宽皮柑橘品种的胚性愈伤组织诱导

以宽皮橘3个品种的幼嫩胚珠和8个品种成熟果实中未发育胚珠为试材,采用EME(MT 500mg/L麦芽浸出物)、MKT(EME 10mg/L KT)、MGS(EME 1mg/L GA3 40mg/L硫酸腺嘌呤)和MDB (EME 0.01mg/L 2,4-D 0.1mg/L BA)4种培养基进行胚性愈伤组织诱导,结果表明EME与MKT培养基配合使用有利于从幼嫩胚珠获得胚性愈伤组织;MGS比MDB更有利于成熟果未发育胚珠胚性愈伤组织的诱导;暗培养有利于此诱导过程。经两种途径获得的胚性愈伤组织,染色体数目稳定,均为二倍体2n=2x=18。     

宜昌橙与伏令夏橙种间体细胞杂种

宜昌橙(Citrus ichangensis SwingIe)试管实生苗叶肉原生质体与伏令夏甜橙(C．Sin-ensis Osbeck)胚性悬浮细胞系原生质体,经聚乙二醇(PEG)诱导融合。再生出的胚状体为畸形,转移到生芽培养基中诱导产生丛芽。丛芽微嫁接在15天龄的枳壳砧上成为完整植株。幼叶染色体检查表明,两亲本均为二倍体2n=2x=18,融合后再生出的植株为四倍体2n=4x=36。过氧化物酶(POX)及谷草酰胺转氨酶(GOT)同工酶分析证明,这些四倍体均为体细胞杂种,它们同时含有双亲的酶带。杂种植株叶片形态更像甜橙。植株移八土壤后生长旺盛。     

海石竹的离体快繁及核型分析

以海石竹 (Armeriamaritima)的叶片为外植体 ,经离体培养诱导产生愈伤组织 ,再分化形成不定芽 ,并经过继代增殖和壮苗生根 ,获得完整的再生植株 ,最后对其再生植株进行核型分析。结果表明 ,海石竹叶片的愈伤组织诱导和分化的适宜培养基为MS +BA 1 .0mg/L +NAA 0 .2mg/L ,诱导初期进行 7d暗培养 ,最佳增殖培养基为MS+BA 1 .0mg/L +NAA 0 .1mg/L ,生根培养基为MS+NAA 0 .2mg/L。以上培养基均含蔗糖3 0 g/L ,琼脂 5g/L ,pH 5 .8。海石竹的核型公式为 2n=2x=1 8=1 0m +8sm ,存在染色体数目变异的现象。     

田埂报春多倍体诱导及其形态学研究

在离体培养条件下,比较不同浓度、不同处理时间的秋水仙素对田埂报春进行染色体加倍的诱导效果。结果表明:0.08%秋水仙素处理48h的诱变效果最佳,诱变率高达56%。经秋水仙素诱导后形成的多倍体植株与原二倍体植株比较,在形态上,四倍体植株表现出多倍体特征,叶片变厚,叶形指数减小,保卫细胞增大,单位面积气孔数减少,叶绿体数明显增多。对变异植株进行细胞学研究发现,体细胞中期染色体数目为2n=4x=36,而原二倍体的染色体数目为2n=2x=18,基数x=9,因此,变异植株(2n=4x=36)为四倍体。前者的核型公式为2n=4x=8L+12M2+4M1+12S,核型属于1A;后者的核型公式为2n=2x=4L+6M2+2M1+6S,核型也属于1A。检测发现少数个体有非整倍体变异。     

柑橘与枳属间体细胞杂种再生及其对脚腐病抗性的评价

Page橘柚 (CitrusreticulataBlanco×C .paradisiMacf.)胚性细胞悬浮系原生质体与枳 (Poncirustrifoliata (L .)Raf.)叶肉原生质体经电场诱导融合 ,4～ 5个月再生 15 0余棵小植株。再生植株根系发达 ,叶片具三叶特征。随机检查 2 0余株再生苗根尖染色体数目 ,表明都为四倍体 (2n =4x =36 )。随机取 7株进行RAPD分析 ,表明被检测植株都为杂种。用引起脚腐病的寄生疫霉菌 (PhytophthoraparasiticaDastar)毒素接种体细胞杂种及双亲叶片 ,结果表明 ,Page橘柚中度感病 ,枳高抗 ,体细胞杂种为抗病类型。     

枸杞胚乳植株的诱导及染色体倍性观察

枸杞[Lycium chinense Mill.var.potaninii(Pojark)A.M.Lu]的未成熟胚乳接种在MS基本培养基上,并添加1毫克/升2,4-D和0.1毫克/升KT,诱导产生愈伤组织,其频率为23.8％。处于色变期的果实为胚乳培养的最适时期。胚在胚乳培养中不是必不可少的,但当有胚存在时可以提高胚乳愈伤组织的诱导频率。胚乳愈伤组织的继代培养对器官分化和植株再生没有明显影响。胚乳植株根尖细胞染色体数目不很一致,甚至有的植株同一根尖的不同细胞染色体数目也有不同。其中有三倍体(2n=3x=36),也有二倍体(2n=2x=24)和非整倍体的细胞,染色体数为9、18等,但从观察的15株胚乳苗来看,三倍体有11株,占73.3％,非三倍体的只有4株,占26.7％。     

中国桔梗多倍体诱导与鉴定

在离体培养条件下,比较了不同浓度、不同处理时间的秋水仙素对中国桔梗（Platycodon grandiflorus A．CD）进行染色体加倍的诱导效果。结果表明：用含0．1％秋水仙素处理40h后诱导频率可达50％,诱导效果最佳。经秋水仙素诱导后形成的多倍体植株与原二倍体植株比较,在形态上,多倍植株叶片变宽变大,叶色变深,茎变粗且节距长,气孔增大而单位叶面积气孔数目减少。对变异植株进行细胞学研究发现,体细胞中期染色体数目为2n=4x=36,而原二倍体的染色体数目为2n=2x=18,基数x=9,因此,变异植株（2n=4x=36）为四倍体。前者的核型公式为2n=4x=14m＋20sm＋2st,核型属于2B;后者的核型公式为2n=2x=7m＋10sm＋1st,核型也属于2B。检测发现有少数个体有非整倍体变异。     

大白菜与结球甘蓝异源三倍体小孢子植株的获得与鉴定

以6个不同基因型的大白菜四倍体(AAAA,2n=4x=40)品系9401、9402、9403、9404、9405、9406为母本,结球甘蓝二倍体(CC,2n=2x=18)自交系9501为父本配制杂交组合得到的6个杂种一代为试材,进行了游离小孢子培养研究,成功诱导出胚状体,获得了再生植株,并对部分再生株进行了染色体数鉴定和性状调查。结果表明:不同组合小孢子胚胎发生能力不同,各组合产胚率均较低;小孢子再生植株中,染色体数为18的个体所占比例最大,达46.7%;小孢子植株减数分裂行为复杂,终变期除二价体和单价体外,还有三价体等联会形式;小孢子植株性状表现各异。     

甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

用花药培养法由马铃薯雄性不育双半日倍体诱导—单倍体植株的研究

用花药培养法由雄性不育材料诱导单倍体的尚未见报道,在马铃薯中,所有双单倍体或一单倍体,也都是由有效花粉百分数较高的雄性可育材料诱导产生的,本文首次报道了用花药培养法由一个典型的雄性不育的双单倍体吕系诱导产生一单倍体的试验结果。从接种的1850个花药中,诱导出了28个胚状体和23块愈伤组织,并从它们分化出了24株绿苗,分化出的小植株生活力大部分较弱,在继代培养过程中逐渐死亡,但也有一些生活力较强的植株存活下来,经L1,L2和L3 3个胚层细胞的检查表明,它们具有典型的一倍体特征,体细胞染色体数目为2n=x=12,植株之间表现出明显的性状分离,说明它们均来自减数分裂后的小孢子,大部分植株的产量较低,但也有少数生活力强,单株块茎产量达0．5公斤左右的抗病,品质好的植株,所有株都具有不正常的减数分裂,无有效花粉形成,个别植株能结实,但无种子形成,用四倍体或双单倍体的可育花粉授粉也不能形成种子,进一步证明了它们具有一单倍体和持性,对放有导马铃薯一单倍体和利用雄性不育材料进行花药培养诱导倍体的意义进行了讨论。     

Triploid Citrus Plants Obtained from Crossing the Diploids with Allotetraploid Somatic Hybrids

Hybridizing the diploid monoembryonic pummelo (Citrus grandis) and polyembryonic tangerine (C. reticulata cv. Huanongbendizao) with allotetraploid somatic hybrids from protoplast fusion were conducted. Seeds of pollinated fruits were found to be abortive 90 days after pollination. The aborted seeds were then cultured on media of MT supplement with 1 mg/L GA3 or with 500 mg/L of malt extract. 25.6% of the seeds germinated or developed into embryoids: The entire plants were transplanted into soil after grafting shoots on the root-stocks of trifoliate orange in vitro, if the germinated embryos have poor roots or no root at all. A total of 73 plants from 6 different combinations were obtained, among which 20 were verified as triploids with 2n= 3x=27 chromosomes, 32 diploids 2n= 2x= 18, 8 a- neuploids and the rest 13 unconfirmed.     

Induction of Haploid Durum Wheat Plants Through Pollination of Maize Pollen

When tetraploid wheat (Triticum durum Desf. ) variety DR147 was crossed with maize (Zea mays L. ) variety suppersweet ss 7700, pollen readily germinated on the stigma and one or more pollen tubes reached the embryo sac in 83.4% of wheat florets. The frequency of fertilization and embryo formation was 44.5% and 42. 6% respectively. The hybrids were karyotypically unstable and the maize chromosomes were eliminated early in the development. Thus haploid wheat embryos were form. Although the double fertilization frequency of durum wheat X maize was high (32.7%) to form embryos and endosperms, yet the endosperms were highly abnormal. It was very difficult to produce viable mature seeds from the mother durum wheat plants. The survival of hybrid embryos produced by durum wheat X maize could be improved or prolonged by treatment with 100 ppm 2, 4-D (either by dipping inflorescences in solution or injecting 0.3 to 0.5 mL 2, 4-D solution into the uppermost internodes of the wheat stem). 9 to 13 days after pollination, caryopsis were excised from the pollinated spikes and surface sterilized for peeling of the embryos in different developing stages. The embryos were plated on MS solid medium containing 3% sucrose, 200 mg/L casein hydrolysate for embryo rescue. The experimental results revealed that the well developed embryos (larger than 0. 5 mm with scutellum structure) were easy to produce calli by callus induction or produce haploid wheat plants by embryo rescue, whereas the poorly developed embryos (globular, pear or torpedo-shaped embryos smaller than 0.3 mm) responsed very poorly. The germination frequencies of well and poorly developed embryos were 83.3 % and 12.5 %, respectively. Chromosome counts of root tip cells of the rescued plants proved their haploid nature (2n= 2x= 14).     

Production of aneuploid melon plants following in vitro culture of seeds from a triploid x diploid cross

Aneuploid melon plants (Cucumis melo L.) were obtained from in vitro cultured seed, which were produced by crossing triploid (3x=36) x diploid (2x=24) plants. Twenty-six fruits were obtained from pollination of 29 bisexual flowers of triploid plants. Seeds were collected from the fruits at 2, 3, 4, 5 and >7 weeks after pollination and germinated in vitro on Murashige & Skoogs (MS) medium. Embryos developed from 0.6 to 1.6% of the cultured seeds after three weeks in culture. Shoots developed from 0 to 47% of embryos after transfer to half-strength MS medium. Some (0 to 50%) of elongated shoots that rooted were subcultured on the same medium. Five rooted plantlets were obtained through culture of 5,353 seeds. Four of the plants were aneuploid, with chromosome numbers of 27, 35, 45 and 46, respectively, and the one was tetraploid (4x=48).     

转HAL1基因番茄的耐盐性

利用农杆菌介导的叶盘法,把HAL1 基因转入番茄,Southern杂交检测得到转基因植株.耐盐实验表明, T1代转基因番茄在150 mmol/L的NaCl胁迫下仍有43%的发芽率,200 mmol/L的NaCl胁迫下发芽率为6%,而对照种子在100和150 mmol/L的NaCl胁迫下发芽率分别为11.0%和0.转基因番茄的电解质相对外渗率小于对照,而根冠比和叶绿素含量大于对照,转HAL1基因显著提高了番茄的耐盐性.盐胁迫下Na 、K 的累积状况表明,转基因番茄根、茎、叶的K /Na 均有所提高,根系的SK/Na增大,茎、叶的RSK/Na和RLK/Na减小,说明根系对K /Na 离子的选择吸收和运输能力加强.不但选择吸收K /Na ,而且表现出整株水平上的有利于耐盐的K /Na 区域化分配.     

An efficient protocol for the production of triploid plants from endosperm callus of neem,Azadirachta indica A. Juss

Triploid plants of neem were obtained by immature endosperm culture. Immature seeds, at the early dicotyledonous stage of embryo development, is the best explant to raise endosperm callus on MS + NAA (5 mumol/L) + BAP (2 mumol/L) + CH (500 mg L-1). Maximum shoot bud differentiation from the endosperm callus occurred on MS + 5 mumol/L BAP. Shoots were multiplied by forced axillary branching and rooted in vitro. The plants were established in soil. Over 66% of the plants were triploid with chromosome number 2n = 3x = 36. A characteristic feature of the shoots of endosperm origin is the presence of a large number of multi-cellular glands.     

普通小麦与鸭茅状摩擦禾的远缘杂交：Ⅱ.未成熟胚的培养

培养了由２６个小麦品种（系）用鸭茅状摩擦禾的花粉授粉１４天后获得６４５个未成熟的胚。结果表明,未成熟胚培养的植株再生频率与胚的小麦基因型的杂交亲和性无关,胚的发育程度直接影响胚培养的结果,离体时分化完全的未成熟胚在无激素的培养基上可以迅速萌发成工轩,而分化未完全的小胚在无激素的培养基上分化进程不能继续,而且在无激素补充的情况下,萌发过程一旦起动,即使将这些胚转至补加了激素的胚分化培养基上,分化过程     

Haploid Plant Regeneration from Protoplast Cultures of Durum Wheat (Triticum durum Desf.)

Haploid suspension callus cultures from embryos of durum wheat (Triticum durum Desf. ) × maize (Zea mays L. ) crosses were used for protoplast isolation. Experimental results from enzyme digestion showed large numbers of viable protoplasts released from both suspension culture and solid culture of callus cut into small pieces of 1 mm in size prior to incubation in an enzyme solution containing 2.0% cellulase RS and 0.5 % pectolyase Y-23. Division frequency of protoplasts isolated from suspension cultured callus was quite different from that of solid cultured callus, however, the former being 5.20%, and the latter less than 1.0% when cultured on KM8p medium containing 1.0 mg/L 2, 4-D using LMP (low melting point) agarose embedding method. Embryogenic ealli could be selected out from protoplast-derived microcalli after 2 to 3 subcultures. Plants could be regenerated from protoplast-derived embryogenic calli after 20 days of culture on differentiation medium I (MS basal medium supplemented with 0.2 mg/L 2, 4-D, 1.0 mg/L BAP, 0. I mg/L NAA, 3 %/4 su- crose, 200 mg/L casein hydrolysate, 146 mg/L glutamine, 300 mg/L aspartic acid) and (components were the same as I without 2, 4-D) respectively. The plant regeneration frequency was about 20%. Chromosome count of root tip cells of 4 plants of the 22 protoplast- derived plants sampled at random revealed haploid in nature (2n= 2x= 14).     

Micropropagation of an elite Darjeeling tea clone

Shoot cultures of Camellia sinensis (L.) O. Kuntz var. T-78, an elite Darjeeling tea clone, were established from cotyledonary nodes and shoot tips of germinated seedlings as well as from nodal explants of field grown plants. Shoot multiplication rate ranged from 4x in nodal explants to 35x in cotyledonary nodes after 18 weeks of culture. Rooting was achieved in 80–90% micro-shoots by either placing them on an inductive medium for 10 d and then transferring shoots to hormone-free medium, or by treating micro-shoots with a chronic dose of IBA (500 mg/l) for 30–40 min. Rooted plants were established in soil under glasshouse condition at 60% frequency after hardening phase of 4–6 weeks. The regenerated plants show a constant chromosome number of 2n=30 and are morphologically true to type. This procedure can be applied for conservation and utilisation of an elite clone of Darjeeling tea.     

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.     

Production and characterization of amphiploids of Aegilops kotschyi and Ae. biuncialis with Secale cereale, and of backcross hybrids of Ae. biuncialis x S. cereale amphiploids with 2x and 4x S. cereale

Amphiploids (2n = 6x = 42) of Ae. kotschyi and Ae. biuncialis with self-compatible S. cereale were produced from F1 sterile hybrids (2n = 3x = 21) through colchicine treatment and callus tissue regeneration. The amphiploids resembled the F1 plants in overall morphology, but were larger in all respects and self-fertile. The spikelets consisted mostly of 3 well-developed florets. Selfed seeds were obtained from some colchicine-doubled sectors and callus regenerates. Most of the produced seeds were well developed. Backcrosses between amphiploids and rye (2x and 4x) resulted in obtaining (Ae. biuncialis x S. cereale amphiploid) x S. cereale hybrids via embryo culture. The BC1 plants (2n = 4x = 28 and 2n = 5x = 35, respectively) were phenotypically intermediate between the parents and vigorous in vegetative growth. Some seeds were obtained only from the 35-chromosome BC1 hybrids.