秋水仙碱对甘蓝型油菜离体小孢子胚胎发生的影响

研究了秋水仙碱不同浓度和处理时间对甘蓝型油菜23个基因型离体小孢子胚胎发生的影响.3个基因型的小孢子被10、50和100mg/L秋水仙碱处理24h或48h,胚产量是2.55～14.75胚/蕾,10～50mg/L处理72h则是0.94～2.43胚/蕾.这表明处理72h对小孢子胚发生有抑制作用.用200、400、500和800mg/L处理2个基因型小孢子16～48h,胚产量为0.6～1.33胚/蕾,未处理对照是6.25和9.36胚/蕾.可见200～800mg/L浓度对胚再生有不同程度的阻碍效应.结果还证明,小孢子对秋水仙碱的反应与其基因型有关.当用10、20、50和100mg/L处理48h时,22B5-6和903-3小孢子的胚产量为37.09～69.47胚/蕾,而F1-29、W592和SF10-12是0.28～1.45胚/蕾,相互之间差异很大.秋水仙碱处理小孢子的目的是使其再生植株的染色体高频率加倍,因此应根据胚产量和染色体加倍率来确定秋水仙碱浓度和处理时间.本试验中,采用10～50mg/L处理48h或者用100mg/L处理24h,约80%基因型的小孢子胚产量在5胚/蕾以上,约70%基因型的再生植株加倍率达60%以上,可有效地用于油菜遗传和育种研究等领域.     

小麦—簇毛麦杂种幼胚无性系的建立及植株再生的研究

普通小麦与簇毛麦杂交十分困难,得到的植株很少。然而通过杂种幼胚无性系培养,不但能获得大量的再生植株,而且利用愈伤组织能较长时间保存杂种种质。经过继代培养十个月共九代,由四个杂种幼胚的愈伤组织获得试管苗1957株,其中秋水仙碱处理1350株,成活1122株。套袋自交后获种子664粒,并获得自然授粉种子508粒。对幼胚愈伤组织和再生植株根尖进行了细胞学观察,绝大多数染色体数目为2n=28,变异率分别为33.04%和12.90%。愈伤组织保存一年后,仍有很强的分化能力,幼苗分化率为85%。     

二甲戊灵对离体大蒜染色体的加倍研究

在MS分化培养基中分别添加不同浓度的秋水仙素和二甲戊灵,以 "改良蒜" 的蒜瓣基部为外植体诱导愈伤组织染色体变异并再生植株,观察根尖细胞染色体数和叶片下表皮保卫细胞特征检测诱变效果.结果表明:大蒜染色体数变异受诱变剂浓度和处理持续时间的影响,诱变剂高浓度和短时间处理均有利于大蒜愈伤组织的分化;添加0.1%秋水仙素处理5 d,愈伤组织存活率为80.7%,分化率为78.1%,四倍体的诱导率为4%;添加 100 μmol/L二甲戊灵处理5 d,愈伤组织存活率为100%,四倍体的诱导率为6%.对根尖细胞染色体和叶片下表皮气孔数目和大小观察显示,诱变株具有四倍体的基本特征.说明二甲戊灵能在离体条件下有效诱导大蒜产生四倍体,可替代秋水仙素对大蒜染色体的加倍作用.     

硬粒小麦—簇毛麦双单倍体愈伤组织染色体加倍技术的研究

对硬粒小麦(Triticum durum)-簇毛麦(Haynaldia vtllosa)杂种幼胚和 F_1幼穗诱导的愈伤组织进行秋水仙素处埋染色体加倍试验,秋水仙素浓度分别为20、50、100和150mg/l;处理时间为8、10、12,14、16和19天;742块愈伤组织在20-27℃温度和每日10小时荧光灯照条件下进行处理。结果表明:不同处理的平均加倍率(结实株数/成活株数×100％)为78.8％,以秋水仙素浓度150mg/l 处理10天的加倍率最高为94.1％。平均处理效果(结实株数/处理愈伤组织数×100％)是15.8％。以秋水仙素浓度150mg/l 处理8天的处理效果最佳为34.1％。试验说明,利用生物技术——幼胚或幼穗培养,使远缘杂种后代快繁和染色体加倍同时进行是可行的。     

离体条件下用秋水仙素处理小麦-黑麦杂种细胞的研究

在液体培养基上用0.05%的秋水仙素处理小麦-黑麦杂种幼穗愈伤组织,含秋水仙素的培养基采用两种方法灭菌：（1）秋水仙素与培养基成分混合在一起用灭菌锅灭菌。处理愈伤后愈伤组织无伤害迹象,推测可能是灭菌过程中秋水仙素浓度降低所致;再生植株的育性得到有效的恢复,48.1%的穗子结实,5.2%的穗子结实率达到34.8%;（2）秋水仙素经超滤灭菌后,在常温下与灭过菌的培养基成分混和,处理愈伤后愈伤组织伤害严重,几乎不能分化苗。     

辣木组织培养与四倍体植株诱导

建立了印度辣木(Moringa oleifera)和非洲辣木(Moringa stenopetala)离体再生体系,并利用秋水仙碱进行了四倍体的诱导。结果表明:离体培养时以茎段为外植体可快速获得无菌苗,愈伤组织诱导与不定芽分化时,印度辣木以MS BA0.6mgL-1 NAA0.1mgL-1的固体培养基为最佳,非洲辣木以MS BA0.5mgL-1 NAA0.2mgL-1的固体培养基为最佳,生根培养基均以MS IBA0.2mgL-1最适。用不同浓度秋水仙碱对染色体进行加倍诱变,以0.2%秋水仙碱处理5d,加倍效果最好,印度辣木达到40.0%,非洲辣木为36.7%。获得了印度辣木和非洲辣木四倍体新种质,四倍体植株初步表现枝粗、叶片变大变厚且叶色更绿等特征。     

秋水仙素对草莓离体叶片再生和多倍体诱导的影响

以草莓(Fragaria×ananassa Duch.)栽培品种'雪蜜'(2n=8X=56)的离体叶片为外植体,研究了不同浓度秋水仙素对愈伤组织诱导率、不定芽再生率以及多倍体植株诱导的影响,并采用流式细胞仪对多倍体植株的倍性进行鉴定.结果显示,用质量体积分数0.1%、0.3%、0.5%和0.7%的秋水仙素浸泡2、4和6 d,草莓离体叶片均能诱导出愈伤组织和不定芽,但随秋水仙素浓度的提高和处理时间的延长,愈伤组织诱导率和不定芽再生率均显著下降.用不同浓度秋水仙素处理均能产生多倍体植株,倍性为9X、10X、11X、12X、14X和16X;随秋水仙素浓度的提高,多倍体诱导率呈现先上升后下降的变化趋势.用质量体积分数0.3%秋水仙素浸泡处理4 d是最佳的草莓离体叶片诱导方法,不定芽再生率达到40.5%,多倍体诱导率为100.0%,并且诱导产生出16X的植株.     

同源四倍体水稻原种制备及影响因素分析

本研究已将从全国各地征集的50份水稻优良种质资源(2n=2x=24)诱变为同源四倍体水稻原种。本讨论不同处理日期、不同秋水仙碱浓度和处理时间、以及不同品种的抗药性等因素对加倍成功率的影响,结果表明：①不同日期处理水稻幼芽,其加倍成功率不同,4月6日～15日加倍效果最好,加倍成功率达19．49／5。②不同浓度秋水仙碱和处理时间其加倍成功率存在一定的差别。0．1％秋水仙碱处理48h和0．2％处理24h为佳,其芽恢复率超过58％,加倍成功率超过16％。③不同水稻品种抗药性不同。在所加倍的50个水稻品种中,中作9025抗药性最强,芽恢复率达92．49%,加倍成功率38．79%。盐州14抗药性最差,芽恢复率仅为10．6％。     

南丹参离体快速繁殖与多倍体诱导

MS附加 6 BA 2 .0mg·L-1和IAA 0 .2mg·L-1的培养基上诱导南丹参丛生芽的频率最高 (达 10 0 % ) ,每一丛生芽数达 2 0～ 30个 ,移栽成活率 90 %。MS附加 6 BA 2 .0mg·L-1和 2 ,4 D 0 .2mg·L-1培养基上 ,叶缘及叶柄上长出绿芽或浅褐色愈伤组织 ,经多次继代仍具有分化植株能力。多倍体诱导以秋水仙碱 15mg·L-1处理愈伤组织和预培养 1周 ,处理时间 1周的叶片诱导效果较好 ,加倍率可达 33.33% ,且多数为整株加倍。加倍植株叶片较肥厚宽大、皱折增多、色深、根粗壮 ,气孔比二倍体明显增大 ,染色体数为 2 8～ 32条不等 ,其中可见部分为同源四倍体     

小麦组织培养再生系统及农杆菌介导的转基因技术研究

通过对晋麦47、鲁麦14、晋阳345、晋麦31等4个小麦品种的组织培养再生系统研究,结果表明,在MS基本培养基中加入Kao＇s有机化合物和T维生素后,小麦幼胚胚性愈伤组织诱导率提高31.4％～42.4％,植株再生能力提高50％～80％。对幼胚愈伤组织进行低温预处理和在农杆菌转化时添加AS处理后,经PPT筛选获得大量正常再生植物,PCR及Southern杂交检测证明其中28株再生植株呈阳性,这些植株明显提高了对basta的抗性。     

Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis

Different explant materials were treated with antimitotic agents to induce chromosome doubling in several Miscanthus sinensis clones. In vitro propagated plants established in soil, in vitro shoots, embryogenic callus, shoot apices and leaf explants were treated with different concentrations of colchicine or oryzalin. No tetraploids were obtained after antimitotic treatment of plants established in soil. The percentage of chromosome doubled plants after antimitotic treatment of single in vitro shoots was genotype dependent. Rooted in vitro plantlets were not a suitable target for antimitotic treatment, due to a high frequency of ploidy chimeras. Many tetraploid plants were regenerated after antimitotic treatment at the callus and explant level, but the efficiency was genotype dependent, primarily due to differences in the ability to form regenerable callus and to regenerate plants from embryogenic callus. Treatment of shoot apices with colchicine was the most efficient and reproducible system in the four genotypes tested. It was possible to repeatedly use the same colchicine-containing medium without any reduction in the induction of regenerable callus or in the percentage of tetraploids, thereby minimising the handling of this very toxic compound.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Establishment of Trisomic Series of Millet (Setaria italica L. Beauv.)

The young-ears of “Yugu No. 1”, a millet (Setaria italica (L.) Beauv. ) variety of good quality, high yielding and stress-resistance, were chosen to induce callus on N6 medium. After 3—5 times of subcultures, the callus was treated with colchicine (0.02%) for 48 hours and then transferred hack to subculture medium to restore growth, after which the callus was transferred to differentiation medium and subsequently, tetraploid plants were obtained. Through crossing, using tetraploid plants as female parent and diploid plant as male parent, 5 triploid plants were found among 2100 offspring plants. The triploid plants were fertilized with pollens from diploid plants. Among the plants developed from the seeds harvested on triploid plants, 9 kinds of trisomics (totally 283 plants) were identified according to chromosomal number and morphologic feature. Each kind of the trisomics has specific marker feature: (Figures in parentheses represent the number of plants obtained) Triplo-1 (52) has rolling leaves, Triplo-2 (18) is dark green, Triplo-3 (43) is bushy and has degenerated spikelets at the tip of panicles: Triplo-4 (58) has long bristle: Triplo-5 (16) has slender stalks: Triplo-6 (36) has twisted panicle neck; Triplo-7 (44) is semi-erect; Triplo-8 (8) has thick panicles; Triplo-9 (8) is pseudonormal (Similar with diploid of “Yugu No. 1”).     

盐度变化条件下芦苇对互花米草的化感效应

以芦苇凋落物为试材,采用溶液浸提法和厌氧腐解法,获得水提物和腐解产物两种浸提物母液;在5‰和10‰盐浓度下以两种浸提物母液和25%母液分别处理互花米草种子、刚萌发的幼苗以及互花米草滩涂中特有的互花米草益生菌,考察盐度变化对互花米草种子的萌发、幼苗生长以及菌落生长的影响.结果表明：盐处理能够显著促进互花米草种子的萌发、幼根以及互花米草益生菌的生长(P＜0.05).较高的盐浓度(10‰)下,芦苇水提物对互花米草的萌发、生长和互花米草益生菌的生长呈促进作用;当盐浓度降低至5‰时,该促进作用消失.在较低的盐浓度(5‰)下,芦苇腐解产物对互花米草萌发产生显著的抑制作用;在较高盐浓度(10‰)时,则对互花米草益生菌产生显著促进作用(P＜0.05);在试验的任一盐浓度下,芦苇腐解产物对互花米草幼苗和幼根的生长均存在显著的抑制效果(P＜0.05).     

Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis

Tetraploid plants are essential for interploid hybridization to create triploid seedless citrus. Here we report a simple and efficient in vitro method for generating autotetraploids for sweet orange (Citrus sinensis). Cell-division activity in ‘Anliucheng’ sweet orange callus was analyzed using flow cytometry to determine the peak frequency of cell division at which time callus in a liquid media and solid media was treated with 1000 mg l⁻¹ colchicine. The percentage of the DNA-content-varied cells in the callus increased markedly from 11.0% to 44.4% and to 59.0% for liquid and solid media respectively. A total of 20 tetraploid plantlets were recovered via embryogenesis from 47 plantlets regenerated from the treated callus. All the autotetraploids were derived from different embryoids. Autotetraploids will be useful parents for interploid hybridization to generate commercially valuable seedless triploid citrus cultivars.     

百日草根尖和花药愈伤组织染色体制片技术

以种子萌发根尖和花药愈伤组织为材料,研究了取样时间、预处理方法对百日草染色体制片的影响。结果表明:根尖上午8:00～9:00,花药愈伤继代3～5d上午9:00～10:00为最佳取样预处理时间;采用三种药剂预处理活体根尖,以4℃下饱和对二氯苯溶液或0.002mol/L的8-羟基喹啉液预处理8h效果最佳,花药愈伤则以饱和对二氯苯溶液预处理6h效果最佳。本实验的预处理温度是固定的,可克服预处理随季节和时间温度的变化而带来的不稳定性,且百日草花药愈伤染色体观察为首次报道。     

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (“Edison de Paula”, EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch’s (VS 50) and Guillard & Ryther’s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L⁻¹) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

In vitro induction of tetraploids in Arachis paraguariensis

In this study, an efficient procedure was established for successful induction of tetraploid Arachis paraguariensis by treating diploid explants with colchicine. Quartered-seed, callus and shoot-tips were treated with colchicine at concentrations of 0.05, 0.1, 0.2 and 0.5?% (w/v) for 4, 8, 16, 20 and 24?h before they were transferred unto modified Murashige and Skoog medium for either callus induction or shoot regeneration. Results showed that quartered-seed displayed the highest frequency of in vitro plantlet regeneration and tetraploid induction, as well as the lowest mortality rate. Flow cytometric analysis also confirmed that the induced tetraploids from quartered-seed were true-to-type. The 0.5?% colchicine treatment for 4 to 8?h gave the best results with 39 and 43?% of the explants yielding tetraploid plants, respectively. Two?months following transfer to ex vitro environment, morphological and growth characteristics of the induced tetraploids were measured. Overall, increasing the ploidy level from 2× to 4× resulted in fewer stomata but more trichomes per unit leaf area. Tetraploid plants obtained in this study should expand the genetic base of Arachis, and can also be used in overcoming the existing hybridization barriers that may be due to ploidy differences within the genus Arachis.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.