Protoplast Fusion in the Genus Medicago and Isoenzyme Analysis of Parental and Somatic Hybrid Cell Lines

Leaf mesophyll protoplasts of Medicago arborea (2n = 32) were electrofused with cell suspension and/or callus protoplasts of Medicago sativa. Heterokaryons were identified in agarose beds by their dual fluorescein isothiocianate—chlorophyll fluorescence and their coordinates were recorded. Hybrid minicalli were manually picked up and grown first in nurse culture and then in callus induction medium. Hybrid nature of the selected calli was confirmed by isoenzyme analyses. In order to verify whether morphogenesis of somatic hybrid calli was affected by cell incompatibility, mesophyll and cell suspension protoplasts, derived from the same plant of M. sativa with high embryogenic capacity, were fused. Only callus tissues derived from mesophyll protoplasts retained the highly embryogenic character of the M. sativa genotype, while hybrid cell lines were non-morphogenic and showed isoenzyme patterns similar to tissues derived from cell suspension protoplasts. The achievement of somatic hybrid plants in the genus Medicago is discussed.     

二倍体马铃薯体细胞电融合的研究

对3种双单倍体马铃薯叶肉原生质体进行自体和异体融合及对二倍体野生种S.phureja实生苗子叶下胚轴原生质体自体融合的实验结果表明:不同基因型马铃薯叶肉原生质体在交变电场中的转动电压、成串电压及细胞拉长电压无明显差异;在1 800 v/ cm的直流脉冲电压、100μs脉冲幅度下,2～3细胞的融合频率最高(24.16 %);实生苗子叶下胚轴原生质体在2 000 v/ cm、100μs的电场作用下融合频率明显高于叶肉组织,其融合频率为43.50 %;实验还显示出,马铃薯叶肉原生质体自体融合频率明显高于异体融合频率。     

龙眼荔枝属间原生质体电融合

由龙眼的幼胚培养诱导、筛选出松散型胚性愈伤组织 ,由之建立了胚性悬浮细胞系 ;用同样的方法建立了荔枝的松散型胚性愈伤组织系 .龙眼的胚性悬浮细胞和荔枝的胚性愈伤组织在含纤维素酶和果胶酶各 10 .0g· L-1的 CPW- 13M的酶解液中酶解 ,经纯化后可得到较为纯净的原生质体 .采用电融合方法进行两种原生质体融合处理 ,获得了适宜的融合参数 ,融合率达 2 0 %以上 .融合产物经初步培养 ,形成了多细胞团     

细胞电融合构建高效蒽降解重组菌株的研究

通过An815与带EGFP标记的铜绿假单胞菌(Pseudomonas aeruginosa/EGFP)原生质体电融合,得到一株融合子R2,该融合子不仅具有An815菌高效降解蒽的能力,而且具有铜绿假单胞菌产生生物表面活性剂的能力,在72 h内将蒽从100 mg.L-1降解至27.9 mg.L-1,降解能力比出发菌An815提高了22%。     

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180–240 mJ cm⁻². Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.     

草地早熟禾种间原生质体电融合条件的研究

为改良培育草地早熟禾(Poa pratensis)新品种,拟建立草地早熟禾商用品种与具有优良性状的野生早地早熟禾种间原生质体融合体系。采用电融合法,研究了电融合仪的电场条件和原生质体密度对草地早熟禾种间原生质体融合的影响。结果表明:在交流电场强度和频率分别为20 V/cm和2000k Hz时,原生质体可在较短的时间内形成2~4个原生质体串;最适于草地早熟禾种间原生质体融合的直流脉冲强度为200 V/cm、DC幅宽为40μs、直流脉冲次数为3次,原生质体密度为3×105个/m L,在此条件下,草地早熟禾种间原生质体总融合率和一对一融合率可分别达到25.39%和12.27%。     

不同电融合参数及融合液对昆明小鼠2-细胞胚胎融合及囊胚率的影响

四倍体胚胎在研究哺乳动物配子发生、发育、四倍体的发生、遗传、免疫机制等方面具有重要作用,因此提高融合及发育效率至关重要。为找出适合小鼠2-细胞胚胎融合的最佳方案,试验采用两种融合液0.3mol/L甘露醇溶液和0.3mol/L蔗糖溶液,两种融合液均采用了120V/mm,20μs×2、100V/mm,40μs×2、80V/mm,80μs×2三种电融合参数。其中采用0.3mol/L甘露醇溶液作为融合液且融合参数采用80V/mm,80μs×2获得了92.5%的融合率和90.6%的囊胚率,是小鼠2-细胞胚胎融合的最佳方案。     

降解蒽遗传重组菌在蒽污染环境中的降解和存活能力研究

为了提高外源蒽降解菌An降解蒽的能力和存活能力，采用原生质体电融合技术，在蒽降解菌An和土生优势菌Tu之间实行了原生质体电融合，并从再生的融合子F中筛选到具有特定性状的后代菌株。把融合子接种于蒽污染的土壤与水环境中，研究其降解蒽的能力与存活能力。结果表明，在蒽污染的土壤中，40d时，融合子F与亲本An对蒽的降解率分别为70．7％、55．5％，融合子比出发菌株高15．2％；100h内融合子F和亲本An对水环境中蒽的降解转化率分别为75％、67．2％。说明在蒽污染的土壤和水环境中．在初始接种量相同的情况下，融合子的存活率明显高于出发菌株。     

小鼠电融合胚胎发育影响因素的研究

电融合技术，是获得四倍体胚胎的主要手段．本试验研究了各种不同因素，对小鼠电融合胚发育的影响．结果表明，电刺激前后，使用不同的操作液对融合胚发育有影响．电刺激前使用Ｍ２液、电刺激后在Ｗｈｉｔｔｅｎ氏液中融合，然后转入ＣＺＢ液中培养，这一操作体系（Ｍ／Ｗ—ＣＺＢ）效果最好，融合胚的囊胚发育率为８０．５％，显著高于Ｍ／Ｗ—Ｗ，Ｗ／Ｗ—Ｗ，Ｗ／Ｗ—ＣＺＢ组，后者的囊胚发育率分别为０，０，５４．５％（Ｐ＜０．０５）．试验还发现，刺激强度为１．０ｋｖ／ｃｍ，８０μｓ；１．０ｋｖ／ｃｍ．４０μｓ和０．８ｋｙ／ｃｍ，８０μｓ时，融合囊胚的发育率分别为７９．０，８１．９和７１．１％．ＨＣＧ注射后４７ｈ、４８ｈ的２—细胞胚胎的融合胚发育率，显著高于（Ｐ＜０．０１）ＨＣＧ注射后４２ｈ、４４ｈ和４６ｈ的，囊胚发育率分别为８２．９，８０．６，０，０和２２．８％．