桃(Prunus persica L.)茎尖培养技术的研究

筛选出了适合于桃品种‘北农早艳’茎尖(0.2 mm 和0.5 mm)培养的培养基为:G培养基附加 IBA 0.25 mg/L+BA 0.5 mg/L+GA_3 1.0 mg/L+LH 500.0 mg/L。茎尖用聚氨酯海绵作支撑物的液体培养比琼脂培养成活率高2～5倍,且生长迅速。获得了0.2 mm 长茎尖的生根植株。     

索拉亚百合茎尖组织培养研究

以索拉亚百合的茎尖为外植体，成功建立了快速无性繁殖体系。茎尖愈伤组织诱导培养基为：MS BA3mg／L NAA0．3mg／L；愈伤组织最佳芽分化诱导培养基为：MS BA1．5mg／L NAA0．5mg／L；芽继代增殖的最佳培养基为MS BA1．5mg／L IBA0．05mg／L。KT对芽的增殖效果不如BA，IBA的效果明显好于IAA。此外，激素的比例对不定芽的生长也有影响，最关键的因素是IBA的使用浓度，以0．05mg／L左右为宜。生根培养基为：MS IAA0．5mg／L NAA0．5mg／L AC1mg／L生根率达100％，平均生根数为5．88/苗。移栽成活率可达95％。     

花生胚小叶体细胞植株再生系统的建立

[目的]研究花生胚小叶体细胞植株再生体系,为利用胚小叶进行外源基因遗传转化提供试验依据。[方法]以花生品种四粒红和改良海花的胚小叶为外植体,2种外植体取材方式(预培养和直接取材)为培养背景,探讨了不同取材方式下各个培养要素(基因型、培养基等)与脱分化、再分化的关系,初步建立了花生胚小叶体细胞植株再生体系。[结果]在预培养取材条件下,预培养6d的四粒红外植体在2号培养基(MS+3mg/L6-BA+1mg/LNAA)上分化得最好,每愈伤组织再生芽2.34个,预培养5d的改良海花外植体在1号培养基(MS+5mg/L6-BA+3mg/LNAA)上分化最佳,每愈伤组织再生芽2.08个。直接取材条件下,改良海花在1号培养基上每愈伤组织出芽数为6个,而四粒红为3个。直接取材在诱导愈伤组织及器官分化方面都好于预培养取材。[结论]直接取材条件下,不同培养基对诱导愈伤组织及芽分化的作用差异较大,而基因型在诱导愈伤组织上的作用有显著差异,在出芽率上的作用差异并不显著。     

棉花耐盐胚性细胞系筛选及其植株再生

 继代1年以上的棉花品种珂字201下胚轴产生的胚性愈伤组织，转入加有不同浓度[0、8.56×10#+(-2)、1.71×10#+(-1)、2.57×10#+(-1)、3.42×10#+(-1)、5.13×10#+（-1）、6.84×10#+（-1）mol/L]NaCl的筛选培养基上培养，经过3代筛选，获得耐盐胚性细胞系和再生植株。NaCl显著抑制了愈伤组织的存活和生长，NaCl半致死浓度在8.56×10#+（-2）～1.71×10#+（-1）mol/L之间，致死浓度为6.84×10#+（-1）mol/L。NaCl浓度影响着体细胞胚的发生和发育，影响着体细胞胚的萌发和植株再生。添加1.71×10#+（-1）mol/L NaCl培养基，筛选出的愈伤组织生长较好，并能分化出体细胞胚和正常再生植株。在本实验中，笔者获得了耐3.42×10#+（-1）mol/L NaCl的体细胞胚和耐1.71×10#+（-1）mol/L NaCl的正常再生植株。     

影响草莓茎尖快繁因素的分析

[目的]分析几种影响草莓茎尖快繁的因素。[方法]以"童子1号"草莓为试材,对草莓茎尖培养快繁中茎尖取材大小、生长调节剂、碳源等影响因素进行分析。[结果]以0.5 mm大小的茎尖接种,脱毒率最高,成活率最理想。诱导"童子1号"草莓茎尖成活与生长的最适培养基、最优增殖培养基均为MS+BA 0.5 mg/L。IBA浓度为800 mg/L时,组培苗瓶外生根不仅有较高的生根率和生根数,而且获得了较壮的生根苗。蔗糖和白砂糖作碳源时,繁殖系数较理想,以白砂糖最为适宜。[结论]草莓茎尖快繁应以0.5 mm大小的茎尖为材料,诱导分化培养基、继代增殖培养基均为MS+BA 0.5 mg/L,组培苗瓶外生根蘸取的最佳生长调节剂为800 mg/L IBA。     

甘薯茎尖脱毒与快速繁殖技术研究

甘薯茎尖分生组织脱毒培养与茎段快速繁殖,对培养条件和几种主要培养因子的反应十分敏感,以MS为基本培养基对甘薯茎尖分生组织与茎段进行不同激素种类种类和浓度试验,结果表明,IAA：6－BA为1：5－20诱导效果好,最佳诱导分化培养基为：MS＋6－BA1mg/L IAA0.1-0.2mg/L GA3 0.1mg/L;试管苗株系经指示植物,NCM－ELISA法检测,获得了6个品种的脱毒苗,脱毒苗茎段试快速繁殖时,不同品种之间有差异,单独使用IAA或与GA3结合使用,在28℃,光照12h/d,液体培养效果好。     

Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary marine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS ,0.1μmol/L Na2SeO3, 0. 1mmol/L β-mercaptoethanol, 1 000ng/ml LIF,10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine), then, we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage marine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0. 125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification, karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.     

支持牛类胚胎干细胞发育的饲养层培养体系的建立

以小鼠胎儿和牛睾丸为材料 ,以含 1 5 % NBS、0 .1 mmol/ Lβ-巯基乙醇、0 .1 μmol/ L Na2 Se O3的DMEM溶液为培养液 ,分离获得了传 1 5代的小鼠胎儿成纤维细胞和 5代牛睾丸成纤维细胞 ,建立了小鼠和牛类 ES细胞培养体系。结果表明 :牛睾丸成纤维细胞和小鼠胎儿成纤维细胞均属附着生长型细胞 ,与小鼠胎儿成纤维细胞相比较 ,牛成纤维细胞直径和长度大 ,生长速度快 ,易于老化 ;1 2～ 1 6日龄的小鼠胎儿最适宜分离与克隆小鼠胎儿成纤维细胞 ;在 2 5℃条件下 ,以 0 .2 5 %胰蛋白酶 0 .0 4% EDTA消化液作用胎儿小块组织分离原代小鼠胎儿成纤维细胞 ,消化液作用时间不应超过 2 0 min,以相同的消化液在 3 7℃条件下 ,离散贴壁成纤维细胞 ,作用时间以 2～ 3 min为宜 ;培养细胞密度与传代时间间隔有密切关系 ,若传代时间间隔为 3～ 4d,培养细胞浓度应为 3× 1 0 5个 / ml～ 5× 1 0 5个 / ml;在成纤维细胞分离与克隆过程中 ,培养基中添加0 .1μmol/ L Na2 Se O3 0 .1 mmol/ Lβ-巯基乙醇 1 5 % NBS,有利于 MEF和 NBTF的增殖     

Metanephric cell microenvironment induces embryonic stem cells to differentiate toward renal cells

AIM: To explore the effects of metanephric cell microenvironment on inducing embryonic stem cells (ESCs) to differentiate toward renal cells.METHODS: Embryoid bodies (EBs) of D3 mouse embryonic stem cells were prepared by hanging drop culture, and the EBs were co-cultured indirectly with metanephric cells derived from E12.5 d mouse embryo. The EBs cell with spontaneous differentiation was used as the control. The proteins of Pax2 and WT-1 were analyzed by immunofluorescence assay. The mRNA expression of Pax2, WT-1, Lim1, Sall1, Emx2, GDNF, Wnt4, BMP7, Nephl, Nephrin, KSP and CD24 genes was detected by RT- PCR.RESULTS: The genes related to kidney development were expressed in the EBs cells after co-culture on day 3, and the mRNA expression of Pax2, WT-1, Emx2, GDNF, Nephl, Nephrin, KSP and CD24 was stronger than those in control group. Pax2 positive cells were found on day 3 in the co-cultured EBs cells, and the positive cells increased on day 5 and day 7. WT-1 protein positive cells were found in the co-cultured EBs cells on day 5. No Pax2 or WT-1 positive cell was observed in control group.CONCLUSION: Metanephric cell microenvironment promotes ESCs differentiation toward renal cells.     

Supportive effects of human AGM region and fetal liver stromal cells on differentiation of murine embryonic stem cells into hematopoietic stem cells and in vivo hematopoietic reconstitution

AIM: To investigate the differentiation of murine embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) by the supportive effects of human aorta-gonad-mesonephros (AGM) region and fetal liver (FL) stromal cells.METHODS: E14 ESCs were induced into embryoid body (EB) first. Then the cells from EB were further co-cultured with human AGM region and FL stromal cells in non-contact system. On day 6, the cells derived from EB were collected for Sca-1⁺c-Kit⁺ cell analysis by flow cytometry, colony forming unit (CFU) assay and teratoma formation checking. BALB/c female mice conditioned with lethal dose of γ-ray irradiation were transplanted with EB cells from different culture systems. The survival rates, engraftment of donor cells, reconstitution of hematopoietic were monitored.RESULTS: Sca-1⁺c-Kit⁺ cells in EB cells co-cultured with human AGM region and FL stromal cells had the value of (21.96±2.54)%, and the total CFU was as (520±52)/10⁵ cells, which were statistically greater than those in EB cells only cultured with human AGM region stromal cells (P＜0.05). No teratoma was found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region and FL stromal cells. In BALB/c female mice transplanted of EB cells co-cultured with human AGM region and FL stromal cells, the survival rate was 77.8%, and the peripheral blood cell count was obviously improved on day 14. PCR results showed the recipients all had sry gene copies from donor in bone marrow. The recipient mice transplanted with EB cells only cultured with human AGM region stromal cells all died within 15 days.CONCLUSION: Stromal cells from human AGM region and FL enhance the directed differentiation of ESCs into HSCs which can reconstruct hematopoiesis in vivo.