马铃薯试管薯诱导及其遗传转化体系的优化

目的：对马铃薯试管薯诱导及遗传转化体系进行优化研究。方法：利用3种培养法对马铃薯品种甘农薯2号和Favorita进行了试管薯的诱导,并用农杆菌介导法对其遗传转化体系进行优化研究。结果：用固体培养、固液培养和液体培养法诱导的甘农薯2号试管薯单瓶平均结薯数分别为4．6、4．4和6．5个,块茎平均直径分别为6．2、5．8和7．7ram;而Favorita单瓶平均结薯数为5．4、5．8和7．4个,平均直径分别达6．2、5．9和7．3mm。用浓度为OD600=0．5的农杆菌菌液侵染8min,共培养2d能够获得较高的转化频率。结论：液体培养法诱导试管薯的效果最好,建立的高频率遗传转化体系使甘农薯2号和Favorita的转化频率分别可达42．6％和36．8％。     

香豆素和寡糖素对马铃薯试管结薯的影响

本研究将生理活性物质香豆素、人参寡糖素和黑节草寡糖素分别加入含有BAP5.0mg/l、蔗糖8％的MS基本培养基中,用于诱导马铃薯试管结薯,并与加入矮壮素诱导的结薯效果作了比较。结果表明,在加入100mg/l香豆素时,试管结薯的数量与使用500mg/l矮壮素效果相近似。使用50mg/l人参寡糖素或10mg/l黑节草寡糖素时,试管结薯的数量明显超过或略超过用500mg/l矮壮素的效果。对4种处理下获得的试管薯进行了过氧化物酶的同工酶及酶活性的测定。结果表明,这4种不同的外源生长调节剂在使用浓度恰当时,所结薯块的过氧化物酶同工酶谱和过氧化物酶活性是近似的。     

“固液双层”培养法诱导马铃薯‘米拉’试管薯的研究

该研究以马铃薯‘米拉’品种的脱毒试管苗为材料,采用"固液双层"的培养方式,通过正交试验对其试管苗壮苗生长阶段和试管薯诱导阶段的培养基进行优化,并通过单因素试验研究蔗糖浓度、光照条件和CCC浓度对试管薯结薯的影响。结果表明:在"固液双层"培养中,‘米拉’壮苗培养阶段优化的培养基为改良MS培养基(硝酸铵为2 000 mg·L~(-1)、硝酸钾为2 000 mg·L~(-1))+500 mg·L~(-1)CCC+0.1%活性炭+0.1mg·L~(-1)DA-6+1 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA+3%蔗糖+6 g·L~(-1)琼脂,pH 5.8。试管薯诱导及生长阶段优化的培养基为MS_1培养基(微量元素和铁盐的用量为MS培养基的2倍)+1.5%活性炭+4 mg·L~(-1)6-BA+8%蔗糖。在试管薯诱导阶段,弱光4 h·d~(-1)培养诱导的试管薯,结薯指数、大薯率、薯块重量均优于暗培养。"固液双层"培养是一种低成本的方法,在组织培养室内就可以大量繁殖‘米拉’试管薯,并且能增加原种的数量,这种方法也能用于马铃薯其他栽培品种试管薯的诱导。     

‘红皮’马铃薯试管苗培养和微型薯诱导

以‘红皮’马铃薯块茎上的芽为材料进行试管苗诱导、增殖及试管微型薯诱导研究。结果表明：在MS + 6-BA 0.5 mg/L +活性炭0.17% + 蔗糖20 g/L培养基中培养30 d,试管苗增殖系数达8.6;MS + 6-BA 0.5 mg/L +活性炭0.17% +蔗糖100 g/L培养基中试管微型薯诱导效果较好,30 d后诱导率达62.5%,试管薯平均直径为4.2 mm。     

香豆素浓度及光照条件对马铃薯试管薯诱导影响初步研究

利用马铃薯脱毒苗直接在三角瓶中诱导马铃薯结薯,试图在更少的空间和更短的时间内诱导出大量试管薯,以便于大规模工厂化生产,获得高质量的原种。在不同香豆素浓度、不同光照条件下对马铃薯试管薯诱导进行研究,结果表明,全黑暗条件对试管薯形成、结薯数和平均单薯重有促进作用;培养基中加入60mg／L香豆素明显提前试管薯形成期,显著增加结薯数;加入30mg／L香豆素显著提高单薯重。     

施加外源抗坏血酸促进马铃薯试管薯形成及其机制初步研究

为研究抗坏血酸(AsA)处理对马铃薯试管薯形成和结薯相关基因表达的影响及敲除结薯关键基因Solanum tuberosum self-prunning 6A(StSP6A)对AsA诱导马铃薯结薯的效应,利用不同浓度(0、1、5、10、20和50 mmol/L)外源AsA处理2个二倍体马铃薯CIP-149(Solanum phureja)、CIP-178(S.ajanhuiri)和四倍体C-88(S.tuberosum)。结果表明,1~5 mmol/L外源AsA处理可显著诱导马铃薯块茎的形成。对10个块茎形成相关基因的表达分析结果表明,外源添加1 mmol/L AsA可显著影响块茎形成相关基因的表达,与对照相比,总体上呈正调控基因表达增强或负调控因子表达被抑制的趋势,特别是StSP6A在AsA处理过程中的块茎形成早期表达量极显著上调,敲除StSP6A可消除外源AsA对马铃薯块茎形成的诱导作用。这些结果表明,AsA诱导马铃薯块茎形成是通过调控与块茎形成相关的基因表达来实现的,而StSP6A在AsA诱导马铃薯块茎形成中起关键作用。     

蔗糖,GA与液体培养基对马铃薯块茎形成的影响

马铃薯是全球第四大粮食作物,粮菜兼用,有丰富的营养价值。马铃薯块茎是马铃薯最具经济价值的部分。马铃薯块茎形成的过程受多种调控途径的调控,其中最重要的是蔗糖途径和赤霉素(GA)途径。本实验设计蔗糖浓度,GA浓度和培养基类型3个因素的正交试验,通过对马铃薯的结薯时间,结薯数,马铃薯均重和大薯率的统计发现高浓度的蔗糖能够使马铃薯结薯提前,液体培养基能够增加马铃薯的薯重和大薯率。添加8%蔗糖和0.1 mg/L GA的液体MS培养基能够使马铃薯提前结薯并且得到的马铃薯数量多,薯重大。     

植物资源开发利用新信息——国外有关植物的专利摘要(十二)

331.提高小麦抗寒和抗旱能力的化合物 据前苏联1991年专利记载。氨基甲酰衍生物2-硫代螺环已基乙内酰脲可提高小麦抗寒和抗旱能力。专利说明对此化合物的化学结构和制备作了叙述。 332.马铃薯茎尖无病毒培养 据前苏联1991年专利记载。马铃薯茎尖为马铃薯植株无病毒部分,用含有分枝甘露聚糖0.15—0.25g/L、氨基胍0.05g/L和三嗪衍生物0.05g/L的vanGoff培养基培养1.5—2天,即可用为无病毒马铃薯种薯繁殖。     

高效马铃薯遗传转化体系的建立及甜蛋白基因的导入

本研究选用了三个马铃薯(Solanum Iuberosum L.)栽培品种“85T-14-3”、“86-2”及“Favorita”的块茎、微型薯和试管薯为起始材料,应用根癌农杆菌 Ti 质粒系统成功地建立了一种方法简单、速度快和频率高的遗传转化体系。其中试管薯薄片的转化速度最快,经根癌农杆菌(Agrobacterium tumefaciens)共培养后,薄片在100mg/L 卡那霉素的分化培养上,2—3周就可产生出抗性小芽,这些小芽进一步仲长后可在50—100mg/L 卡那霉素的无激素MS 培养基上生根。从共培养到转化植株的获得只需6—7周。微型薯和试管薯的转化频率较高,最高可达67.5%。大多数抗性植株均能检测到胭脂碱合成酶或 GUS 基因的表达。把带有甜蛋白基因和胭脂碱合成酶标记基因的Ti质粒导入马铃薯,获得大量转化植株。叶片抗性检测和 nopaline 检测可推断外源甜蛋白基因已进入马铃薯基因组。     

水杨酸对马铃薯试管微薯形成的影响研究

研究了不同浓度水杨酸（ＳＡ）对马铃薯脱毒试管苗生长,分化及试管微薯诱导和发育的 浓度ＳＡ显著抑帛式管苗主茎和根的生长,促进侧枝和匍匐茎分化。高浓度（０．１－１．０ｍｍｏｌ／Ｌ）ＳＡ能诱导试管微薯形成并显著提高结薯率。ＳＡ浓度为０．５ｍｍｏｌ／Ｌ时,结薯率最高,且成薯集中,薯块大小整齐一致。     

Hydroxycinnamoylputrescines are not causally involved in the tuberization process in potato plants

The possible role of hydroxycinnamoylputrescines in the tuberization process of potato plants was studied using in vitro tuberization systems. Minitubers in shoot cultures of Solanum tuberosum ssp. andigena and S. tuberosum ssp. tuberosum were obtained in vitro within 3 weeks of dark incubation after increasing the sucrose concentration in the Murashige-Skoog (T. Murashige and F. Skoog. 1962. Physiol. Plant. 15: 473–497.) medium (without hormones) from 60 to 240 m M . both in the presence and absence of benzylaminopurine (BAP). Feruloylputrescine (FP) and caffeoylputrescine (CP) increased with tuberization, with a sharp maximum at day 9 in the shoot, but only when the medium contained BAP. When inhibitors of phenylalanine ammonia-lyase (PAL) and of polyamine biosynthesis were added to the medium containing BAP, the levels of FP and CP were reduced to values lower than those observed in the absence of BAP, but there was no significant effect on the number and dry weight of tubers formed. Addition of BAP without increasing the sucrose content also resulted in CP and FP accumulation. but failed to induce tuberization of the cultures. Experiments with in vitro stolon cultures and leaf cuttings also supported the conclusion that CP and FP accumulated as a response to the application of BAP, without having any effect on optimal tuberization. These results indicate that the increase of hydroxycinnamoylputrescines during tuber formation is unlikely to be causally involved in the tuberization process in potato plants.     

Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Study on Regulation of 5′ Flanking Regions of Granule-bound Starch Synthase Gene of Potato

Binary vectors were constructed by fusing 0.4, 0.8, 1.6, and 2.9 kb 5' flanking regions of GBSS gene with GUS (β-glucuronidase). Transient GUS expression was observed in in vitro tuber slices bombarded with 0.8 kb GBSS-GUS construct. These constructs were then transferred into potato ( Solanum tuberosum L. cv. Desiree) via Agrobacterium tumefaciens transformation. Transgenic potato plants were confmned by X-Gluc staining and PCR. Using in vitro tuberization system, GUS expressions were assayed with fluorescence, it was shown that 0.8, 1.6 and 2.9 kb GBSS-GUS expressions were higher than 0.4 kb GBSS-GUS. 1.6 and 2.9 kb GBSS-GUS expressions were about 2 to 10-folds higher in tubers than in stems. In cultured shoots, GBSS-GUS expression could be induced by increased sucrose concentration but inhibited by light.     

文心兰试管苗丛生芽高效增殖体系的建立

分别采用细胞分裂素6-BA和Ad对文心兰试管苗增殖的作用进行研究,结果表明：在与0．2mg／L的NAA配合使用时,6-BA含量在2．0～4．0mg／L水平下的培养基较适合试管苗的增殖,最大增殖系数可达7．27,苗生长健壮,叶色鲜绿,适合进一步生根移栽;Ad在1．0～6．0mg／L的范围内增殖系数较小,苗生长缓慢、矮小。但有形成丛生苗的倾向;以2．0mg／L的6～BA和1．0mg／L的Ad组合使用,易形成丛生芽苗且苗体相对较小,增殖系数也较高;接种单株小苗较大苗易形成丛生芽苗;接种连体小芽苗,全部能形成丛生芽苗,增殖系数可达10．07,适合于进一步增殖使用。6-BA与Ad配合使用结合接种连体小芽苗可建立高效的丛生芽苗增殖体系,培养基最佳激素组合为MS 6-BA2．0mg／L Ad1．0mg／L NAA0．2mg／L 3％蔗糖 0．4％琼脂粉。