192.王多佳,苍晶,牟永潮等,2009,植物抗寒基因研究进展[J].东北农业大学学报,40(10)：134-138.
193.王关林,李铁松,方宏筠等,2004,番茄转果聚糖合酶基因获得抗寒植株[J].中国农业科学，37：1193-1197.
194.王延玲,丰震,赵兰勇等,2004,仙客来花药培养外植体消毒研究[J].山东林业科技，4：8-9.
195.王子成,李忠爱,邓秀新,2005,柑橘成年态茎段外植体消毒方法研究[J].河南大学学报：自然科学版,35(2)：57-60.
196.韦善君,孙振元,巨关升等,2005,冷诱导基因转录因子CBF1的组成型表达对植物的抗寒性及生长发育的影响[J].核农学报，19(6)：465-468.
197.吴坤林,曾宋君,张志胜等,2008,番木瓜外植体消毒方法研究[J].福建农林科技，35(3)：76-79.
198.杨晓红,陈晓阳,2006,果聚糖对植物抗逆性的影响及相应基因工程研究进展[J].华北农学报,21(1)：6-11.
199.姚庆荣,2007,木薯遗传转化体系的建立与优化及转AGPase基因的研究[T].华南热带农业大学博士学位论文.
200.尹明安,崔鸿文,樊代明等,2001,抗冻蛋白及其在植物抗冻基因工程中的应用[J].西北植物学规21(1)：8-13.
201.于晓红,朱勇清,陈晓亚等,2000,种子特异表达ipt转基因棉花根和纤维的改变[J].植物学报，42(1)：59-63.
202.袁维风,徐德聪,钱玉梅,2009,基因抗寒基因及基因工程研究[J],宿州学院学报，24(6)：109-112.
203.张鹏,2008,能源木薯种质资源面临的问题与解决策略[J].生物产业技术，5：25-30.
204.张赛群,叶志彪,吴昌银等,1999,异戊烯基转移酶基因导入番茄及转基因植株再生[J].园艺学报，26(6)：376-379.
205.张玉红,曲伟娣,2008,培养条件对黄檗快速繁殖影响的研究[J].植物研究，28(2)：236-239.
206.张钰,吴加林,黄永芬等,1998,转美洲拟蝶抗冻蛋白基因(afp)番茄过氧化物酶、过氧化氢酶、超氧歧化酶活性测定[J].哈尔滨师范大学自然科学学报，14(4)：188-193.
207.赵微平,1996,植物基因组构建、表达和调控[M].北京：首都师范大学出版社.
208.甄伟,陈溪,孙思洋等,2000,冷诱导基因的转录因子CBF1转化油菜和烟草及抗寒性鉴定[J].自然学进展，10(12)：1104-1108.
209.郑丽,李名扬,晁岳恩等,2005,根癌农杆菌介导ipt基因对切花菊的遗传转化[J].农业生物技术学报，13(1)：26-31.
210.钟蓉,刘玉乐,朱峰等,1996,1TA29Bar基因导致油菜雄性不育的研究[J].植物学报，38(7)：582-585.
211.钟胜,2008,抗寒相关基因CBF3、ICE1及AtGolS3转基因拟南芥耐寒效果评价[T].华南热带农业大学硕士学位论文.
212.周丽娟,王丹,黄海涛等,2008,费约果外植体灭菌及愈伤组织的诱导[J].热带亚热带植物学报，16(2)：179-183.
213.朱淑颖,陆颂规,梁承邺等,2007,草珊瑚组培中外植体消毒方法的研究[J].福建林业科技，34(4)：82-89.
214.朱文丽,2006,木薯胚胎发生再生植株与离体保存技术的初步研究[T].华南热带农业大学硕士学位论文.
215.朱晔荣,达来,2003,抗冻蛋白基因转化植物的研究展望[J].内蒙古大学学报(自然科学版),2003,34(1)：97-101.
2. Alves AAC,2002, Cassava Botany and Physiology. In:Cassava Biology, Production and Utilization (eds.) Hillocks RJ, Thresh JM and Bellotti AC. CABI Publishing, Oxon, New York.
3.Anderson MD,Busk PK, Svendsen I et al.,2000, Cytochromes P450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin [J].JBiol Chem,275:1966～1975.
4. Angelov MN, Sun J, Byrd GT et al.,1993,Novel characteristics of cassava (Manihot esculenta Crantz),a C3-C4 intermediate photosynthesis species [J].Photosyn Res,38:61～72.
5.Apel K and Hirt H,2004, Reactive oxygen species:Metabolism, oxidative stress, and signal transduction[J].Annu Rev Plant Biol,(55):373～379.
6. Ariizumi T, Kishitani S,Inatsugi R et al.,2002, An increase in unsaturation of fatty acids in phosphatidyl glycerol from leaves improves the rates of photosynthesis and growth at low temperatures in transgenic rice seedlings [J].Plant Cell Physiol,43(7):751～758.
7.Arruda SCC, Souza GM, Almeida M et al,2004, Anatomical and biochemical characterization of the calcium effect on Eucalyptus urophylla callus morphogenesis in vitro [J].Plant Cell, Tissue and Organ Culture,63(2):142-154.
8.Bachmann M, Matile P, Keller F.,1994, Metabolism of the raffinose family oligo saccharides in leaves of Ajuga reptans L. cold acclimation translocation, sink to source transition: discovery of chain elongation enzyme [J].Plant Physical,(105):1335～1345.
9. Barrios EA, Bressani R,1967, Composicion quimica de la rainy de la hoja de algunas variedades de yucca Manihot [J].Turrialba,17:314～320.
10. Bastow R, Mylne JS, Lister C et al.,2004, Vernalization requires epigenetic silencing of FLC by histone methylation [J].Nature,427:164～167.
11.Bellotti AC,2002, Arthopod pests. In:Hillocks RJ and Thresh JM (Eds.)Cassava:Biology, production and Utilization, CABI, Oxon, pp:209-235.
12. Brenac P, Horbowicz M, Downer SM et al.,1997,Raffinose accumulation related to desiccation tolerance during maize (Zeamays L.) seed development and maturation [J].Plant Physiol,150:481-488.
13.Cerqueira YM,1989, Efeito da deficiencia de agua na anatomia foliarde cultivares de mandio ca (Manihot esculenta Crantz). MSc Thesis, UniversidadeFederal da Bahia, Cruz das Almas, Brazil.
14. Chafouleas JG, Bolton WE, Boyd III AE et al.,1982, Calmodulin and the cell cycle: involvement in regulation of cell-cycle progression [J].Cell,28,41～50.
15.Chafouleas JG, Lagace L, Bolton WE et al.,1984, Changes in calmodulin and its mRNA accompany reentry of quiescent cells into the cell cycle [J].Cell,36:73～81.
16. Cheng M, Fry JE, Pang S,1997,Genetic Transformation of wheat mediated by Agrobacterium tumefaciens [J]. Plant Physiol,115(3):971～980.
17.Cheung WY,1980, Calmodulin plays a pivotal role in cellular regulation [J].Science, 207:19-27.
18.Conde P, Sousa A, Costa A et al,2008,A protocol for Ulmus minor Mill. Micropropagation and acclimatization[J].Plant Cell Tiss Organ Cult,92:113～119.
19. Cooke RD, Coursey DG,1981,Cassava:a major cyanidecontaining food crop. In. Vennesland B,Conn EE and Knowles CJ (eds) Cyanide in Biology. Academic Press, New York.
20. Dey PM,Dixon RA,1985,Biochemistry of storage carbohydrates in green plants [M].New York:Academic Press.
21.Dhandapani M, Hong SB, Aswath CR et al.,2008,Regeneration of zoysia grass (Zoysia matrella L. Merr) cv. Konhee from young inflorescences and stem nodes [J].In Vitro Cell Dev Biol-Plant,44:8-13.
22. Dickau R, Ranere AJ, Cooke RG,2007,Starch grain evidence for the preceramic dispersals of maize and root crops into tropical dry and humid forests of Panama [J]. Proc Natl Acad Sci USA,104:3651-3656.
23.Edwards GE, Sheta E, Moore BD et al.,1990, Photosynthetic characteristics of cassava (Manihot esculenta)a C3 species with chloronchymatous bundle sheath cell [J].Plant Cell Physiol,31:1199～1206.
24. Eklund L, Eliasson L,1990, Effects of calcium ion concentration on cell wall synthesis [J]. Journal of Experimental Botany,41:863～7.
25.El-Sharkawy MA, Cock JH,1987,C3-C4 intermediate photosynthesis characteristics of cass ava (Manihot esculenta Crantz).I [J].Gas Exchange Photosyvethesis Research,12:219～236
26. El-Sharkawy MA and Cock JH,1990, Photosynthesis of cassava (Manihot esculeuta) [J].Exeprimental Agriculture,26:325～340.
27. Etienne H, Lartaud M, Carron MP et al.,1997, Use of calcium to optimize long term proliferation of friable embryogenic calluses and plant regeneration in Hevea brasiliensis [J]. JExp Bot,48:129～137.
28.Faiss M, Zalubilova J, Strnad M et al.,1997, Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants [J]. Plant J,12:401～415.
29. Fan Y, Liu B,Wang H et al.,2002, Cloning of an antifreeze protein gene from carrot and its influence on cold tolerance in transgenic tobacco plants [J].Plant Cell Rep,21:296～301.
30. Frischmuth T, Stanley J,1991,African cassava mosaic virus DI DNA interferes with the replication of both genomic components [J].Virology,183:539～544.
31.Georges F, Saleem M, Cutler AJ,1990, Design and cloning of a synthesis gene for the flounder antifreeze protein and its expression in plant cells [J].Gene,91(2):159～165.
32. Ghosh SP, Ramanujam T, Jos JS et al,1988, Tuber Crops. Oxford & TBH Publishing Co., New Dehli,pp:3～146.
33.Gilmour S, Sebolt AM,2000, Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation [J].Plant Physiol,124:1854～1865.
34.Gilmour SJ,Zarka DG, Stockinger EJ et al.,1998,Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptionalactivators as an early step in cold-induced COR gene expression [J].The Plant Journal,16(4):433-442.
35.Gleeson D, LeluWalter MA, Parkinson M.,2005,Over production of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frost [J].Molecular Breeding,15:21～29.
36. Griffth M and Ala P,1992, Antifreeze protein produced endogenously in winter rye leaves [J]. Plant Physiol,100:593～596.
37.Gupta AS, Webb RP, Holaday AS et al.,1993,over expression of superoxide dismutase protects plants from oxidature stress [J].Plant Physiol,103:1067～1073.
38.Haake V, Cook D, Riechmann JL et al.,2002, Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis [J].Plant Physiol,130(2):639～648.
39. Hayashi H and Alia ML,1997, Transformation of Arabidopsis thaliana with the codA gene for choline oxidase:accumulation of glycinebetaine and enhanced tolerance to salt and cold stress [J].Plant,12:133.
40. Heather MW, Louise VM, Olga S et al,2003, Functional characterization of two cytochrome b5 fusion desaturases from Anemone leveillei:the unexpected identification of a fatty acid A6 desaturase[J].Planta,217:983～992.
41.Hewelt A, Prinsen E, Schell J et al.,1994, Promoter tagging with a promoterless ipt gene leads to cytokinin-induced phenotypic variability in transgenic tobacco plants:implications of gene dosage effects [J].Plant J,6:879～891.
42. Highttower R, Baden C,1991,Expression of antifreeze proteins in transgenic plant [J].Plant Mol Biol,17:1013～1021.
43.Highttower R, Baden C,1991,Expression of antifreeze proteins in transgenic plant [J].Plant Mol Biol,17:1013～1021.
44.Hillocks RJ,2002, Cassava in Africa. In:Hillocks RJ, Thresh JM, Bellotti AC (eds) Cassava: biology, production and utilization. CABI Publishing, New York, pp:41-54.
45.Hinesley LE, Pharr DM, Snelling LK et al.,1992, Foliar raffinose and sucrose in four conifer species:relationship to seasonal temperature [J]. Am Soc Hortic Sci,(117):852～855.
46.Holmberg N,2001,Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco [J].Gene,275(1):115～124.
47. Hong Y and Stanley J,1996, Virus resistance in Nicotiana benthamiana conferred by African cassava mosaic virus replication associated protein (ACI)[J].Mol Plant Microbe Interact, 9:219～225.
48.Horsch RB,Fraley RT, Rogers SG et at.,1984, Inheritance of function foreign genes in plants [S].Science,223:496～498.
49. Howeler KH,Cadavid LF,1983,Accumulation and distribution of dry matter and nutrients during a 12 month growth cycle of cassava [J].Field Crop Research,7:123～139.
50. Hsieh TH, Lee JT, Yang PT et al.,2002, Heterology expression of the Arabidopsis C-repeat/dehydration response element binding Factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato [J].Plant Physiol,129:1086～1094.
51.Hu T, Metz S, Chay C et al.,2003,Agroobacteriuim-mediated large-scale transformation of wheat(Triticum aestivum L.) using glyphosate selection [J].Plant Cell Rep,21:1010～1019.
52.Huang J, Hirji R, Adam L et al.,2000, Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants:metabolic limitations [J].Plant Physiol, 122:747～756.
53. Huang T, Nicodemus J, Zarka DG et al.,2002, Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature [J].Plant Molecular Biology,50:333～344.
54. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG et al,1998,Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance [J].Science,280(5360):104～106.
55.Jansen MAK, Booij H, Schel JHN et al.,1990, Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures [J].Plant Cell Reports,9:221～223.
56. Jo UA, Murthy HN, Hahn EJ et al.,2008,Micropropagation of Alocasia amazonica using semisolid and liquid cultures [J].In Vitro Cell Dev Biol-Plant,44:26～32.
57.Johanson U, West J, Lister C et al,2000, Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time [J]. Science,290:344～347.
58.John D,Plant Genetic Transformation and Gene Expression A Laboratory Manual. Blackwell Scientific Publications Oxford London Edingburgh.
59. Kasuga M, Liu Q, Miura S et al,1999, Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress inducible transcription factor [J]. Nature Biotechnology,17: 287～291.
60. Katia H,Katia and A B,1993,Terence Enhance transformation of tomato co-cultivated with Agrobacterium tumefaciens C58el Rifr:pGSR.1161 in the presence of acetosyringone [J]. Plant cell report,12:422～425.
61.Keating BA, Evenson JP, Fukai S,1982b, Environmental effects on growth and development of cassava (Manihot esculenta crantz).Ⅲ.Assimilate distribution and storage organ yield. Field Crops Research,5:293～303.
62. Keating BA, Evenson JP, Fukai S,1982, Environmental effects on growth and development of cassava (Manihot esculenta Crantz) I. Crop development [J].Field Crops Research, 5:271～281.
63.Khodakovskaya M, McAvoy R, Peters J et al.,2006, Enhanced cold tolerance in transgenic tobacco expressing a chloroplast ω3 fatty acid desaturase gene under the control of a cold inducible promoter [J]. Planta,223:1090-1100.
64. Kim JH,Cetiner S, Jaynes JM,1992, Enhancing the nutritional quality of crop plants: design and expression of an artificial plant storage protein gene. In:Bhatnager D and Cleveland TE (Eds.) Molecular Approaches to Improving Food Quality and Safety, Avi Books,New York,1-36.
65.Kirsten J,Saren B,Peter K,2005,Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers.Distribution of cyanogenic glucosides, their site of synthesis and transport and blockage of the biosynthesis by RNA interference technology [J].Plant Physiol, 139:363～374.
66. Kochetov AV,2004, Suppressor of proline dehydrogenase Gene, Are characterized by higher proline content and cytoplasm osmotic pressure [J].Russian Journal of Genetics,2:216～218.
67.Kodama H,1995,Fatty acid desaturation during chilling acclimation is one of the factors involved in conferring low temperature tolerance to young tobacco leaves [J].Plant Physiol, 107(4):1177～1185.
68.Kumar S, Dhingra A, Danlel H,2004, Plastid expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confer enhanced salt tolerance [J].Plant Physiol,136:2843～2854.
69. Ladino J, Echeverry M, Mancilla L et al.,2002, Genetic transformation of cassava: confirmation of transgenesis in clone 60444 and analysis of CRY1Ab protein in transgenic lines. Preliminary data on transformation of farmer preferred cultivars SM12199 and CM33064. Annual Report, CIAT, Cali,Colombia.
70. Li Q, Robson PR, Bettany AJE et al.,2004, Modification of senescence in ryegrass transformed with ipt under the control of a monocot senescence-enhanced promoter [J].Plant Cell Rep,22:816～821.
71.Li Y, Hagen G, Guilfoyle TJ,1992, Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs [J].Dev Biol,153:386～395.
72.Li,HQ, Sautter C, Potrykus I,1996, Genetic transformation of cassava (Manihot esculenta Crantz)[J].Nature Biotech.14:736～740.
73.Lilius C,Holmberg N, Bulow L,1996, Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase [J].Biotechnology,14:177.
74. Liorente F, Lopezc RM,Catala R et al,2002, A novel cold inducible gene from Arabidopsis, RCI3,encodes a peroxidase that constitutes a component for stress tolerance [J].Plant J, 32:13～24.
75.Liu JJJ, Krenz DC, Galvez AF et al.,1998,Galactinol synthase increased enzyme activity and levels of mRNA due to cold and desiccation [J].Plant Sci,134:11～20.
76. Liu Q, Kasuga M, Sakuma Y et al.,1998,Two transcription factors:DREB1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in droughtand low temperature responsive gene expression, respectively, in Arabidopsis [J].Plant Cell,10:1391～1406.
77.Lorenzi JO,1978,Absorcao de micronutrients a acumulacao de material seca para duas cultivares do mandioca.MSc Thesis Universidade do Sao Paulo, Escola Supcrior de Agricultura Luis de Queiroz, Piracicaba,Brazil,92.
78.Ma J and Liu D,1996, Cloning of Spinach SAD gene, its construction and transformation to tobacco [J].Supplement to Plant Physiol,111(2):814.
79. Mahon JD, Lowe SB,Hunt LA et al.,1977,Envionmental effects on photosynthesis and transpiration in attached leaves of cassava (Manihot esculenta Crantz) [J].Photosnthetica, 11:121-130.
80. Makunga NP and Staden J,2008,An efficient system for the production of clonal plantlets of the medicinally important aromatic plant (Salvia africanalutea L.) [J]. Plant cell Tiss Organ Cult,(92):63～72.
81.Martinea UB,1995,Highsolids tomato shows promise in the field [J].Agbiotech News and Information,7(5):104～105.
82.Martineaub,1995,High-solid stomato shows promise in the field [J].Agbiotech News and Information,7(5):104-105.
83.MC Kersie BD,Chen Y, Beus M et al.,1993,Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.) [J].Plant Physiol,
103(4):1155～1163.
84. Mckersie BD, Chen Y, Beus M et al.,1993,Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.) [J].Plant Physiol,103(4):1155-1163.
85.McMahon JM, White WLB, Sayre RT,1995,Cyanogenesis in cassava (Mahihot esculenta Crantz) [J].Journal of Experimental Botany,46:731～741.
86. Medina J, Bargues M, Terol J et al.,1999, The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration [J].Plant Physiol,119:463～470.
87. Meyer K,1999, A leucinerich repeat protein of carrot that exhibits antifreeze activity [J]. FEBS Letters,447:171～178.
88.Miceli FAG, Estudillo AD, Archila MA et al.,2008,Optimization of Renealmia mexicana (Klotzsch ex. Petersen)cultivation in vitro[J].In Vitro Cell Dev Biol-Plant,44:32-39.
89. Mitten DH, Redenbaugh MK, Sovero M et al.,1992, Safety assessment and commerciali-zation of transgenic fresh tomato products and transgenic rape seed oil products [R].
90. Mkpong O, Yan H, Chism G et al.,1990, Purification, chatacterization and localization of linamarase in cassava [J].Plant Physiol,15:176～181.
91.Monger WA, Seal S, Cotton S,2001,Identification of different isolates of cassava brown streak virus and development of a diagnostic test [J].Plant Path,50:768～775.
92. Montoro P, Etienne H, Micbaux-Ferriere N et al.,1993,Callus friability and somatic embryogenesis in Hevea brasiliensis [J].Plant Cell Tissue and Organ Culture,33:331～338.
93.Morgan AJ, Cox PN, Turner DA et al.,1987, Transformation of tomato using an Riplasmi dvector [J].Plant science,49:37～49.
94. Motohashi T, Toda MI, Kondo K,2008,Adventitious embryo formation derived from zygotic embryos in Cycas revoluta [J].Plant biotechnology,25:589～591.
95.Munyikwa T, Raemakers C, Schreuder M et al.,1998,Pinpointing towards improved transformation and regeneration of cassava [J].Plant Sci,135:87～101.
96. Murata N,Ishizaki NO, Higashi S et al.,1992, Genetically engineered alteration in the chilling sensitivity of plants[J].Nature,356:710～713.
97.Nanjo T, Kobayashi M, Yoshiba Y et al.,1999, Antisense suppression of proline degradation ilmproves tolerance to freezing and salinity in Arabidopsis thaliana [J], FEBS Lett,461: 205～210.
98.Nilgun, Mkeith ET, Richard SN et al.,1987, Expression of alfalfa mosaic virus coat protein gene confers cross protection in transgenic tobacco and tomato plant [J]. The EM BO Journal, 6(5):181～188.
99. Nishizawa A, Yabuta Y, Shigeoka S,2008,Galactinol and raffinose constitute a novel function to protect plants from oxidative damage [J].Plant Physiol,147:1251～1263.
100. Nomura K, Komamine A,1986, Somatic embryogenesis in cultured carrot cells [J]. Development Growth and Differentiation,28:511～517.
101.Nweke FI, Spencer D, Lynam JK,2002, Cassava Transformation:Africa's Best Kept Secret East Langsing. Michigan Sate University Press.
102.Obrien G, Taylor A, Poulter N,'1991,Improved enzymatic assay for cyanogens in fresh and processed cassava [J].JSci Food Agric,56:277～289.
103.Oelsligle D,1975, Accumulation of dry matter, nitrogen, phosphorus and potassium in cassava(Manihot esculenta Crantz)[J].Turrialba,25:85～87.
104. Oh SJ, Song SI, Kim YS et al,2005, Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth [J].Plant Physiol, 138:341～351.
105.Olsen KM,Schaal BA,1999, Evidence on the origin of cassava:phylogeography of Manihot esculenta [J].Proc Natl Acad Sci USA,96:5586～5591.
106.Panikulangara TJ, Eggers-Schumacher G, Wunderlich M et al.,2004, Galactinol synthasel:a novel heat shock factor target gene responsible for heatinduced synthesis of raffinose family oligosaccharides in Arabidopsis [J].Plant Physiol,136:3148～3158.
107. Parvanova D,Popova A, Zaharieva I et al.,2004, Low temperature tolerance of tobacco plants transformed to accumulate proline, fructans or glycine betaine.Variable chlorophyll fluorescence evidence [J].Photosynthetica,42(2):179～185.
108.Pierrobon D,Virgilio FO, Pozzea T,1990, Structural and functional aspects of calcium homeostasis in eukaryotic cells [J].European Journal of Biochemistry,193:559～622.
109. Pilonsmits EAH, Ebakamp MJM, Jenken MJW et al.,1995,Improved performance of transgenic fructan accumulating tobacco under drought stress [J].Plant Physiology, 107:125-130.
110. Pita JS,Fondong VN,Sangare A,2001,Recombination, pseudore combination and synergism of geminiviruses are determinant keys to the epidemic of severe cassava mosaic disease in Uganda [J].J Gen Virol,82:655～665.
111.Plumbley RA, Rickard JE,1991,Postharvest deterioration of cassava [J].Tropical Sci, 31:295～303.
112. Polashock J, Lipson D,1993,Expression of yeast Δ9 fatty acid desaturase activity enhances chilling resistance in tobacco [J].Supplement to Plant Physiol,102:160.
113.Poovaiah BW, Reddy ASN,1987, Calcium messenger system in plants [J]. CRC in Plant Science 6:47～103.
114. Raemakers CJJM,1993,Primary and cyclic embryogenesis in cassava.PhD thesis, Wageningen Agricultural University, Wageningen, The Netherlands.
115.Raemakers CJJM, Jacobsen E, Visser RGF,1997, Micropropagation of Manihot esculenta (Crantz) cassava. In:Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 39. High tech and micropropagation. Springer, Berlin Heidelberg New York, pp 77～102.
116. Raemakers C, Schreuder M, Pereira I et al,2003,Production of amylose-free cassava plants by genetic modification. In:Fauquet CM, Taylor NJ (eds.) Cassava:An Ancient Crop for Modern Times, Proceedings of the 5th International Meeting of the Cassava Biotechnology Network.
117. Reilly K, Gomez Vasquez R, Buschmann H et al,2003,Oxidative stress responses during cassava postharvest physiological deterioration [J].Plant Mol Biol,53:669～685.
118.Rogers DJ,1965,Some botanical and ethnological considerations of Manihot esculenta [J]. Econ Bot,194:769～773.
119. Roosens NH, Al Bitar F, Loenders K et al.,2002, Over expression of ornithine-δ-aminotrans-ferse increases proline biosynthesis and confers osmotolerance in transgenic plants [J].Mol Breed,9:73～80.
120. Rupp HM, Frank M, Werner T et al,1999, Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem [J].Plant J,18:557～563.
121.Samis K, Bowley S, Mckersie BD,2002, Pyramiding Mrisuperoxide dismutase transgenes to improve persistence and biomass production in alfalfa [J].Jexpbot Oxford:Oxford University Press,53(372):1343～1350.
122. Saravitz DM,Pharr DM, Carrer TE,1987, Galactinol synthase activity and soluble sugarsin developing seeds of four soybean gene types [J].Plant Physical, (83):185～189.
123. Sarria R, Torres E, Angel F et al,2000, Transgenic plants of cassava (Manihot esculenta) with resistance to Basta obtained by Agrobacterium-mediated transformation [J].Plant Cel Rep,19:339～344.
124. Sato Y, Murkami T, Funatsuki H et al,2001,Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings [J].J Expe Bot,52:145～151.
125.Schopke C, Taylor N, Carcamo R et al,1996,Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures [J]. Nature Biotech,14:731～735.
126. Shinwari ZK, Nakashima K, Miura S et al,1998,An Arabidopsis gene family encoding DRE/CRT binding proteins involved in low temperature responsive gene expression [J]. Biochem Biophys Res Commun,250:161～170.
127.Silva P, Ricardo CPP,1992, β-Fructosidases and in vitro dedifferentiation-redifferentia-tion of carrot cells[J].Phytochemistry,31:1507～1511.
128.Siritunga D, AriasGarzon D,White W et al,2004, Overexpression of hydroxynitrile lyase in transgenic cassava roots accelerates cyanogenesis and food detoxification [J].Plant Biotech J, 2:37-43.
129. Siritunga D, Sayre RT,2003,Generation of cyanogens-free transgenic cassava [J].Planta, 217:367～373.
130.Smigocki A, Neal JWJ,McCanna I et al,1993,Cytokinin-mediated insect resistance in Nicotiana plants transformed with the ipt gene [J].Plant Mol Biol,23:325～335.
131.Smigocki AC,1991,Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene [J].Plant Mol Biol,16:105～115.
132.Stamp JA, Henshaw GG,1982, Somatic embryogenesis in cassava [J].Z P glanzen physiol, 105:183～187.
133.Stocking EJ, Gilmour SJ, Thomashow MF,1997,Arabidopsis thaliana CBF1 encodes an AP2 domaincontaining transcriptional activator that binds to Crepeat/DRE, a cisacting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proc Nat Acad Sci USA,94:1035～1040.
134. Su J, Hirji R, Zhang L et al.,2006, Evaluation of the stressinducible production of choline oxidase in transgenic rice as a strategy for producing the stress protectant glycine betaine [J]. J Expe Bot,57(5):1129-1135.
135.Taji T, Ohsumi C, Hchi S et al.,2002, Important roles of drought and cold inducible genes for galactinol syntheses in stress tolerance in Arabidopsis thaliana [J].Plant Jour, 29(4):417-426.
136.Tavora FJAT, Melo FIO, Pinho JLN et al,1995,Yield crop growth rate and assimilatory characteristics of cassava at the coastal area of Ceara [J].Revista Brasileira de Fisiologoa hegetal,7:81～88.
137.Thomashow M F,1998,Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance [J].Science,280:104～106.
138.Thomashow MF,1999, Plant cold acclimation:freezing tolerance genes and regulatory mechanisms[J].Annu Rev Plant Physiol Plant Mol Biol,50:571～599.
139. Thresh JM, Fargette D,1994, Effects of African cassava mosaic geminivirus on the yield of c assava [J].Trop Sci,34:26～42.
140. Tian HQ, Yuan T,2000, Calcium function in fertilization process in angiosperms [J].Acta Phytophysiol Sin,26:369～380.
141.Timmers ACJ, Schel JHN(1991).Localization of cytosolic Ca2+ during carrot somatic embryogenesis using confocal scanning laser microscopy. In:Karssen CM, van Loon LC, Vreugdenhil D (eds).Progress in plant growth regulation. Dordrecht:Kluwer Academic Publishers,347～353.
142.Touchell DH, Chiang VL, Tsai CJ,2002, Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification [J].Plant Cell Rep,21:118～224.
143.Ueno 0 and Agarie S,1997,The intercellular distribution of glycine decarboxylasre in leaves of cassava in relation to the photosynthetic mode and leaf anatomy [J].Japanese Journal of crop Science,66:268-278.
144. Ugent D, Pozorski S, Pozorski T,1986, Archaeological manioc (Manihot) from coastal Peru [J].Econ Bot,40:78～102.
145.Urrutia ME, Duman JG, Knight CA,1992, Plant themal hysteresis proteins [J].Biochim Biophy Acta,1121(1):199～206.
146. Vargas Bonilla HL, Bolivar LR, Arias V et al.,2002, Nataima31 variedad de yucca (Manihot esculenta Crantz) resistant a mosca blanca (Aleurotrachelus socialias Bondar) para el valle calido del Alto Magdalena. Publication CORPOICA Regional Seis.Codigo 28306322002. A. Postal 064, El Espinal, Tolima, Colombia.
147. Verdus MC, Dubois T, Dubois J et al.,1993,Ultra structural changes in leaves of Cichorium during somatic embryogenesis [J].Annals of Botany,11:375～383.
148.Vladinir S,Larry G, Prince A et al.,2006, Agrobacteriummediated transformation of seeding derived maize callus [J].Plant Cell Reports,25:320～328.
149.Wada H, Gombos Z, Murata N,1990, Enhancement of chilling tolerance of facyano bacterium genetic manipulation of fatty acid desaturation [J].Nature,347:13,200.
150. Wallis JG, Wang H, Guuerra DJ et al.,1997, Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures [J].Plant Molecular Biology, 35(3):323～330.
151.Wallis JG, Wang HY, Daniel J,1997, Structure, function and evolution of antifreeze proteins [J].Plant Molecular Biology,35:323～330.
152. Wheatley CC, Chuzel G,1993,Cassava:the nature of the tuber and use as a raw material. In: Macrae R, Robison RK and Saddler MJ (Eds.) Encyclopedia of Food science, Food technology and Nutrition. Academic Press, San Diego, CA. pp:734～743.
153.Wheatley CC, Orrego JI, Sanchez T et al.,1993,Quality evaluation of cassava core collection at CIAT. In:Roca WM, Thro AM (eds). Proceedings of the Girst International Scientific Meeting of the Cassava Biotechnology Net, work. CIAT, Cartegena, Colombia. pp:255-264.
154. White W, AriasGarzon D, Mahon JM et al.,1998,Cyanogenesis in cassava:the role of hydroxynitrile lyase in root cyanide production [J].Plant Physiol,116:1219～1225.
155. Worrall D, Elias L, Ashford D et al.,1998, A carrot leucine rich repeat protein that inhibits ice recry stallization [J]. Science,282(2):115～117.
156. Yamaguchi-Shinozaki K,Shinozak K,2006, Transcriptional regulatory networks in cellular re sponses and tolerance to dehydration and cold stresses[J].Annu Rev Plant Biol,57:781～803.
157.Yang HY,1999, The role of calcium in the fertilization process in flowering plants [J].Acta Bot Sin,41:1027～1035.
158.Yokio S, Higashi SL, Kishitani S,1998, Introduction of the cDNA for Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acid and chilling tolerance of photosynthesis on rice [J].Mol Breeding,4:269～275.
159.Zhang P, Gruissem W,2004, Production of Transgenic cassava (Manihot esculenta Crantz). In:Cuitis IS.(ed.),Transgenic crops of the world-essential protocols,301～319.
160. Zhang P, Jaynes J, Potrykus I et al,2003,Transfer and expression of an artificial storage protein(ASP1)gene in cassava (Manihot esculenta Crantz) [J].Transgenic Res,12:243～250.
161.Zhang P, Vanderschuren H, Futterer J et al.,2005,Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes [J].Plant Biotechnol J,385～391.
162.Zhao J, Ren W, Zhi D et al,2007,Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress [J].Plant Cell Rep,26(9):1521～1528.
163.陈儒钢,巩振辉,逯明辉等,2008,植物抗寒基因工程研究进展[J].西北植物学报,28(6)：1274-1280.
164.陈旭微,杨玲,章艺,2004,类脂对植物生长和发育的作用[J].植物生理学通讯,40(3)：373-378.
165.陈永波,赵清华,2005,魔芋试管苗批量生产过程中外植体消毒灭菌技术研究[J].氨基酸和生物资源，27(3)：27-29.
166.德庆措姆,布穷,2000,银白杨组织培养中外植体消毒时间初探[J].辽宁林业科技，4：29,37.
167.段永波,赵德刚,赵丰兰等,2007,低温胁迫下ipt基因水稻某些生理特性研究[J],山地农业生物学报,26(2)：95-98.
168.樊军锋,2002,mtlD/gutD双价耐盐基因转化杨树、猕猴桃的研究[T].西北农林科技大学博士学位论文.
169.冯金玲,陈辉,杨志坚等,2006,锥栗组织培养外植体消毒和选抒[J].福建林学院学报，26(1)：22-26.
170.付永彩,刘新仿,曹守云等,1999,水稻中抑制衰老的嵌合基因的基因枪转化和表达分 析[J].农业生物技术学报，(1)：17-22.
171.付永彩,孙传清,李自超等,2000,农杆菌介导的抑制衰老的嵌合基因转化籼稻的研究[J].农业生物技术学报，8(1)：28,32.
172.耿飒,麻密,李国凤等,2000,ipt基因定位表达对转基因烟草育性的影响[J].植物学报，42(2)：217-220.
173.黄洁,李开绵,叶剑秋等,2008,我国的木薯优势区域概述[J].广西农业科学，39(1)：104-108.
174.黄永芬,汪清胤,付桂荣等,1997,美洲拟鲽抗冻蛋白基因(afp)导入番茄的研究[J].生物化学杂志，13(4)：418-42.
175.贾庚祥,2002,BADH基因转化番茄提高耐盐性的研究[T].中国科学院研究生院植物研究所博士学位论文.
176.焦芳婵,2001,转基因改良植物的胁迫耐性[J].生物技术通讯，12(2)：137.
177.金建凤,高强,陈勇等,2005,转移拟南芥CBF1基因引起水稻植株脯氨酸含量提高[J].细胞生物学杂志，27(1)：73-76.
178.金万梅,董静,尹淑萍等,2007,转移拟南芥CBF1基因引起水稻植株脯氨酸含量提高[J].西北植物学报，27(2)：223-227.
179.赖玉洁,路丙社,陈书明等,2007,青榨槭外植体消毒方法初步研究[J].河北林果研究，22(2)：143-146.
180.李开绵,林雄,黄洁,2001,国内外木薯科研发展概况[J].热带农业科学，(1)：56-60.
181.林茂,闫海霞,眭顺照等,2008,植物CBF转录因子及其在基因工程中的应用[J].广西农业科学，39(1)：21-25.
182.林雄,李开绵,1992,海南岛木薯种质资源考察报告[A].华南热带作物科学研究院,中国农业科学院作物品种资源研究所.海南岛作物(植物)种质资源考察文集[C].北京：农业出版社.
183.刘雪红,刘俊华,张孝霖,2009,冬枣组织培养的消毒方法[J].北方园艺，2：97-98.
184.罗小英,崔衍波,邓伟等,2004,超量表达苹果酸脱氢酶基因提高苜蓿对铝毒的耐受性[J].分子植物育种，2(5)：621-626.
185.马国华,许秋生,羡蕴兰,1998,从木薯嫩叶直接诱导初生体细胞胚胎发生和芽的形成[J].植物学报，40(6)：503-507.
186.马华升,孙玉强,童建新等,2008,千层塔组织培养中外植体消毒灭菌研究初报[J].杭州农业科技，3：15-18.
187.毛自朝,于秋菊,甄伟等,2002,果实专一性启动子驱动ipt基因在番茄中的表达及其对番茄果实发育的影响[J].科学通报，47(6)：444-448.
188.酶、超氧歧化酶活性测定[J].哈尔滨师范大学自然科学学报，14(4)：188-193.
189.孟凤,郁松林,郑强卿等,2008,甜菜碱与植物抗逆性关系之研究进展[J].中国农学通 报，24(4)：225-228.
190.沈漫,王明麻,黄敏仁,1997,植物抗寒机理研究进展[J].植物学通报，14(2)：1-8.
191.汤绍虎,孙敏,周启贵等,2004,采用正交设计快速获得梨无菌外植体的研究[J].西南师范大学学报(自然科学版),29(2)：282-284.
192.王多佳,苍晶,牟永潮等,2009,植物抗寒基因研究进展[J].东北农业大学学报,40(10)：134-138.
193.王关林,李铁松,方宏筠等,2004,番茄转果聚糖合酶基因获得抗寒植株[J].中国农业科学，37：1193-1197.
194.王延玲,丰震,赵兰勇等,2004,仙客来花药培养外植体消毒研究[J].山东林业科技，4：8-9.
195.王子成,李忠爱,邓秀新,2005,柑橘成年态茎段外植体消毒方法研究[J].河南大学学报：自然科学版,35(2)：57-60.
196.韦善君,孙振元,巨关升等,2005,冷诱导基因转录因子CBF1的组成型表达对植物的抗寒性及生长发育的影响[J].核农学报，19(6)：465-468.
197.吴坤林,曾宋君,张志胜等,2008,番木瓜外植体消毒方法研究[J].福建农林科技，35(3)：76-79.
198.杨晓红,陈晓阳,2006,果聚糖对植物抗逆性的影响及相应基因工程研究进展[J].华北农学报,21(1)：6-11.
199.姚庆荣,2007,木薯遗传转化体系的建立与优化及转AGPase基因的研究[T].华南热带农业大学博士学位论文.
200.尹明安,崔鸿文,樊代明等,2001,抗冻蛋白及其在植物抗冻基因工程中的应用[J].西北植物学规21(1)：8-13.
201.于晓红,朱勇清,陈晓亚等,2000,种子特异表达ipt转基因棉花根和纤维的改变[J].植物学报，42(1)：59-63.
202.袁维风,徐德聪,钱玉梅,2009,基因抗寒基因及基因工程研究[J],宿州学院学报，24(6)：109-112.
203.张鹏,2008,能源木薯种质资源面临的问题与解决策略[J].生物产业技术，5：25-30.
204.张赛群,叶志彪,吴昌银等,1999,异戊烯基转移酶基因导入番茄及转基因植株再生[J].园艺学报，26(6)：376-379.
205.张玉红,曲伟娣,2008,培养条件对黄檗快速繁殖影响的研究[J].植物研究，28(2)：236-239.
206.张钰,吴加林,黄永芬等,1998,转美洲拟蝶抗冻蛋白基因(afp)番茄过氧化物酶、过氧化氢酶、超氧歧化酶活性测定[J].哈尔滨师范大学自然科学学报，14(4)：188-193.
207.赵微平,1996,植物基因组构建、表达和调控[M].北京：首都师范大学出版社.
208.甄伟,陈溪,孙思洋等,2000,冷诱导基因的转录因子CBF1转化油菜和烟草及抗寒性鉴定[J].自然学进展，10(12)：1104-1108.
209.郑丽,李名扬,晁岳恩等,2005,根癌农杆菌介导ipt基因对切花菊的遗传转化[J].农业生物技术学报，13(1)：26-31.
210.钟蓉,刘玉乐,朱峰等,1996,1TA29Bar基因导致油菜雄性不育的研究[J].植物学报，38(7)：582-585.
211.钟胜,2008,抗寒相关基因CBF3、ICE1及AtGolS3转基因拟南芥耐寒效果评价[T].华南热带农业大学硕士学位论文.
212.周丽娟,王丹,黄海涛等,2008,费约果外植体灭菌及愈伤组织的诱导[J].热带亚热带植物学报，16(2)：179-183.
213.朱淑颖,陆颂规,梁承邺等,2007,草珊瑚组培中外植体消毒方法的研究[J].福建林业科技，34(4)：82-89.
214.朱文丽,2006,木薯胚胎发生再生植株与离体保存技术的初步研究[T].华南热带农业大学硕士学位论文.
215.朱晔荣,达来,2003,抗冻蛋白基因转化植物的研究展望[J].内蒙古大学学报(自然科学版),2003,34(1)：97-101.