提高木薯循环培养的次生体胚再生植株频率研究

用木薯成熟体胚子叶作外植体诱导次生体胚,接种7d或15d后分别在诱导培养基或成熟培养基中加入AgNO3,可明显提高植株再生频率。前者可使体胚再生植株频率由对照的40．5％提高到58．1％;后者可使植株再生频率由对照的36．1％提高到61．3％。用ABA进行上述处理,效果更加显著。诱导期处理,可使植株再生频率由对照的40．5％提高到72．6％;成熟期处理,植株再生频率由对照的36．1％提高到81．3％。若诱导7d后,将2,4－D浓度由4．0mgL-1降至2．0mgL-1,并加AgNO3,继续诱导15d,转到附加0．25mgL-1ABA的成熟培养基中培养至30d,能极显著地促进体胚的发育和成熟,使植株再生频率达到一个相当高的水平,最高达95％,平均每个外植体产生的植株数达39．6个,分别相当于对照的2．38倍和2．46倍。     

黄瓜成熟胚离体培养中的胚状体诱导和植株再生（简报）

以10个黄瓜品种为材料,取成熟胚外植体先经MS 2,4—D 1.0mg/L(单位下同) KT0.5诱导后转入MS IAA 0.1 KT0.5上分化,胚状体发生频率高并有植株再生。材料的基因型差异显著,“农大春光”分化率最高(40％)。双层培养有效地促进畸形胚状体的正常发育,5％蔗糖和1.0mg/L ABA促进胚性愈伤组织的继代增殖和胚性保持。     

几种影响木薯芽器官发生及植株再生的因素

基本培养基中,ＭＳ在诱导木薯芽器官发生及植株再生方面表现最好,ＧＤ表现最差;麦芽糖和蔗糖在诱导芽器官发生及植株再生方面优于其它碳源;乙烯发生抑制剂ＡｇＮＯ３有促进芽器官发生的作用,但长时间培养在含高浓度ＡｇＮＯ３培养基上的组织生长则发生钝化。     

麻栎成熟合子胚外植体体胚发生和植株再生

为探索麻栎快速繁殖技术新途径,以成熟合子胚为外植体诱导体胚发生,进一步培养形成幼苗。结果表明:体胚诱导以MS+1.0mg.L-1 6-BA+1.0mg.L-1 IBA+1.0g.L-1谷氨酰胺+0.5g.L-1脯氨酸为最优,培养30d诱导率达70.0%;体胚成熟以1/2MS+2.0mg.L-1 6-BA+0.5mg.L-1 IBA+2.0mg.L-1 ABA+4.0g.L-1谷氨酰胺+2.0g.L-1脯氨酸为佳,培养60d,体胚完全成熟;体胚萌发最适培养基为1/2MS+0.2mg.L-1 6-BA+10g.L-1山梨醇,且冷处理有利于体胚的萌发,萌发率高达100%,萌发培养80d,形成再生植株。     

胡杨器官和体胚发生方式的植株再生

目的：为以胡杨为亲本的体细胞杂交育种奠定基础。方法：以胡杨苗叶片为外植体,通过器官和体胚两种不同发生方式建立了离体再生体系。结果：附加0．75mg/L BA、0．5mg/L NAA基本培养基及3w暗培养是愈伤组织诱导的最佳条件;附加0．25mg/L BA和0．1mg/L NAA的基本培养基上不定芽的诱导率最高;1／2大量元素的MS培养基附加0．1mg/l NAA、0．05mg／L和1．5％蔗糖对不定芽生根效果最好;诱导并筛选出的胚性愈伤组织在附加了0．5mg/L BA、0．5mg／L NAA的基本培养基上诱导获得大量胚状体,干化处理后大部分能经子叶胚期萌发成苗。结论：外植体的采集周期和培养条件影响胡杨离体叶片的形态发生途径。     

木薯体细胞胚胎发生及植株再生研究

对木薯体细胞胚胎发生的影响因素进行了优化研究。结果表明,基因型对木薯体细胞胚胎发生影响很大,在供试的六个品种中,“华南 8 号”的体细胞胚胎发生率和产胚量最高,分别为 65% 和19个;侧芽茎尖为最佳外植体,体细胞胚胎发生的最佳培养基为MS +0.5mg/L CuSO4 + 4 mg/L 2,4-D。同时,对木薯体细胞胚再生成完整植株的主要影响因素作了分析,建立了一个高效的植株再生体系。     

温度对枳椇次生胚发生和植株再生的影响

在含有2,4-D的培养基上所诱导和增殖的枳棋体细胞胚只能形成无再生能力的畸形胚。通过次生胚发生途径可以在不加植物生长调节物质的培养基诱导出发育正常的体细胞胚,子叶期胚的次生胚诱导能力高于早期体胚。相对高的温度(30℃)有利于次生胚的诱导,相对低的温度(20℃)更有利于次生胚的发育、植株再生和移栽成活。     

影响体胚发生途径香蕉(Musa spp.,AAB Group)植株再生的因素

香蕉品种‘Agbagaba'和‘Orishele'的胚性细胞悬浮系(ECS)在液体培养基中分别预培养1和2周后,将其接种在RD1和M3培养基上,于光照或黑暗条件下进行体胚的再生.从沉积细胞体积(SCV)为1 mL(1 mL SCV)的ECS获得的再生体胚数量因预培养时间、再生培养基的种类及培养条件的不同而异.植株的再生率及从1 mL SCV的ECS获得的再生植株数量也受上述体胚再生条件的间接影响.     

Effect of medium salt concentration on differentiation and maturation of somatic embryos of cassava (Manihot esculenta Crantz)

Culture of cassava somatic embryos on media with an altered macro- and micro-nutrient salt concentration affected embryo development and germination capability. In the tests, quarter-, half-, full- or double-strength Murashige and Skoog (MS) media were compared. The maximum number of somatic embryos differentiated from a proliferative nodular embryogenic callus (NEC) on either half- or full-strength MS medium, and the greatest numbers of cotyledonary stage embryos were formed on full-strength MS medium. Developed somatic embryos were then desiccated above a saturated K2SO4 solution for 10 d. After transfer to germination medium, embryos that had developed on half- and full-strength MS medium yielded 8.3 and 8.6 germinants g(-1) NEC tissue, respectively. For this important but often disregarded culture factor, either half- or full-strength MS medium is recommended for both the differentiation and development of cassava somatic embryos that are capable of germination.     

Microsatellites in Cassava (Manihot esculenta Crantz): discovery, inheritance and variability

 Fourteen microsatellites containing GA-repeats were isolated and characterized in cassava (Manihot esculenta Crantz, Euphorbiaceae). Microsatellite heterozygosity (h) was estimated in 48 accessions using (³²P)-end-labeled primers and in more than 500 accessions using fluorescence-based genotyping. Heterozygosity values ranged from 0.00 to 0.88 and the number of alleles detected varied from 1 to 15. The reproducibility of allele sizing was also assessed using fluorescence-based genotyping. The average inter-gel size difference was 1.03 nucleotides. Chi-square tests (χ²) were performed to analyse segregation distortion and the linkage between alleles segregating from either or both parents in an F₁ mapping population. Most microsatellite loci segregated in the expected 1 : 1, 1 : 2 : 1 or 1 : 1 : 1 : 1 ratio. Linkage was detected between loci segregating from either parent, and segregation distortion from the male parent was detected for locus GA-131. Approximately 80% of the microsatellites detected one or two alleles per accession, suggesting a low degree of microsatellite locus duplication, an unexpected finding for a putative allopolyploid, highly heterozygous species. The high h values of most microsatellites, their amplification in other Manihot taxa and their suitability for high-throughput, fluorescence-based genotyping, make microsatellites the marker of choice for germplasm characterization and saturation of the cassava map. Received: 4 September 1997 / Accepted 16 March 1998     

Isozyme Diversity in Cassava Cultivars (Manihot esculenta Crantz)

Isoenzyme electrophoresis was used as a method to determine genetic diversity in various M. esculenta cultivars collected in the Southwestern (SW) and Northwestern (NW) regions of the State of Parana, in the South region of Brazil, and in cultivars produced at the Agronomic Institute of Campinas (IAC), S~ao Paulo State, Southeastern region of Brazil. The cultivars have been maintained by vegetative propagation for 5 years and are useful in production programs. A total of 28 loci in the acid phosphatase (ACP; EC 3.1.3.2), esterases (EST; EC 3.1.1.1), malate dehydrogenase (MDH; EC 1.1.1.37), and shikimate dehydrogenase (SKDH; EC 1.1.1.15) isozymes was analyzed. The proportion of polymorphic loci for NW, SW, and IAC cultivars was 57.14, 50.0, and 53.6%, respectively. Genetic diversity calculated by Nei's genetic identity (I) showed high I values for the three M. esculenta subpopulations. The high degree of polymorphism expressed by cassava cultivars is highly relevant to stimulate breeding programs with M. esculenta species.     

Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid     

A molecular genetic map of cassava (Manihot esculenta Crantz)

 A genetic linkage map of cassava has been constructed with 132 RFLPs, 30 RAPDs, 3 microsatellites, and 3 isoenzyme markers segregating from the heterozygous female parent of an intraspecific cross. The F₁ cross was made between ‘TMS 30572’ and ‘CM 2177-2’, elite cassava cultivars from Nigeria and Colombia, respectively. The map consists of 20 linkage groups spanning 931.6 cM or an estimated 60% of the cassava genome. Average marker density is 1 per 7.9 cM. Since the mapping population is an F₁ cross between heterozygous parents, with unique alleles segregating from either parent, a second map was constructed from the segregation of 107 RFLPs, 50 RAPDs, 1 microsatellite, and 1 isoenzyme marker from the male parent. Comparison of intervals in the male-and female-derived maps, bounded by markers heterozygous in both parents, revealed significantly less meiotic recombination in the gametes of the female than in the male parent. Six pairs of duplicated loci were detected by low-copy genomic and cDNA sequences used as probes. Efforts are underway to saturate the cassava map with additional markers, to join the male- and female-derived maps, and to elucidate genome organization in cassava. Received: 5 July 1996/Accepted: 22 November 1996     

影响根癌农杆菌介导的木薯遗传转化因素分析

采用根癌农杆菌介导的叶盘转化法,以我国南方地区主栽木薯品种—华南8号的胚状体子叶为受体,对影响木薯遗传转化效率的主要因素进行了分析。研究结果表明,在木薯的遗传转化中,选用GV3101作为浸染外植体的农杆菌菌株,将感染时间和共培养时间分别控制在30~45 min和3~4 d、菌液浓度（OD₆₀₀）采用0.45、并添加200 μmol·L^-1的乙酰丁香酮（AS）均可明显提高其转化效率,但若对外植体进行预培养反而会降低其转化效率。利用该体系从453块外植体中共转化获得10株抗性再生植株,经PCR和Southern杂交检测,有8株木薯的基因组中已整合进了外源基因glgC336,转化率为1.77%。     

Adventitious shoot regeneration from leaf explants of an African clone of cassava (Manihot esculenta Crantz) and analysis of the conformity of regenerated plants

A protocol for regeneration of adventitious shoots from immature leaf lobes from in vitro plants of cassava has been developed. Induction occurred in the presence of 2,4-D for ten days which was followed by transfer onto the regeneration medium containing 23 μM zeatin. Induction for more than 10 days in 2,4-D resulted in reduced regeneration and delayed shoot formation. A friable callus developed after a longer induction phase. A shorter induction resulted in the formation of foliar structures which did not develop further. Following subculture calluses produced shoots with improved frequency: 1.0 shoot/explant after 4 subcultures and 1.8 shoots/callus after 14 subcultures. When transplanted into soil, the regenerated plants were apparently normal and similar to the stock plants. A sample of 203 regenerants was examined for isozymes and DNA content using flow cytometry. Glutamate oxaloacetate transaminase (GOT) banding pattern was identical to that of the stock plants. Small changes were however detected in the phosphoglucomutase (PGM) pattern for 3.4% of regenerated plants, showing an additional band in the fast migration zone. The DNA content of the regenerated plants was homogeneous and was similar to that of the stock plants. The ploidy level was unchanged (2n = 36 chromosomes). This revised version was published online in June 2006 with corrections to the Cover Date.     

Antholysis in Cassava (Manihot esculenta Crantz) Possibly Caused by Mycoplasma-Like Organisms

Phyllody and apostasis of cassava plants were frequently observed during recent disease surveys in the Cauca Valley of Colombia. Many valuable cassava clones have been affected rendering them unsuitable for hybridization. Light and electron microscopic observations have revealed the presence of mycoplasma-like organisms in the diseased phloem tissues. The causal agent is sensitive to tetracycline and streptomycin at 1000 ppm a. i., but not to penicillin.