牡丹(Paeonia suffruticosa)胚胎培养研究进展

种胚是牡丹组织培养的重要外植体,胚胎培养技术是牡丹遗传转化及生物技术育种的重要手段之一。本研究从基因型、胚龄、解除休眠、培养基成分等方面分析了不同因子对牡丹胚胎培养的影响,总结了牡丹胚胎培养的研究进展。并根据存在的问题,对牡丹胚胎培养未来研究的重点方法、领域及发展的方向进行了探讨,旨在建立以牡丹胚胎为外植体的高效、稳定的再生体系,为牡丹转基因研究提供参考。

In Vitro Development of Tobacco Primary Endosperm Cells in Microculture

用酶解_研磨法分离出烟草 (NicotianatabacumL .)受精后胚囊和初生胚乳细胞进行微室饲养培养。培养基为Km8p附加各种其他成分 ,饲养细胞为分裂旺盛的烟草叶肉原生质体 ,在 2 5℃下静止暗培养。培养 3d后 ,初生胚乳细胞开始第一次分裂 ,继续分裂至 14d时形成大的细胞团。首次报道了双子叶植物初生胚乳细胞的离体发育。

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice, the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF). Compared with the feeder layer of MEF cells, medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyo-typic normality of ES cells only in short term cell propagation. Besides, ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras. Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more convenient and efficient than the conventional microdrop method.

In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepato- cytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxan- induced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/ progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/ progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.

Induction and Characterization of Embryogenic Callus in Cotyledons Leaves of Tabebuia roseo-alba

Seeds from Tabebuia roseo-alba lose viability very fast. Moreover, the seed germination rate is very low, reaching approximately 40%. This study aimed at the in vitro induction of embryogenic callus. This technology allows subsequent plant regeneration as an alternative for the production of T. roseo-alba seedlings. Seeds were germinated in vitro and after 20 days, cotyledonary leaves, hypocotyls and root segments excised from these seedlings were used as explants. They were inoculated on MS medium supplemented with sucrose （30 g/L）, agar （5.0 g/L） and different auxins. The effect of 2,4-D, picloram and NAA at concentrations 0.0, 0.5, 1.0, 2.0 and 4.0 mg/L was evaluated. For the analysis of callus with embryogenic characteristics, ultra-structural study by scanning electron microscopy and cytochemical test with carmine were performed. The results showed that the culture medium supplemented with 4 mg/L NAA presented induction of callus with embryogenic characteristics in all explants used, with cotyledonary leaves showing the highest percentage （70% of explants with embryogenic characteristics）. The use of 2, 4-D and picloram was efficient for callus formation in different explants, but no embryogenic characteristics were observed. From the ultra-structural analysis of callus with embryogenic characteristics, it was found that cells from different explant sources had isodiametric format. This format is similar to somatic embryos in globular stage. The cytochemical analysis confirmed the presence of pro-embryogenic cells in callus mass. Callus induced from cotyledonary leaves presented 46% positive reaction to carmine acetic.

Dicamba Growth Regulator Promotes Genotype Independent Somatic Embryogenesis from Immature Zygotic Embryos of Tropical Maize Inbred Lines

Maize is one of the most important cereal crops in Sub-Saharan Africa and an important source of energy for humans. However, the difference in the dedifferentiation frequency of immature embryos among various genotypes indicates that callus induction and genetic transformation is dependent on the genotype. This phenomenon is an impediment in the fundamental process of improving tropical maize germplasm especially through genetic engineering. Here, five tropical maize （Zea mays L.） genotypes, CML 216, CML 144, A 04, E 04 and TL 21, were evaluated for callus induction on MS medium supplemented with the growth regulator dicamba. Embryogenic and non embryogenic callus induction was independent ofgenotype when young immature embryos, 12 days after pollination （DAP） were used for tissue culture in combination with dicamba. The optimal concentration of dicamba for induction ofembryogenic callus in all the genotypes was 3 mg/L, which was also the concentration at which non embryogenic callus formation was lowest. The frequency of embryogenic callus induction ranged from 35% to 79% among the five genotypes and somatic embryos regenerated R0 shoots that produced normal R1 progenies. This regeneration method is expected to facilitate the development of a more efficient genotype independent Agrobacterium- mediated transformation system for tropical inbred lines.

Regulation Culture on Cytological, Biochemical and Physiological Characteristics of Somatic Carrot Embryos

Cytological,biochemical and physiological charcteristics of quiescent,high-vigor carrot somatic embryos obtained with the use of regulation culture were studied. As shown in cytological observation, quiescent somatic embryos obtained by regulation culturing for 70 days displayed small compacted cells with thick wall, dense cytoplasm and large amount of starch granules. Biochemical tests revealed a starch content and a soluble sugar content 1.9 and 1.7 times respectively as much as those of the control embryos(normal culture) while physiological study demonstrated that the respiration was maintained stally at a very low level,only 1/4 that of the control at the end of the 70-day period. These results provide further evidences that regution culture so after the morphology, biochemistcy, physiologr and metabolism of somatic embryos that they became quiescent,highly synchronized, and had their roots ungrown, a sate facilitating safe storage.

In Vitro Picloram-lnduced Somatic Embryogenesis from Leaflets of Cherry （Prunus incisa Thunb,）

Cherry regeneration via somatic embryogenesis is a powerful tool to breeding. In this way, the embryogenic capacity of Prunus incisa specie has been tested from leaves under different interactions of picloram concentrations and darkness exposures. Induction culture was achieved on MS medium supplemented with picloram concentrations at 0.5, 1 and 1.5 mg.L1 and submitted to 10, 20, 30 and 40 days of darkness. The best rate of embryogenic leaves was obtained with the interaction of 30 days darkness exposure＊ 1 mg.L^-1 picloram. According to their age, leaves were differently reacted to somatic embryogenesis; indeed, the 2nd expanded leaf from the apex was the most embryogenic one. Concerning the effect of additional auxin to picloram （1 mg·L^-1）, IAA at 0.1 mg·L^-1 and IBA at 0.1 mg·L^-1 gave significantly higher induction rates than all other concentrations, but regenerating somatic embryos showed some teratological abnormalities probably due to seconda;y embryogenesis. At the opposite, NAA at 0.5 mg·L^-1 didn＇t improve embryogenic rate but affected positively embryo development. Furthermore, embryogenesis preferentially took place on the basal part of leaf. Satisfactory rates of somatic embryogenesis are obtained but further improvement remains possible.

Direct Differentiation of Plants from Small Cell Clusters of Indica Rice in Suspension Culture

The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.

Exogeneous energy supply and excitability of cells in embryonic atypical epidermis of Cynops cultured in vitro

Cells of in vitro cultured epidermis explants of ectoderm isolated at early gastrula stage,showed only weak excitability or even non-excitable at 6V when examined electrophysiologically.If non-excitable explants were treated with 100 mM glucose,the action potential (AP) appeared and within 1 hr reached its maximum.At the same time,their stimulus threshold became lowered gradually.And,if the glucose was washed out,AP gradually disappeared.If explants were treated with glucose of different concentrations,the percentage of explants which displayed AP increased with the increase of glucose concentration.When explants with approximately the same original stimulus threshold were treated with glucose of different concentrations,the stimulus threshold became lowered more in the more concentrated solution.If explants with different original stimulus thresholds were treated with glucose of the same concentration,the lowering of stimulus threshold was more obvious in those with higher original stimulus threshold.Other energy supplying substances used showed similar effect.

Complete Cytogenetic Insight of Tho-Tho Cattle

To investigate the cytogenetic parameters and characterise the chromosomal banding pattern of Tho-Tho cattle, a breed of indigenous cattle found in the Northeastern states of India were reared for meat purpose. Lymphocyte culture technique was carried out in 28 Tho-Tho cattle and culture was harvested for good metaphase spread. Good metaphase spreads were selected for analysis, such as relative length, centromeric index and arm ratio. Centromeric banding （C-banding） and reverse banding （R-banding） methods were done for detail and better understanding of the chromosome morphology. The chromosome number in Tho-Tho cattle was observed to be 2n = 60 in all complete metaphase. The mean relative length of the autosomal chromosomes varied from 5.48% ：~ 0.107% to 1.79% ＋ 0.105% in male and 5.31% ：E 0.148% to 1.86% ＋ 0.055% in female, respectively. The chromosome banding showed C-positive dark band heterochromatin in all the acrocentric autosome. However, in sex chromosome, the Y-chromosome showed negative C-band and also the X-chromosome did not show any stain at the centromeric region. The numbers of R-band pattern were observed to be 490 and 499 band in male and female, respectively. One of the X-chromosome showed light banding pattern, confirming the inactivation during the embryonic development in female. The fundamental chromosome number and banding pattern of Tho-Tho cattle did not vary from the other breed of the Bos indicus. However, it is necessary to start a cytogenetic screening of the Tho-Tho cattle and expand upon more number to be kept at different villages of Nagaland in order to identify animals with chromosomal abnormalities, so that it can be excluded from future breeding strategies for conservation of Tho-Tho genetic resource.

In Vitro Regeneration of Commercial Sugarcane （Saccharum spp.） Cultivars in Nigeria

The effect of different concentrations of 6-benzylaminopurine （BA） with or without 0.2 mg/L NAA on in vitro regeneration of sugarcane （Saccharum spp.） cultivars SP726180, B47419, M1176/77 and M2119/88 were evaluated. Leaf base explants were cultured on Murashige and Skoog （MS） basal medium supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid （2,4-D） for 4 weeks. Thereafter, induction of somatic embryogenesis was observed following the transfer of resulting calli to 2,4-D-free medium for another 4 weeks. Regeneration was achieved by transfer of the embryogenic calli to regeneration media fortified with different concentrations of BA ＋ tt-naphthylacetic acid （NAA）. The number, length and vigor of shoots produced in all the genotypes were highest on media supplemented with 1.0 and 1.5 mg/L BA with and without 0.2 mg/L NAA. Among the genotypes tested, B47419 and M1176/77 recorded highest number of shoots, while maximum shoot length and crop vigor was obtained with M1176/77. Induction of callus with 3.0 mg/L 2,4-D and its subsequent incubation on 2,4-D-free media, followed by regeneration on media supplemented with 1.0 or 1.5 mg/L BA with 0.2 mg/L NAA was found to be efficient for in vitro regeneration of the sugarcane genotypes used in this study. This protocol could be applied for micropropagation of other elite genotypes.