抗生素对山茶愈伤组织诱导和生长的影响

研究了卡那霉素、潮霉素对浙江红山茶、大花红花油茶愈伤组织的生长及对攸县油茶幼苗叶片诱导愈伤的影响,确定作为选择压力的卡那霉素质量浓度为50～60mg·L-1,潮霉素为25～30mg·L-1。在3种抑菌性抗生素中,羧苄青霉素促进愈伤组织诱导,对愈伤组织的生长没有影响,头孢霉素则有抑制作用,而羟氨苄青霉素轻微抑制愈伤组织的生长,但对愈伤组织的诱导影响不大。考虑到抑菌效果,适于在转基因研究中使用的羧苄青霉素的抑菌质量浓度为250mg·L-1,羟氨苄青霉素为125mg·L-1。     

青钱柳组织培养研究

以青钱柳幼嫩叶片为外植体进行组织培养,观察青钱柳愈伤组织形成及再分化形成不定芽的过程,研究不同培养基和不同生长调节物质组合对青钱柳愈伤组织诱导及植株再生的影响。结果表明:(1)MS、WPM和B5培养基均能诱导出愈伤组织,激素及其浓度对愈伤组织诱导有显著影响。愈伤组织在WPM+2.0 mg/L2,4-D+1.0 mg/L 6-BA培养基组合上诱导率最高,达97.6%;(2)青钱柳愈伤组织能分化形成不定芽,激素不同浓度组合及外源物对愈伤组织分化有显著影响。青钱柳叶片愈伤组织在WPM+1.0 mg/L 6-BA+2.0 mg/LZT+0.5 mg/L2,4-D+10%椰乳的培养基上分化不定芽的频率最高,达66.33%;(3)青钱柳诱导生根的最佳培养基组合为1/2 MS+1.5 mg/L IBA。     

木槿组织培养及无性系建立的研究

以开花早、花期长的木槿嫩茎为材料,进行了愈伤组织诱导、愈伤组织和不定芽分化与继代,试管苗的生根、移栽和移植研究。结果表明:MS+BA0.3 mg/L+2,4-D 0.6 mg/L是诱导嫩茎形成具有分化能力愈伤组织的理想培养基;MS+AgNO3 1.2 mg/L+BA 0.6mg/L+NAA 0.1 mg/L是诱导愈伤组织和不定芽分化的理想培养基;1/2MS+IAA0.2 mg/L+NAA0.1～0.2 mg/L是试管苗生根培养的理想培养基;河沙和炉灰渣是试管苗移栽的理想基质。移植成活的试管苗生长整齐、长势旺盛,保持了开花早、花期长的特点。     

楸叶泡桐杂种无性系9503的组织培养技术研究

以楸叶泡桐和白花泡桐杂交无性系的枝条为外植体,开展了离体芽增殖技术,以及愈伤组织诱导、芽分化和不定芽生根等组织培养技术的研究。结果表明,增殖培养基采用MS+8mg/L6-BA+0.3mg/LNAA,不定芽的增殖倍数可达到9;利用其叶片进行愈伤组织诱导的最佳培养基为MS+12mg/L6-BA+0.4mg/LNAA,诱导率高达95%;愈伤组织分化的最佳培养基为MS+10mg/L6-BA+0.7mg/LNAA,不定芽生根的最佳培养基为1/2MS+0.5mg/LIBA+0.3mg/LNAA,生根率100%。     

巨桉无性系EG5叶片高效再生体系的建立

以巨桉栽培无性系EG5无菌苗的叶片为外植体进行愈伤组织诱导与植株再生研究。结果表明:愈伤组织高效诱导和不定芽分化的最适培养基为改良MS+0.12 mg·L-1TDZ+0.25 mg·L-1NAA;硝酸铵对EG5叶片愈伤组织生长及植株分化的影响较大,最适宜质量浓度为0.412 5 g·L-1;最佳伸长培养基配方为改良MS+0.3 mg·L-16BA+0.05 mg·L-1IBA+0.3 mg·L-1IAA;暗培养10 d能促进不定芽分化,延缓愈伤组织老化和褐变速度。生根培养基为改良MS+0.4 mg·L-1NAA时生根率最高,为65.47%,移植成活率为90%以上。     

透骨草组织培养的研究

以透骨草的嫩茎为材料,进行了愈伤组织诱导、愈伤组织分化、不定芽生根、试管苗的移栽、试管苗扦插、试管苗移植的研究,成功的建立起透骨草的嫩茎无性系。结果证明：1/2MS＋BA0.5mg/L＋2,4-D1.2 mg/L＋NAA0.2-0.8 mg/L为嫩茎愈伤组织的的理想培养基;1/2MS＋AgNO31.2 mg/L＋BA0.6 mg/L＋NAA0.1 mg/L为愈伤组织和不定芽分化的理想培养基;1/3MS＋IAA0.4 mg/L为不定芽生根培养和生根继代培养的理想培养基;炉灰渣是试管苗移栽和扦插的理想基质。移植试管苗后期出现生长旺盛、长势整齐的性状,翌年春季出现萌发早5d、根系相当于野生植株2倍的性状。     

紫穗槐茎段愈伤组织诱导及再生体系的建立

以紫穗槐茎段为研究对象,确定了影响其愈伤组织诱导的关键因子、不定芽分化及生根培养的最佳条件,建立了紫穗槐稳定高频再生体系。试验结果表明：紫穗槐茎段愈伤组织的最适诱导培养基为MS＋6-BA4.0mg/L＋NAA2.0mg/L＋2,4-D0.5mg/L,诱导率为100%,经愈伤组织分化不定芽的最佳培养基为MS＋6-BA0.5mg/L＋NAA0.1mg/L＋KT2mg/L,分化率为96.2%,增殖倍数为7.3;不定芽最佳生根培养基为1/2MS＋酵母提取物0.5mg/L;再生植株移植在选用田园土、蛭石（5∶1）混合的基质上,光强3000lx,光照时间14h/d下生长,3周后成活率达85.67%。     

湿地松不定芽分化生根与解剖观察

以湿地松器官性愈伤组织为材料,开展不定芽分化、生根与组织解剖学的研究.试验结果表明:不定芽分化最佳培养基为TX+BA 0.5 mg·L-1+IBA 0.5 nag·L-1+30 g·L-1蔗糖;不定芽同步发育最佳培养基为TX+IBA0.5 mg·L-1+30 g·L-1蔗糖;生根培养,初期以无大量元素培养基诱导生根、25天后添加大量元素的培养程序可有效缩短生根培养时间,提高生根率;亮白色、半紧实结构的外部形态及"拟分生组织"的内部结构是器官性愈伤组织的重要标志.     

黑荆树不同外植体愈伤组织诱导与芽分化的研究

为建立高效的黑荆树快速繁殖体系,以黑荆树叶片、茎段和下胚轴为外植体,在添加不同植物生长调节剂的MS培养基上进行了愈伤组织的诱导和不定芽的分化研究。结果表明:综合最高诱导率结果和愈伤组织的生长状况,外植体的愈伤组织诱导能力顺序为叶片(44.83%)下胚轴(36.35%)茎段(30.05%);愈伤组织诱导最佳培养基为MS+1.5 mg/L 6-BA+0.005 mg/L TDZ+1.0 mg/L IBA。叶片和下胚轴所诱导的愈伤组织均可分化出不定芽,但芽诱导率差异达极显著水平;不定芽分化最佳培养基为MS+0.01 mg/L TDZ+0.2 mg/L IBA,叶片愈伤组织的芽分化率最高可达32.60%。     

红千层愈伤组织诱导及植株再生

以茎段、芽和叶片为材料,研究了红千层愈伤组织诱导及植株再生的方法。结果表明:红千层的茎段、芽和叶片均可诱导出愈伤组织,通过继代培养愈伤组织可发育成绿色和粉红色2种类型,其中绿色、致密的愈伤组织可以分化出不定芽。培养基1/2M S 6-BA 1.0 m g/L NAA 0.1 m g/L适宜愈伤组织不定芽的诱导,在培养基1/2M S IBA 0.25 m g/L上试管苗的生根率可达95%。     

Influences of antibiotics on plantlet regeneration via organogenesis in Ioblolly pine （Pinus taeda L.）

Three antibiotics ampicillin, carbenicillin, and cefotaxime were evaluated for their effects on induction, growth, and differentiation of organogenic calli, as well as rooting of regenerated shoots of three Ioblolly pine (Pinus taeda L.) genotypes. Of the antibiotics administered, cefotaxime maximally increased the frequency of callus formation and growth rate of organogeni ccalli, carbenicillin maximally increased the frequency of shoot regeneration and the average number of adventitious shoots per piece of organogenic callus, ampicUlin maximally decreased the rooting frequency of regenerated shoots and mean number of roots per regenerated shoot, in comparison with antibiotic-free media. Compared with the control, ampicillin minimally increased the frequency of callus formation, cefotaxime minimally increased the frequency of shoot regeneration, and carbenicillin minimally decreased the rooting frequency of regenerated shoots in three Ioblolly pine genotypes tested. All three antibiotics increased the frequencies of callus formation and shoot regeneration, and reduced the rooting frequency ot regenerated shoots suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into Ioblolly pine need to select a suitable antibiotic. This investigation could be useful for optimizing genetic transformation of conifers.     

Mevalonate kinase activity during different stages of plant regeneration from nodular callus cultures in white pine (Pinus strobus)

Mevalonate kinase (MK) catalyzes a step in the isoprenoid biosynthetic pathway, which leads to a huge number of compounds that play important roles in plant growth and development. Here, we report on changes in MK activity in white pine (Pinus strobus L.) during plant regeneration by adventitious shoot organogenesis from cotyledons of mature embryos, including nodular callus induction, shoot formation and rooting. Nodular calli were induced from Pinus strobus (PS) embryos by culture in nodular callus induction medium in a 0-, 8- or 16-h photoperiod. Mevalonate kinase activity peaked in nodular calli after three weeks of culture on nodular callus induction medium in a 16-h photoperiod, whereas frequency of nodular callus formation peaked after 4 weeks of culture on nodular callus induction medium in darkness. During adventitious shoot formation, MK activity peaked in shoots derived from dark-grown nodular calli after 3 weeks on bud formation medium, and frequency of shoot formation was highest in dark-grown nodular calli cultured on bud formation medium for 4 weeks. During rooting, MK activity peaked 2 weeks after transfer of adventitious shoots to rooting medium and rooting frequency was highest in adventitious shoots after 3 weeks on rooting medium. Although during nodular callus induction in darkness MK activity was inversely related to frequency of nodular callus formation, MK activity was highly correlated with frequency of shoot formation and with rooting frequency. The observed increase in MK activity preceding rooting suggests that MK could serve as a marker for rooting of white pine shoots in vitro.     

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Introduction1Genetictransformationinconifershasthepotentialtoallowtheselectiveimprovementofindividualtraitsinelitecloneswhilestillmaintainingtheexistingcombinationofgenesresponsibleforthesuperiorphenotype(Charestetal.1991;Jamesetal.1996;Walteretal.1999).Atpresent,althoughconsiderableresearchefforthasbeendevotedtothegeneticengineeringofconiferspecies(Sederoffetal.1986;Bekkauoietal.1988,1990;Robertsonetal.1992;Bomminenietal.1993;Shinetal.1994;Klimaszewskaetal.1997),ithaslaggedbehindadvancesma…     

Genetic transformation of loblolly pine using mature zygotic embryo expiants byAgrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of β-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected withAgrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of β-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficientAgrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.     

Effects of antibiotics on plantlet regeneration via organogenesis in Populus euphratica

Four antibiotics, kanamycin, geneticin, carbenicillin and cefotaxime,were evaluated for their effect on the regeneration of adventitious buds, shoot differentiation, rooting from regenerated shoots of Populus euphratica as well as on their control of Agrobacterium -mediated transformations. Results showed that the optimum concentration ranges of kanamycin and geneticin were 15-20 and 10-15 mg·L-1 at the stage of transgenic plantlet selection. The inhibitory effects of cefotaxime and carbenicillin varied among different genotypes of Agrobacterium. The inhibition of cefotaxime on Agrobacterium C58 was stronger than that of carbenicillin. The optimum concentration of cefotaxime was 100 mg·L-1. Cefotaxime and carbenicillin had similar effects on Agrobacterium LBA4404 and their optimum concentrations were both 150 mg·L-1.     

In vitro plant regeneration from cotyledon-derived callus cultures of leguminous tree Gleditsia caspica Desf.

An in vitro regeneration system was developed using organogenic callus derived from in vitro grown cotyledonary explants of Gleditsia caspica Desf., an important leguminous tree. Murashige and Skoog (MS) basal medium augmented with 0.2 g L^?1 myo-inositol and various concentrations of either 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid, or indole-3-butyric acid (IBA) alone as well as combined with cytokinins was used for callus induction. The highest frequency of organogenic yellowish-white and nodular callus (93 %) was obtained from explants grown on medium supplemented with 13.5 μM 2,4-D and 4.4 μM benzyladenine (BA). The yellowish-white and nodular callus when transferred to MS medium supplemented with BA (2.2–17.7 μM) or kinetin (KT; 2.3–18.8 μM) solely or in combination with 2.3 μM 2,4-D produced several microshoots after 5 weeks culture. The calli cultured on MS medium with 4.4 μM BA singly showed superior growth response and produced both maximum shoot regeneration (94 %) and the highest mean number (4.3) of microshoots per callus. Transfer of regenerated microshoots onto modified MS basal medium fortified with 5.8 μM gibberellic acid and 4.4 μM BA resulted in the maximum number of internodes per shoot and the highest shoot elongation after a period of 6 weeks. Optimum rooting of 90 %, an average 6.1 roots per shoot, and a mean root length of 3.6 cm was observed when half-strength MS medium was supplemented with 9.8 μM IBA and 0.92 μM KT. The regenerated healthy plants with well-developed shoots and roots showed a survival rate of 77 % after acclimatization and transplanting to garden soil for a 10-week hardening period under ex vitro conditions.     

Preliminary studies on establishment of genetic transformation system in loblolly pine

A transformation system was established for loblolly pine (Pinus taeda L.) mature zygotic embryos usingAgrobacterium tumefaciens. The gene coding for the β-glucuronidase (GUS) gene was introduced into loblolly pine tissues and its transient expression was detected with histochemical staining. The influences of different genotypes.Agrobacterum concentrations. and cocultivation time on GUS expression and kanamycin resistant callus and shoot regeneration were investigated. The results showed that the highest GUS expression frequency (16.3%) and shoot regeneration frequency (78%) were obtained from genotype 9–1003 withAgrobacterium concentration decreased 9 times and cocultivation time of 56 hours respectively. GUS expression was obtained in all genotypes tested. The successful expression of the GUS gene in different genotypes suggested that it will be a useful transformation system for loblolly pine. (Responsible Editor: Chai Ruihai)     

微弹介导的火炬松遗传转化

本文建立了一个微弹介导的火炬松遗传转化系统。这个系统解决了火炬松遗传转化过程中存在的许多困难。运载抗虫基因的质粒载体经微弹转化法进入火炬松成熟合子胚,然后在添加了卡那霉素的培养基上从转化的成熟胚上诱导出有器官发生潜力的愈伤组织,再从转化的愈伤组织上产生转基因植株。利用这一系统生产的转基因植株已经被随机扩增技术、Southern杂交技术和虫试验所证实,并且转化的植株已在土壤中成活。图3表2参28。     

Preliminary Studies on Establishment of Genetic Transformation System in Loblolly Pine

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Effect ofl-phenylalanine on rooting of shoots from axillary buds of adultLarix leptolepis

Rooting of shoots derived from axillary buds was examined to establish an efficient shoot culture system of clonal micropropagation in adult tree ofLarix leptolepis Gord. (Japanese larch). Nine out of ten shoots induced calli (90%) on their shoot bases, and the two of them formed root primordia with a red pigment (20%) on the calli surface within 5 weeks after culturing on modified Murashige and Skoog (MS) medium supplemented with 1.5 μM of indolebutyric acid (IBA) and 1.5 μM of naphthaleneacetic acid (NAA). However, the primordia did not elongate actively. The addition of 10 mMl-phenylalanine in the MS medium with the auxins resulted in the formation of roots at high frequency, about 80%, and they elongated actively. Although callus was formed in all the shoots cultured on the medium withl-phenylalanine, it appeared that the callus development was less as compared to the medium withoutl-phenylalanine. Consequently, the rooting might be associated with the suppression of the induced callus.