黄瓜无菌苗雌花诱导体系的建立

研究Spm和IAA对无菌黄瓜苗雌花诱导的协同作用,及不同外植体、培养基中琼脂含量和KH2PO4含量对雌花诱导的影响,由此建立了有效的雌花诱导体系。黄瓜去根苗接种在MS培养基上,单独添加Spm、IAA时的雌花诱导率、雌花数偏低或为0,同时添加12mg.L-1Spm与0.01mg.L-1IAA时达21%、29枚,对照组未见雌花,说明Spm和IAA对雌花诱导的协同作用显著。实验证明,在全苗、去根苗、去根去顶苗、顶芽四种外植体中,及在0.5%～0.9%琼脂含量和1.0～2.0mmol.L-1KH2PO4含量的培养条件下,采用0.7%琼脂含量和1.75mmol.L-1KH2PO4含量培养黄瓜去根去顶苗的雌花诱导效果最好,其诱导率和雌花数分别达46%、54枚。     

外源激素KT对黄瓜去根苗雌花形态建成中生化物质变化的影响

在建立了离体黄瓜去根苗雌花诱导体系的前提下,研究了在外源激素KT影响下离体黄瓜去根苗嫩枝的生理生化变化及雌花的形态建成。结果表明,在雌花形态建成过程中去根苗嫩枝的可溶性糖(C)、可溶性蛋白含量(N)、淀粉含量和C/N与雌花诱导率呈正相关关系;嫩枝中较高的POD、CAT、SOD活力水平明显有利于雌花的形态建成;诱导组和对照组的RNA含量差别明显,其含量高低顺序与雌花诱导率大小一致,而DNA含量差异很小,说明激素对雌花的诱导作用发生在转录水平。可见在激素影响下去根苗嫩枝出现的生化物质变化有利于雌花的形态建成。     

濒危物种绒毛皂荚组织培养快繁技术的初步研究

以绒毛皂荚(Gleditsia vestita Chun et How ex B.G.Li)种子为外植体,通过愈伤组织诱导、不定芽分化增殖、生根和驯化移栽,建立了绒毛皂荚组织培养的快速繁殖体系。结果表明:1)绒毛皂荚子叶和胚轴在MS+6-BA3.0 mg/L+KT 0.4 mg/L+IAA 0.3 mg/L培养基上同步诱导出愈伤和芽,愈伤组织诱导率达到90.5%,同步分化率高达77.9%;2)不定芽增殖的最佳培养基为1/2MS+6-BA 1.0 mg/L+IBA 0.3 mg/L+NAA 0.2 mg/L,培养20 d后,增殖倍数为4.0;3)MS+NAA 0.5 mg/L+IAA 0.25 mg/L条件下,生根率高达100%,且移栽后植株长势好,成活率达90%以上。     

甘蓝型油菜子叶和下胚轴再生植株无性系建立

以甘蓝型油菜(Brassica napus L.)豫油2号和6257的子叶和下胚轴为材料,在不同激素配比的MS培养基上诱导出了愈伤组织。将经过继代的部分愈伤转入分化培养基,分化结果表明:除基因型、外植体和分化培养基的激素配比对分化率有影响外,诱导愈伤培养基的激素配比对分化率也至关重要。豫油2号的子叶和下胚轴在最适诱导培养基(ZT 1+NAA0.5+2,4-D 0.2 mg/L)和最适分化培养基(ZT4+IAA 0.2 mg/L)组合中的愈伤分化率分别为12.5%和75%;6257的子叶和下胚轴在其最适诱导培养基(KT 2+NAA1+2,4-D 0.2 mg/L)和最适分化培养基(6-BA 4+IAA 0.02 mg/L)组合中的愈伤分化率分别为50%和37.5%。将其最适诱导培养基中的愈伤组织继代达8个月以上,建立了不同继代愈伤的再生植株无性系。     

离体培养黄瓜子叶诱导雌花的研究

采用添加Spd和IAA的MS培养基培养离体黄瓜子叶,研究了Spd和IAA对雌花诱导的协同作用,及昼夜温差、培养基中N素和pH值对雌花诱导的影响。结果表明,分别添加Spd、IAA时的雌花诱导率和单株雌花数偏低或为0,12 mg·L^-1 Spd与0.01mg·L^-1 IAA 配合时的诱导效果明显高于单独处理的,而对照组未见雌花,说明Spd和IAA对雌花诱导的协同作用显著。在0、2、6、10℃昼夜温差,60、70、80、90 mmol·L^-1的N素含量和pH 5.4、5.8、6.2、6.6的培养条件下,70 mmol·L^-1 N、6℃温差和pH 6.2时的雌花诱导效果较好,表明适当提高昼夜温差、培养基中N素和pH值有利于黄瓜子叶的雌花诱导。     

沙打旺原生质体培养再生植株

用1%半纤维素酶,0.4%纤维素酶,0.1%果胶离析酶,CPW9M酶液分离沙打旺无菌苗下胚轴和子叶原生质体。K8P原生质体培养基悬滴培养。下胚轴原生质体形成小细胞团后用琼脂糖包埋培养,形成小块愈伤组织后转入增殖培养基M1、M2(改良MS培养基)上形成大块愈伤组织。经过两次诱导分化,在分化培养基M3(MS 0.7mg/L BA 0.2mg/L NAA),M4(MS 0.5mg/L BA 0.5mg/L KT 0.5mg/L ZT 0.2mg/L NAA)和M6(MS 3mg/L ZT 0.2mg/L IAA)上分化出苗,再生植株。由子叶分离的原生质体未能形成愈伤组织。     

甘蓝型油菜高效离体再生体系的建立

以甘蓝型油菜(Brassica napusL.)HC8为材料,从无菌苗苗龄、预培养基激素浓度、预培养天数、6-BA及NAA的浓度等方面对影响油菜组织培养的因素进行了分析研究,建立了甘蓝型油菜品系HC8的离体再生技术体系。结果表明,该油菜组织培养的最佳苗龄为5 d;最佳预培养时间为5 d,最佳2,4-D浓度为1.0 mg/L;子叶最佳诱导培养基为MS+2.0 mg/L 6-BA+0.05 mg/LNAA+3.5 mg/L AgNO3或MS+3.0 mg/L 6-BA+0.1 mg/L NAA+3.5 mg/L AgNO3,该条件下子叶愈伤组织诱导率最高可达100%,再生频率及分化频率分别可达88.0%和108.33%;下胚轴最佳诱导培养基为MS+4.0 mg/L 6-BA+0.05 mg/L NAA+3.5 mg/L AgNO3,子叶愈伤组织诱导率最高可达95.24%,再生频率及分化频率分别可达81.82%和104.55%;最佳生根培养基为MS+0.5 mg/L NAA,生根率最高为90.0%。     

不同辣椒材料离体再生及其影响因素的研究

以8个辣椒(Capsicum annuumL.)纯系为材料,对不同材料、外植体种类和激素组合等因素对辣椒植株离体再生的影响进行了研究。结果表明,较高的6-BA/IAA值有利于辣椒外植体的分化再生,而6-BA/IAA值较低则适合于再生芽的伸长;不同辣椒材料的再生能力差别较大;辣椒带柄子叶再生能力比下胚轴强,是较好的外植体材料;12~16 d苗龄的外植体分化频率较高;在供试的8个辣椒材料中‘2096’、‘B4’和‘B7’的再生能力较强。高频率的不定芽分化培养基为MB(MS无机成分 B5有机成分) 0.8 mg/L IAA 5.0 mg/L 6-BA 4.0 mg/L Ag-NO3;不定芽伸长的培养基为MB 0.8 mg/L IAA 2.0 mg/L 6-BA 2.0 mg/L GA3 4.0 mg/L AgNO3;高效生根诱导培养基为MB 0.2 mg/L IAA 0.1 mg/L NAA。     

非洲茉莉组织培养(简报)

以非洲茉莉顶芽为外植体,在MS 6-BA3.0mg/L NAA0.2mg/L培养基上诱导产生不定芽;在MS 6-BA2.0～3.0mg/L KT0.1mg/L NAA0.2mg/L增殖培养基上分化率达3～4倍;在1/2MS KT0.2mg/L NAA0.5mg/L生根培养基中生根率达100%。     

小麦遗传转化受体系统建立的研究

选用‘小偃22’和‘宁春16’小麦品种的成熟胚和幼胚进行培养,研究不同种类的胚和培养因子对愈伤组织诱导和分化的影响。结果表明,幼胚和成熟胚的愈伤组织诱导率无明显差异,但较高浓度的2,4-D有利于成熟胚的诱导,而幼胚培养时2,4-D浓度的影响效果因品种而异;两种外植体分化率的高低与KT/IAA的配比均有密切关系,但高浓度的激素水平不利于成熟胚的分化;诱导培养基中低浓度的2,4-D有利于所诱导的愈伤组织的分化。同时,在诱导培养基中添加低浓度的KT能显著提高两品种成熟胚愈伤组织的分化率;各种培养基处理与品种间都存在显著的互作效应,‘小偃22’成熟胚培养的最佳培养基组合为MSD 3.0 mg/L 2,4-D和MSD 0.5 mg/LIAA 1.0 mg/L KT,幼胚培养为MSD 4.0 mg/L 2,4-D和MSD 0.5 mg/L IAA 1.0 mg/L KT;‘宁春16’成熟胚培养为MSD 4.0 mg/L 2,4-D和MSD 1.0 mg/L IAA 1.0 mg/L KT,幼胚培养时为MSD 1.0 mg/L 2,4-D和MSD 2.0 mg/L IAA 2.0 mg/L KT。     

荞麦高频离体再生及发根农杆菌转化体系的建立

荞麦无菌苗下胚轴切段在不同激素配比的MS培养基上诱导愈伤组织,出愈率均为100％。在2．0mg/L2,4-D和1．5mg/L 6-BA组合下诱导产生的愈伤组织;转入2．0mg/L 6-BA和1．0mg/L KT的MS培养基,再生苗分化率在80％以上。根尖色体分析表明再生植株具一定的遗传稳定性。发根农杆菌A4转化荞麦下胚轴和子叶获得发状根,纸电泳检测所有随机取样测定的发状根均有相应冠瘿碱的存在。     

Endogenous Phytohormones and Regulation of Morphogenesis of Arabidopsis thalianaby Blue Light

We studied the effects of blue light (BL) on the levels of endogenous phytohormones (IAA, ABA, gibberellins, and cytokinins) and morphogenesis of the 7-day-old Arabidopsis thaliana(L.) Heynh seedlings of wild type (Ler) and its hy4mutant with a disturbed synthesis of cryptochrome 1 (CRY1), which is a receptor for BL. In darkness, the mutant contained considerably less free IAA and zeatin, but much more ABA as compared to the wild-type seedlings. BL retarded the hypocotyl growth in the wild-type seedlings but stimulated it in the mutant. Elongation of mutant hypocotyls was accompanied by accumulation of free IAA and a decrease in the content of free ABA; the level of cytokinins did not change. We believe that the response of the hy4hypocotyls to BL is mediated by a BL receptor distinct from cryptochrome 1. The conclusion is that light and hormonal signals interact in the control of the hypocotyl growth in A. thalianaseedlings.     

Influence of genotype and explant source on the in vitro regeneration ability of different melon varieties

Nine genotypes of melon (Cucumis melo L.) were selected for the investigation of regeneration. Most of the tested varieties showed regeneration ability on medium containing 0.5 mg l?1 or 1 mg l?1 BA, but following the appearance of shoot buds, only six varieties produced leafy shoots. The effect of combinations of BA with different auxins (IAA, NA, 2,4-D) and ABA in the culture medium on shoot regeneration was tested on cotyledon explants of 'Hógolyó' and 'Hale's Best'. To establish optimal conditions for the adventitious shoot induction six types of seedling-derived explants were prepared from seedlings of four different ages. The best results for shoot forming capacity were achieved with cotyledons followed by decapitated seedlings and hypocotyls derived from 4-day-old seedlings. Cotyledon segments of 'Hógolyó' and 'Hale's Best' were also cultivated on media with different concentrations of IAA and BA supplemented with 0.26 mg l?1 ABA. The highest number of well-formed plantlets was counted for 'Hógolyó' on the medium supplemented with 0.9 mg l?1 BA+ 0.6 mg l?1 IAA+ 0.26 mg l?1 ABA. This is the first report on the in vitro regeneration of 'Hógolyó' from decapitated seedling and hypocotyl explants and of 'Javított Zentai', 'Muskotály', 'Hógolyó', 'Tétényi csereshéjú' and 'Magyar Kincs' from cotyledon explants.     

Effect of ABA-beta-D-glucopyranosyl ester and activity of ABA-beta-D-glucosidase in Arabidopsis thaliana

Exogenously applied ABA-beta-d-glucopyranosyl ester (ABA-GE) inhibited hypocotyl growth of Arabidopsis seedlings at concentrations greater than 0.3mumol/L, and the concentration for 50% inhibition of hypocotyl growth was 1.8mumol/L. ABA-beta-d-glucosidase activity in Arabidopsis seedlings was 17nmol/mg protein/mim and increased by exogenously applied ABA-GE. The pH optimum of this enzyme in crude extract of Arabidopsis seedlings was 6.0 for the assay in the ABA-GE to ABA direction and its K(m) value for ABA-GE (pH 6.0) was 0.41mmol/L. These results suggests that exogenously applied ABA-GE may be absorbed by roots and hydrolyzed by ABA-beta-d-glucosidase and librated free ABA may induce growth inhibition in Arabidopsis hypocotyls.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.     

Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum

Protoplasts were isolated and cultured from hypocotyl embryogenic callus tissue of Gossypium hirsutum L. cv. "Lumian 6". The highest yields of viable protoplasts were obtained from a vigorous embryogenic callus 7 to 9 d old subcultured on MS medium supplemented with 2 mg/L IAA and 1 mg/L KT using a solution of 1% cellulase Onozuka R-10, 1% pectinase, 0.7 mmol/L KH2PO4, 2.5 mmol/L Ca2+ , and 0.5 mol/L osmoticum (mannitol), at pH 5.8 and at a temperature of 30 ℃. After separation and purification (in 21% sucrose floatation medium), the protoplasts were laid up in a quiet liquid protoplast culture medium containing K3 salts, NT vitamins with 0.1 mg/L 2,4-D, 0.2 mg/L KT and 0.45 mol/L glucose for 10 to 15 min. The protoplasts were fractioned into an upper and a lower layer in the centrifugal tube. Most of the protoplasts in the lower layer were smaller, round and rich in cytoplasts in which contain many granular substances. When this kind of protoplasts were cultured in the thin liquid protoplast culture medium with a density of 1 x l0s to 5 x los protoplasts/mL, the division and the callus formation of the regenerated cells were easily observed. The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with decreased osmoticum once or twice is needed for the continuous protoplasts division to form calli. Regenerated calli, 3 to 5 mm in diameter, were transferred in succession on MS medium with 2 mg/L IAA and 1 mg/L KT for the initiation of embryogenesis. The embryoids germinated on the hormonefree MS medium and a number of plantlets were obtained. It seems that using vigorous embryogenic callus and decreasing osmoticum are the two critical factors for plant regeneration of cotton protoplasts.     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside