Establishment of an Autotetraploid Rice Anther Clone with High Frequency and Long Term Plant Regeneration

Results of study on clone A87203 4C-1-5 from anther culture of homologous tetraploid rice were reported. The trials showed that the clone had a bud multiplication rate of 150–200 times, Under stereoscope, buds were differentiating on buds, forming bud masses. It had a callus induction frequency about 5 times when the bud masses were clipped into pieces and cultured on MS medium for dedifferentiation. Buds with the characteristics of high multiplication were able to be produced from about 50% of the calli when they were transferred onto MS medium for differentiation. It still maintained a vigorous multiplying ability though it had been subcultured for more than 30 generation during the past 1 year or more. Numerous plantlets were able to be produced by culture under regulated temperature and light conditions. A number of plantlets deriving from the buds had been planted in the fields this year. Most of them were normal, however, few plants had morphological variations, with 6 trisomic (2n+1 =25) plants observed during meiotic division of their pollen mother cells.     

Variation of phenotype,ploidy level,and organogenic potential of in vitro regenerated polyploids of <Emphasis Type="Italic">Pyrus communis</Emphasis>

A wide range of phenotypic variation was observed among neopolyploids obtained from the diploid pear cultivar ‘Fertility’ by in vitro colchicine treatment. The variant plantlets had alterations in leaf characteristics. Neopolyploids had significantly different ratios of leaf length to leaf width compared to the diploid control. Shoot regeneration from leaf explants and rooting ability from in vitro shoots of neopolyploids was examined. Regeneration frequencies of shoots from leaf explants of seven of the nine neopolyploids were significantly decreased compared to the diploid control. The organogenic potential of neopolyploids was highly genotype-dependent for both shoots and roots. Tetraploid clone 4x − 4 failed to regenerate shoots from leaf explants and the pentaploid clone 5x − 2 failed to root from in vitro shoots. The results suggest that polyploidization caused the decrease in or loss of in vitro organogenic potential. Regenerated shoots derived from neopolyploids showed different phenotypes, depending on the ploidy of the donor plant.     

Epiphylly in a Variant of Helianthus Annuus x H. Tuberosus Induced by In vitro Tissue culture

A variant clone (EMB-2) derived by in vitro tissue culture of the interspecific hybrid Helianthus annuus x Helianthus tuberosus shows a particular deviation from the usual pattern of plant development in that it produces, both in vitro and in vivo, epiphyllous embryos and/or shootlike structures. Ectopic structures, which are usually arranged in clusters or rows along preexisting veins, originate asynchronously from epidermal cells of the adaxial surface of the leaf blade. Sometimes embryos and buds are also detected on the adaxial plane of the petioles and at the nodes of the stem. EMB-2 individual plants differ greatly in terms of the timing and extent of phenotypic expression of epiphylly. Leaves precociously affected by ectopic structures show a more drastic alteration in the differentiation process. Growth is arrested, the spongy parenchyma and air spaces are absent, and the mesophyll cells do not enlarge. Excluding the veins and epidermis, the leaves are wholly composed of isodiametric cells that are regularly arranged in parallel rows that have dense cytoplasm and prominent nuclei. Ectopic structures isolated from leaves and cultured in vitro mostly produce plantlets with the same phenotype as the original clones. In vivo, the EMB-2 plants are propagated by tubers. Often, the shoot-meristems that originate from tubers exhibit a teratological appearance and die without further development. However, several normal shoots grow and produce plants that display epiphyllous structures like those of the parent plants. Alterations of the endogenous hormonal levels or mutations in genes involved in the switch from indeterminate to determinate cell fate may be responsible for the ectopic development of shoots and embryos on leaves of EMB-2 variant.     

发根农杆菌转化怀地黄再生植株

利用发根农杆菌15834菌株感染怀地黄组培苗子叶、叶柄和茎切段,建立了有效的毛状根培养及其植株再生体系。毛状根可直接从受伤的外植体产生,能在无外源激素的1/2MS固体和液体培养基上自主生长,表现出典型的发根特征。用100μmol/L乙酰丁香酮处理对数生长期的农杆菌菌液感染子叶获得了46.7%的最高转化频率;在附加0.2mg/L KT和3.0mg/L 6/BA的1/2 MS培养基上,毛状根能100%形成愈伤组织,51.49%分化出芽;分化芽在1/2 MS培养基上100%生根,形成具有矮化、节间短和根系发达等特征的转化再生植株且移栽后生长旺盛;1个转化毛状根克隆的梓醇含量为0.557mg/g,是鲜地黄梓醇含量的48.5%,是生地鲜重梓醇含量的18%。rolB基因PCR、Southem blot分析、冠瘿碱纸电泳和RT-PCR扩增检测证明农杆菌Ri质粒上的T-DNA整合到怀地黄基因组中并表达。     

The relationship between polyploidy and organogenetic potential in embryo- and root-derived tissue cultures of Vicia faba L.

The cell cycle was examined in embryo and root explants of Vicia faba in culture to test whether or not polyploidy and aneuploidy affected organogenetic potential. Nuclear DNA contents and the mitotic index were measured in the 0–1 mm apical segment of primary roots of 5-day old seedlings and at various times following transfer to modified MS in darkness or Chu's N6 medium in an 8 h light/16h dark cycle (N6-MS programme) at 20°C. Mature embryos were dissected and cut longitudinally. Each half was cultured on the N6-MS programme. Root explants grown on MS in darkness developed into callus but there was no subsequent organogenesis. Only on the N6-MS programme were new roots initiated from root-derived callus. Using the N6-MS programme, embryo-derived callus became green and after 3 to 4 months, produced roots and shoots. Approximately 40% of these cultures regenerated plantlets. Polyploidy occurred within 24 h of culture irrespective of both tissue source and culture protocol. Variations in chromosome number from 2n=2x=12 were also routinely observed. Thus, calluses had the ability to initiate roots and shoots regardless of persistent polyploidy and aneuploidy. Compared with the baseline of cell cycle data for roots in vivo, the proportions of cells in the different cell cycle phases remained constant. Thus, in V. faba induction of organogenesis seems more related to culture protocols than to specific changes to the cell cycle. The mitotic index was significantly lower in vitro compared with meristems of intact roots.     

Meristem culture of Ophiopogon japonicus and production of embryo-like structures

Meristems of Ophiopogon japonicus (Liliaceae) were grown on a modified MS medium without auxins or cytokinins and plantlets and embryogenic callus were obtained at a low frequency. When meristems growing on modified basal medium were briefly soaked in 0.54 mM naphtaleneacetic acid (NAA) or temporarily grown on medium that contained NAA or NAA and benzyladenine, a larger proportion of meristems developed into plantlets or produced callus and additional plantlets following their return to basal medium. Calluses grown in liquid culture without auxins or cytokinins produced abundant single cells and cell aggregates. Larger cell aggregates formed embryo-like structures that produced roots, cotyledons, and then plantlets following transfer to soilid medium. Prolonged liquid culture produced embryo-like structures directly in liquid medium. These structures met many of the criteria for somatic embryos and developed into normal plantlets when placed on solid medium.     

The effect of aluminium and media on the growth of mycorrhizal and nonmycorrhizal highbush blueberry plantlets

A factorial experiment was conducted to determine the effect of aluminium (0 and 600M) and media (sand, and 1:1 sand:soil) on mycorrhizal (M) and non-mycorrhizal (NM) highbush blueberry plantlets. There were no differences in nutrient uptake and total plant dry weight between M and NM plantlets. However, more root growth, as determined by dry weight, was observed in M than NM plantlets. The plantlets growing in sand had more dry weight than did those in the soil medium. Although the root growth and shoot growth were reduced by the 600M Al treatment, the direct effect of Al on plantlet growth was not clear due to Al and P interactions. Plant nutrient uptake was reduced by high concentrations of Al, suggesting that high Al concentration limited the ability of roots to acquire most of the nutrients. Mycorrhizal cortical cell infection levels of 15–20% wene maintained in the roots in soil medium but decreased to about 5% over the 6 weeks of the experiment in the sand medium. Although M plantlets accumulated more Al in their roots, Al was readily transported to the leaf tissues of M and NM plantlets.     

Transformation of Populus tomentosa by Agrobacterium and Regeneration of Transformed Plantlets

The establishment of efficient transformation system of Populus tomentosa by Agrobacterium is reported. The strains of Agrobacterium used in experiments were: 1. A. rhizogenes R1000, which harboured the Ri plasmid pRiA4b. 2. A. rhizogenes R1000 (pTVK85), which carried the plasmids pRiA4b and pTVK85 Containing supervirulent region. 3. A. tumefaciens C58C1 (pBZ693), the plasmid pBZ693 containing genes 1 and 2. After being cocultured with the bacteria on media containing 0.5 ppm kinetin for 2 days, explants of P. tomentosa were transferred to MS medium containing 500 ppm cefotaxime. Roots appeared on the explants in a week. The roots induced by A. tume[aciens were morphologically different from those induced by A. rhizogenes. The frequency of the explants transformed by A. rhizogenes R1000 (pTVK85) was nearly up to 60%. Some Ri plasmid transformed roots could spontaneously produce adventitious shoots or calli. By adding appropriate plant growth regulators in the media, we could have all of the root lines transformed produce adventitious shoots which would develop into intact plantlets on a hormone-free medium. Some phenotypical differences were observed among clones of the transformed plantlets. Some clones had short internodes, large number of leaves, reduced apical dominance, rich root systems with a great quantity of branches and root hairs, whereas in other clones aboveground parts of plantlets were morphologically normal and only their root systems were different from those of untransformed plantlets. None of the plantlets transformed by A. rhizogenes had the phenomenon of wrinkle leaves and shapes these leaves were analogous to normal plantlets. It was often observed that roots were regenerated from stems above the medium surfaces. Southern analysis on three clones of the putative transformed plantlets by A. rhizogenes R1000 (pTVKS5) showed that two of them were hybridized positively with the probe covering the TL-DNA region of the plasmid pRiA4b.     

Hairy root induction and plant regeneration of <Emphasis Type="Italic">Rehmannia glutinosa</Emphasis> Libosch. f. <Emphasis Type="Italic">hueichingensis</Emphasis> Hsiao via <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation

The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time. This text was submitted by the authors in English. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 247–255.     

Comparisons of alfalfa somaclonal and sexual derivatives from the same genetic source

Summary Tetraploid alfalfa (Medicago sativa L.) clones were derived from the same diploid genetic background by four different methods. A phenotypically superior clone was selected from each method and compared for herbage yield and fertility. The four methods and their best clones were: a) In vitro somatic chromosome doubling of one diploid hybrid (HG2-4x); b) selection within a two allele tetraploid synthetic population derived from HG2-4x (HAG); c) somaclonal variant selection from cell suspension culture of the diploid hybrid (NS1); and d) sexual polyploidization of a sibling hybrid (HXG). Clones HG2-4x, HAG, and NS1 were likely diallelic or monoallelic at all loci. Clone HXG was probably tetrallelic or triallelic at most loci. Experiments measured fertility, clonal herbage yield, and herbage yield of test cross progeny for each selected clone. Fertility rankings were HXG = HAG > NS1 > HG2-4x. Clonal herbage yield rankings were HXG = HAG > NS1 > HG2-4x. Test cross progeny herbage yield rankings varied depending on the tester, but, in general, HXG HAG NS1 HG2-4x. Overall the best clones from the sexual methods exceeded the best somaclonal variant which, in turn, was better than the chromosome doubled clone.     

新疆雪莲毛状根的诱导及其植株再生体系的建立

利用发根农杆菌R1601、R1000、LBA9402感染新疆雪莲的叶片、叶柄和根段外植体,诱导产生毛状根。毛状根接种量为2.8 g/L(FW)时,20d生长量可达66.7 g/L,黄酮含量达到干重的10.23%。冠瘿碱的检测和rolB基因的PCR分析表明,Ri质粒中的T_DNA片段已经整合到毛状根细胞的基因组中。预培养时间、外植体类型以及发根农杆菌的菌株属性对毛状根诱导有着重要的影响。其中预培养2 d的新疆雪莲根段外植体,经过R1601感染后,毛状根的诱导率可达100%。诱导产生的毛状根在附加生长素的液体培养基中,有少量愈伤组织产生。由毛状根再生的植株与雪莲外植体再生的植株在形态上无明显区别,但前者的黄酮含量仅为后者的53%。     

Ri T-DNA对盾叶薯蓣的遗传转化及薯蓣皂甙元产生的影响

利用农杆菌介导法成功地将Pd T-DNA转入药用植物盾叶薯蓣,产生了毛状根,经分子信标探针检测农杆菌Pd质粒上的T-DNA已整合进植物基因组中。研究建立了毛状根大量快速繁殖技术,基本技术要求为：1／2 MS液体培养基,28℃培养温度,350lux弱光条件下有利于毛状根的增殖培养,提高生物量。HPLC测定结果显示,转基因获得的毛状根其薯蓣皂甙元的含量分别是微块茎、愈伤组织和植物体合成量的5．68倍、6．12倍和2．68倍。     

滇黄芩毛状根的诱导及其黄芩苷含量测定

本文利用发根农杆菌A grobacterizum rhizogenes1.2556感染滇黄芩再生苗的茎段和叶片,建立了毛状根培养及其植株再生体系。毛状根可直接从受伤的茎、叶外植体表面产生,在无外源激素的MS固体和液体培养基上自主生长,表现出典型的发根特征。毛状根茎段的诱导率较叶片高,最高可达到14.44%;经rolB基因PCR分析和甘露碱纸电泳检测,证明Ri质粒T-DNA已整合到滇黄芩基因组中并表达;毛状根在附加6-BA2mg/L和NAA0.2mg/L的MS固体培养基上直接诱导不定芽,并在MS培养基上生根,形成再生植株。获得的毛状根系经MS液体培养基培养30d后通过HPLC都能检测到黄芩苷,其中1个转化系黄芩苷含量为2.59%,是药材黄芩的0.20倍,而从3年单位时间黄芩苷生成量计算,毛状根是药材黄芩的7.18倍。本研究建立的毛状根培养体系,将对滇黄芩转基因技术的完善和利用毛状根生产黄芩苷的生物转化提供了实验基础。     

Genetic stability of micropropagated almond plantlets,as assessed by RAPD and ISSR markers

Almond shoots produced by axillary branching from clone VII derived from a seedling of cultivar Boa Casta were evaluated for somaclonal variation using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) analysis. To verify genetic stability we compared RAPD and ISSR patterns of plantlets obtained after 4 and 6 years of in vitro multiplication. A total of 64 RAPD and 10 ISSR primers gave 326 distinct and reproducible band classes, monomorphic across all 22 plantlets analysed. Thus, a total of 7,172 bands were generated, exhibiting homogeneous RAPD and ISSR patterns for the plantlets tested. These results suggest that the culture conditions used for axillary branching proliferation are appropriate for clonal propagation of almond clone VII, as they do not seem to interfere with the integrity of the regenerated plantlets. These results allowed us to establish the use of axillary branching plantlets (mother-plants) as internal controls for the analysis of somaclonal variation of shoots regenerated from other in vitro culture processes performed with clone VII (adventitious regeneration, regeneration from meristem culture, virus sanitation programs and genetic engineering).M. Martins and D. Sarmento contributed equally to this paper     

草木樨状黄芪抗甲硫氨酸变异系的离体筛选及鉴定

通过组织培养体系进行了草木樨状黄芪(Astragalus melilotoides Pall.)抗甲硫氨酸变异系的筛选。无菌苗幼茎切段诱导的愈伤组织经NaN_3诱变后,经过筛选获得了抗14mmol/L甲硫氨酸的变异细胞系,并分化出大量植株。再生植株已经在大田中开花结实。经分析表明:抗性细胞系脱离选择压6个月后,放在含15mmol/L甲硫氨酸的培养基上培养25天后,其相对增长率是对照的10.2倍。抗性系再生植株游离甲硫氨酸是对照的2.12倍,并且天冬族氨基酸都有明显增加。SDS-PAGE及过氧化物酶同工酶分析表明:在蛋白质及同工酶酶谱上抗性系再生植株均出现与对照不同的差异。     

黄花菜不同外植体形成的愈伤组织再生苗观察

研究了黄花菜不同外植体愈伤组织的形成与植株再生。结果表明,出愈率是花柄>花茎>叶片。愈伤组织在MS+6-BA 2mg/L的培养基上出现致密愈伤组织颗粒,经增殖→分割→增殖的程序逐渐形成“球状体”似的愈伤组织。叶片、花茎形成的球状体经20代继代培养,再生能力没有减退,苗形态正常,根尖细胞染色体数目为2n=2x=22。该球状体经秋水仙素处理获得多种形态的四倍体苗和少数重复加倍现象。花柄球状体再生苗中出现了叶片花瓣状或类似花蕾状的形态异常苗,其频率与花柄所处的花期及花蕾大小有密切关系。再生异常苗的球状体继代培养,或者由异常苗叶片重新形成的球状体仍然再生异常苗。花柄球状体经秋水仙素处理,获得的变异株其染色体数目类型较多。因而不宜用花柄作黄花菜快速繁殖的外植体。     

辣椒花药培养的胚状体分化及其与成苗率的关系

12个辣椒品种的花药培养的结果表明,所有品种均可诱导出胚状体,其中9个品种可获得健壮的再生植株。每一辣椒品种的适宜植物生长调节利配比不同。不同品种的胚状体诱导率和成苗率有差异。成熟的胚状体均能分化成苗,根先分化或停止发育的胚状体很少成苗。     

发根农杆菌对骆驼刺的转化及转化组织的形态发生

将骆驼刺离体再生苗的茎切段经发根农杆菌A4菌株感染后,在含500mg/L头孢霉素的MS无激素培养基上培养,产生了转化的发根和愈伤组织.转化根在附加2mg/L2,4-D和0.5-1mg/L6BA的MS培养基上培养后,亦可诱导出愈伤组织.在含3mg/L6BA和0.5mg/LNAA的培养基上诱导出了苗的分化.冠瘿碱分析表明,在95%以上的发根和75%的转化愈伤组织及再生植株中都显示了T-DNA的整合和表达.染色体检查发现,约81%的发根细胞具有正常染色体数(2n=18),其余则存在染色体数目的变化,在继代培养一年之后,转化体仍维持旺盛的再生能力.     

The effect of carbohydrate on the development of a Cattleya hybrid in association with its mycorrhizal fungus

To investigate beneficial effects of mycorrhizal fungi to advanced leafy orchids, growth studies on the development of symbiotic seedlings of the orchid Cattleya (aclandiae x schoeffeldiana) x aclandiae were conducted in vitro over a period of 18 months using split plates with minerals and carbohydrates on one side and water agar on the other. Mycorrhizal infection and shoot and root growth of seedlings on the nutrient side were compared to growth on the water agar side with nutrient uptake by the orchid only possible via external mycorrhizal hyphae. Seed germination was followed by mycorrhizal infection and rapid development of protocorms on both nutrient and non-nutrient sides of the plates. With 0.5% starch, development of protocorms was sustained for a least 12 weeks, compared to only 6 weeks with 0.1% starch. Advanced protocorms with two small leaves and a smoll root were transferred at week 22 to new fungal plates. When harvested at week 43, plantlets on 0.5% starch (both nutrient and water agar sides) had 2.7 times the dry weight of plantlets on 0.1% starch. Shoot-root ratios were higher on the lower level of carbon. In all plantlets, mycorrhizal infection involved less than 5% of the root length. With zero, 0.1% or 0.5% starch, the roots were re-infected on transfer to fresh fungal plates but young roots that developed following the transfer stayed free of infection, Plantlets on 0.5% starch (nutrient and water agar side) after 18 months had longer roots than plantlets grown in the absence of starch or on 0.1% starch. Shoots were small but significantly larger on the nutrient side than on the water agar side, independent of the carbohydrate level. The shoot-root ratio was highest on the nutrient side with no starch present. In this latter case, plantlet development was steady but plantlets on the non-nutrient side developed slowly; thus there was little evidence of nutrient translocation by the mycorrhizal fungus from the nutrient to the non-nutrient side in the absence of carbohydrates. Mycorrhizal infection is discussed as a mechanism for heterotrophic carbon assimilation. In advanced leafy orchids of Cattleya, external carbon resulted in increased root growth, decreased shoot/root ratio and sometimes yellowish-green plantlets.