马铃薯四倍体栽培种茎段组织的EMS诱变研究

用1%的EMS溶液处理“中薯2号”试管苗茎段材料,通过对诱变材料再生过程的观察、再生苗试管培养观察及棚栽阶段的生理指标统计分析,研究了EMS对马铃薯四倍体栽培种茎段组织化学诱变的作用。试验结果表明:EMS能够有效渗入生长点细胞;经EMS处理后试管苗生长量显著小于对照,再生植株和组织器官发生变异;试验证实1%(v∶v)浓度EMS处理4h以上具有明显的诱变效应,可以应用于今后马铃薯诱变育种。     

马铃薯品种‘并薯6号’遗传转化体系的建立

以马铃薯品种‘并薯6号’无菌试管苗茎段作为外植体,通过对遗传转化过程中预培养时间、植物激素的种类及配比、筛选剂浓度和抗生素的种类及浓度进行研究,初步建立了马铃薯品种‘并薯6号’的愈伤再生转化体系。结果表明,预培养时间为2 d时,愈伤组织诱导率、出苗率及转化频率最高。最适外植体愈伤组织诱导的培养基配方为MS+2.0 mg/L 6-苄氨基嘌呤(6-BA)+0.5 mg/L 2,4-二氯苯酚代乙酚(2,4-D)+0.4 mg/L激动素(KT),愈伤组织的诱导率为86.7%,愈伤组织形态较紧密,为深绿色,茎段两端膨大呈哑铃状,表面的白色须状物和毛状根较少。最适愈伤组织分化的培养基配方为MS+1 mg/L 6-苄氨基嘌呤(6-BA)+1 mg/L赤霉素(GA_3)+1 mg/L玉米素(ZT),幼苗比较粗壮,长势良好。最适的植株再生及生根筛选剂为10 mg/L的潮霉素(Hygromycin B,Hyg),转化频率较高,为20.3%。农杆菌抑制剂的最适浓度为200 mg/L的特美汀(Timentin,TMT),且在愈伤组织诱导、分化及生根阶段都需要加入TMT。综上所述,通过对农杆菌介导的愈伤再生转化过程中各个因素的研究,初步建立了‘并薯6号’的遗传转化体系,为马铃薯分子育种发展提供一定的基础。     

马铃薯两个基因型不同外植体的组织培养与植株再生

以Favorita和东农303两个马铃薯基因型的幼叶、茎段、微型薯和种薯的块茎为外植体,在6种培养基中诱导愈伤组织和植株再生。试验中观察到马铃薯的分化率在不同外植体间差异较大,ZT有可能是诱导马铃薯芽分化的理想激素。     

发根农杆菌介导抗菌肽Shiva1基因转化马铃薯的初步研究

利用已构建载体530Shiva,转化马铃薯普通栽培种甘农薯1号、澳布、HLS和夏波蒂的试管苗及块茎盘的研究。结果表明,以Kan50mg·L-1和100mg·L-1作为筛选转基因细胞和植株的剂量。对于发根农杆菌侵染马铃薯试管苗应采用新鲜菌落直接涂抹伤口进行侵染,这种方法诱导生根率高且易于操作,根诱导率、成愈率及分化率远高于浸泡法;发根农杆菌转化试管苗,后期愈伤组织分化产生5个幼苗。发根农杆菌菌液侵染甘农薯1号和夏波蒂两品种的块茎盘,7d后就有很小的毛状根突起产生,2周后即有大量毛状根产生,3周后毛状根产生量有所减少,产生的毛状根3周时其成愈率分别为31.0%和27.0%,愈伤组织分化成苗比较困难,分化出3个幼苗,分化率分别为6.45%和3.70%。转化材料进行冠瘿碱检测表明,分化苗均为胭脂碱阳性反应。环腐病菌接种试管苗结果表明,分化植株与对照相比抗性明显增强。     

发根农杆菌介导抗菌肽Shival基因转化马铃薯的初步研究

利用已构建载体530 Shiva,转化马铃薯普通栽培种甘农薯1号、澳布、HLS和夏波蒂的试管苗及块茎盘的研究.结果表明,以Kan 50 mg·L-1和100 mg·L-1作为筛选转基因细胞和植株的剂量.对于发根农杆菌侵染马铃薯试管苗应采用新鲜菌落直接涂抹伤口进行侵染,这种方法诱导生根率高且易于操作,根诱导率、成愈率及分化率远高于浸泡法;发根农杆菌转化试管苗,后期愈伤组织分化产生5个幼苗.发根农杆菌菌液侵染甘农薯1号和夏波蒂两品种的块茎盘,7 d后就有很小的毛状根突起产生,2周后即有大量毛状根产生,3周后毛状根产生量有所减少,产生的毛状根3周时其成愈率分别为31.0%和27.0%,愈伤组织分化成苗比较困难,分化出3个幼苗,分化率分别为6.45%和3.70%.转化材料进行冠瘿碱检测表明,分化苗均为胭脂碱阳性反应.环腐病菌接种试管苗结果表明,分化植株与对照相比抗性明显增强.     

农杆菌法转化棉花茎尖组织的初步研究

农杆菌法是当今转基因植物研究中应用最为广泛的技术,因具有转化工作不受季节限制、转化机理较为清楚、转基因植株多为单拷贝和转化效率较高等优点而备受青睐。采用农杆菌介导法和基因枪法进行棉花遗传转化时,一般都需经过离体培养再生植株的过程,实验周期长,且受到基因型限制,只有少数品种的离体培养材料能再生植株。转化茎尖分生组织的方法具有直接产生不定芽和基因型依赖性弱等优点,因此,建立棉花茎尖组织遗传转化体系具有重要的现实意义。1材料和方法1.1材料品种为中棉所35,农杆菌菌株为LBA4404,转化载体为pCAMBIA-3300。1.2方法1.2…     

根癌农杆菌介导的大麦茎尖转化研究

为了建立农杆菌介导的大麦茎尖遗传转化技术,以我国大麦主推品种鄂大麦9号、鄂大麦32122为材料,以农杆菌介导法转化大麦茎尖,经PPT筛选获得了转抗除草剂基因的大麦株系。PCR鉴定表明,以鄂大麦9号茎尖为受体,筛选获得4个转基因植株,阳性率为0.59%;以鄂大麦32122茎尖为受体,筛选获得10个阳性植株,阳性率为1.96%。转基因T1代Southern杂交分析证实,外源基因确已整合到大麦基因组中,已鉴定的转基因株系均含单个转基因拷贝,不同转基因株系整合的位点不同。因此大麦茎尖可作为农杆菌转化的受体。本研究结果为拓宽大麦遗传转化受体提供了技术和方法。     

Bt基因导入马铃薯品种大西洋的研究

通过基因工程手段,利用农杆菌介导法,将Bt基因导入马铃薯品种大西洋,同时对马铃薯品种大西洋的叶片再生体系进行优化,探讨了影响植株再生频率和遗传转化效率的因素,确立了最佳植株再生体系和遗传转化条件,获得了转基因植株。     

花生基因工程研究进展

９０年代以来,花生基因工程研究重点放在建立花生遗传转化和再生体系上。随着花生遗传转化和再生技术的进步,美国已分别获得抗病和抗虫转基因花生植株,取得突破性进展。目前,农杆菌介导的遗传转化是以花生幼苗嫩叶为外植体,通过卡那霉素筛选,器官发生再生转化植株;基因枪介导的遗传转化是以胚愈伤为转化材料,通过潮霉素筛选转化体胚,再生转化植株。     

大豆生物工程研究进展

90年代以来，大豆的生物工程研究重点放在建立遗传转化和再生体系上，随着遗传转化和再生技术的发展，我国已获得了抗病、抗虫转基因大豆植株，取得突破性进展。目前，大豆遗传转化主要采用农杆菌介导法、花粉管通法、基因枪及PEG法。农杆菌介导的遗传转化是以大豆子叶、胚轴为外植体，通过卡那霉素筛选，器官发生再生转化植株；花粉管介导法是以植株整体为受体导入外源原因，获得稳定遗传品系；基因枪介导的遗传转化是以大豆幼胚的胚轴、用基因枪轰击生长点，可以从任意一个基因型获得转基因植株；PEG介导的遗传转化是将质粒导入大豆原生质体，通过潮霉素进行筛选，获得转基因植株。     

Growth and yield of potato plants grown from microtubers in fields

Despite many reports of thein vitro production of microtubers, little is known about plant growth and yield from microtubers planted in the field. This study clarified differences in growth and yields between potato plants grown in the field from microtubers and from conventional seed tubers. The experiments were performed at Hokkaido University, Japan, over four years. Conventional seed tubers of about 50 g and microtubers of two sizes (0.5–1.0 g and 1.0–3.0 g) of the latematurity cultivar Norin 1 were planted, and plant growth and tuber yields were analyzed. The microtuber plants had a lower initial increase in root and leaf area index than conventional seed tuber plants, but had the same leaf area index after about 40 days from emergence. The first tuber formation in microtuber plants was about 7 days later than in conventional seed tuber plants, while tuber bulking occurred about 14 days later in microtuber plants. Consequently, the onset of tuber weight increase was later in microtuber plants, but the rate of increase thereafter was similar between conventional seed tuber and microtuber plants. At harvest the tuber fresh weight of microtuber plants was 82% that of conventional tuber plants, suggesting a potential for using microtubers for field planting.     

Soil water stress and the growth and yield of potato plants grown from microtubers and conventional seed tubers

Tuber yields of potato plants grown from microtubers in fields are more variable than yields from conventional seed tubers (CT). One reason could be their higher susceptibility to water stress. This study clarified the effect of soil water stress from 1 month after emergence on the growth and yield of plants grown from conventional seed tubers and microtubers in fields. Microtubers (0.5–3 g) and conventional seed tubers (50 g) were grown in Hokkaido, Japan, over three field seasons. One month after emergence, poly-shelters were placed over the plots to prevent rainfall, and either irrigated (wet plot) or non-irrigated (dry plot) treatments were formed. At mid-flowering (about 50 days after emergence) leaf area index (LAI) in microtuber plants was decreased relatively more due to soil water stress than LAI in conventional seed tuber plants. However, at maximum shoot growth (about 80 days after emergence) both microtuber and conventional seed tuber plants had a similar relative decrease in LAI due to soil water stress. At mid-flowering and maximum shoot growth microtuber and conventional seed tuber plants had reduced stomatal conductance due to soil water stress, but the reduction in stomatal conductance was greater in conventional seed tuber plants than in microtuber plants. Microtuber and conventional seed tuber plants had similar root development at maximum shoot growth. Tuber production from mid-flowering until plant maturity was similarly affected by soil water stress in microtuber and conventional seed tuber plants. At harvest, plants affected by soil water stress had about 87% of the tuber dry weight of irrigated plants. We conclude, that the greater variation on tuber yield of microtuber plants cannot be attributed to soil water stress from 1 month after emergence.     

Effect of cultivar maturity period on the growth and yield of potato plants grown from microtubers and conventional seed tubers

Potato plants of early cultivars grown from microtubers have been reported to have a much lower growth vigor and produce lower yields than microtubers of late cultivars. This study intended to clarify the field performance of plants grown from directly planted microtubers of cultivars with different maturity periods, with a special attention to early cultivars. The experiments were conducted at Hokkaido University, Japan, over four years. Microtubers and conventional seed tubers of the early cultivar Kitaakari, late cultivars Konafubuki and Norin 1, and very late breeding line IWA-1 were planted, and the plant growth and tuber yields were analyzed. The microtuber plants of Kitaakari had a lower initial increase in leaf area index than conventional seed tuber plants, but at the maximum shoot growth had the same leaf area index. This pattern was also observed in the other cultivars. Tuber initiation and tuber bulking occurred on average five days later in microtuber plants than in conventional seed tuber plants of cultivar Kitaakari. At maximum shoot growth, microtuber plants had on average 65% of tuber dry weight of conventional seed tuber plants, with small variation among cultivars. Irrespective of maturity period, microtuber plants showed a higher tuber increase after maximum shoot growth, achieving around 86% of tuber dry weight of conventional seed tuber plants at harvest. From the results of this study we conclude that microtuber plants of early and late cultivars have a similar yield potential relative to conventional seed tuber plants, and microtubers of both early and late cultivars might be used as an alternative seed tuber source for potato production, if necessary.     

用组培法诱导试管微型薯的研究

用各种不同培养基对马铃薯四倍体、双单倍体栽培种及有结薯习性的野生种进行了试管微型薯诱导。在其它条件不变时,用食用白糖替代蔗糖可以获得同样的诱导效果,能大大降低成本。液体培养较固体培养效果好。BAP 的诱导效果随基因型不同而异,但并不一定是必需的。香兰素补加到液体培养基中对提高诱导效果有一定作用。试验表明用 MS 加10%食用白糖或 MS 加10%白糖再补加一定浓度香兰素的液体培养基,在黑暗条件下室温于20～28℃培养是高效率生产试管微型薯较好的方法。     

农杆菌介导的花生遗传转化条件优化

为建立花生快速高效的遗传转化体系,以浸泡吸水后的花生半粒种子为转化受体,利用根癌农杆菌介导法,以除草剂Basta抗性筛选及基因组DNA的Bar基因PCR检测为指标,优化花生遗传转化体系。研究结果表明,在侵染悬浮液中加入1mmol/L二硫苏糖醇（DTT）提高了农杆菌介导的转化效率（最高提升36.5 %）;与YEB培养液相比,AB重悬液的使用显著提高了农杆菌的转化效率（最高提升19.3 %）;农杆菌菌液OD600为0.7时转化效率最高;基因型显著影响转化效率,EXP27-1516的转化效率为69.03%,显著高于14AU01（56.67 %）;以花生半粒种子为转化受体,从种子培养到形成茁壮根系仅需8周,明显短于子叶节转化所需的13周。优化后的农杆菌介导的花生遗传转化体系为花生转基因研究提供了重要的研究工具。     

光照长度、强度及温度对试管薯诱导的影响

通过一步法进行试管薯的诱导,不更换培养基,不添加任何外源激素,研究试管苗培养阶段光照周期、光照强度及温度对Favorita、Atlantic和克新1号3个马铃薯品种试管薯诱导的影响。结果表明:短光周期培养有利于试管薯的诱导,但产生的试管薯较小,适当的延长光照时间有利于诱导较大的试管薯;不同品种需要不同的适宜试管薯诱导的光照强度;变温处理最适于试管薯的诱导。不同品种需要做培养环境的筛选和品种结薯性评价,以筛选出最佳的诱导条件。     

白糖浓度与马铃薯试管苗长势及试管薯产量的相关性

为了获得健壮的试管苗及高质量的试管薯,采用两个品种(早大白和克新13号),通过设定不同的白糖浓度,调查试管苗长势、试管薯均粒重、每瓶粒数,研究它们之间的相关性。白糖浓度与马铃薯试管苗长势及试管薯均粒重之间显著正相关。若要生产健壮的试管苗和较大的试管薯,可以通过提高白糖浓度来实现,本研究中较适合的白糖浓度为5%～6%。试管苗长势与试管薯均粒重显著或极显著正相关,培育健壮试管苗是生产较大试管薯的基础条件。     

马铃薯试管薯诱导培养基6-苄基腺嘌呤与蔗糖浓度的筛选

试管薯的诱导在马铃薯种薯繁殖体系中起重要作用。试验以早熟鲜食品种早大白为材料,研究诱导培养基中6-苄基腺嘌呤(6-BA)浓度和蔗糖浓度对试管薯数和试管薯重的影响。结果表明:MS的液体培养基+活性炭浓度0.15%+6-BA浓度2 mg/kg+蔗糖浓度8%的效果最好,应用该诱导培养基提高马铃薯早大白的单株结薯数。     

Infection of Early and Young Callus Tissues of Indica Rice BPT 5204 Enhances Regeneration and Transformation Efficiency

A rapid and reproducible method to develop transgenic plants with enhanced transformation efficiency using Agrobacterium has been developed for the elite indica rice variety BPT 5204. Different rice calli aged from 3 to 30 d were co-cultivated with pre-incubated Agrobacterium suspension culture （LBA4404： pSB1, pCAMBIA1301） and incubated in dark for 3 d. Based on the transient GUS gene expression analysis, 6-day-old young calli showed high transformation frequency followed by 21-day-old ones. Thus, both 6-and 21-day-old calli were used for assessing the stable transformation efficiency. It was observed that the 6-day-old young transformed calli showed about 2-fold higher regeneration frequency when compared with 21-day-old calli. The transformation efficiency was enhanced for young calli to 5.9% compared with 0.8% of the 21-day-old calli. Molecular and genetic analysis of transgenic plants （To） revealed the presence of 1-2 copies of T-DNA integration in transformants and it follows Mendalian ratio in T1 transgenic plants. From the present study, it was concluded that the development of transgenic rice plants in less duration with high regeneration and transformation efficiency was achieved in BPT 5204 by using 6-day-old young calli as explants.     

通过农杆菌介导把Harpin_(Ea)基因导入马铃薯的初步研究

应用农杆菌介导的叶盘法进行马铃薯遗传转化研究。通过D3、D4培养基筛选法 ,把HarpinEa蛋白基因导入马铃薯四倍体栽培种大西洋 (Atlantic) ,获得 10 7个转化株系。并经过卡那霉素的抗性鉴定和PCR分子检测 ,有 5 9株PCR检测呈阳性 ,占所有转化植株的 5 5 14%。结果表明HarpinEa基因已整合到马铃薯的基因组中。转化植株的抗病性鉴定正在进行中。