Apical Shoot Meristem Culture in Red Pepper (Capsicum annuum L.)

A protocol has been developed to obtain whole plants from apical shoot meristems of red pepper (Capsicum annuum L. cv. Bhivapuri), susceptible to viral infections. The meristems (～ 0.8 mm long), from aseptically grown seedlings (one-month-old), cultured on filter paper bridge in liquid Murashige and Skoog medium supplemented with 2 mg/l benzylaminopurine produced multiple shoots (5–7 per explant). The differentiated shoots developed further upon transfer to agar-solidified medium. Complete plantlets were obtained after rooting of shoots on MS medium fortified with 1 mg/l naphthaleneacetic acid.     

Optimization of bulked AFLP analysis and its application for exploring diversity of natural and cultivated populations of red clover.

Landraces and wild populations of red clover (Trifolium pratense L.) may represent a significant yet poorly characterized genetic resource of temperate grasslands. A bulking strategy with amplified fragment length polymorphism (AFLP) markers was optimized to characterize 120 red clover populations in 6 different groups: Swiss wild clover populations, Mattenklee landraces, Mattenklee cultivars, field clover cultivars, Dutch wild clover populations, and Dutch landraces. Analysis of 2 bulked samples/population consisting of 20 plants each with12 AFLP primer combinations was found optimal for determining genetic diversity and relationships within and among red clover populations and groups. Swiss wild clover populations were clearly separated from all other red clover groups and variability within and among populations was shown to be particularly high in wild clover populations and Mattenklee landraces, emphasising their value as genetic resources for improvement of red clover cultivars, as well as for conservation and restoration of biodiversity. This study shows that the ancestry of red clover landraces is primarily found in introduced cultivars rather than in natural wild clover populations. In addition, the methodological considerations presented here may help improve diversity analyses using bulked samples.     

Induction of embryogenic callus of loose skin mandarins]

Young ovules from 3 cultivars and undeveloped ovules in mature fruits from 8 cultivars of loose skin mandarin of Citrus were cultured on 4 different media respectively to induce embryogenic calli. Results showed that the combination of EME(MT + 500 mg/L malt extract) and MKT (EME + 10 mg/L KT) media performed well in the induction of embryogenic calli from young ovules; MGS(EME + 1 mg/L GA3 + 40 mg/L sulfate adenine) medium was better than MDB (MT + 0.01 mg/L 2,4-D + 0.1 mg/L BA) medium in inducing calli from the undeveloped ovules, and the darkness was conducive to the induction of embryogenic calli. There was no chromosome number variation in the induced calli. All of the examined cells were diploid with 2n = 2x = 18 chromosomes.     

Development of an Agrobacterium-mediated transformation method for pear (Pyrus communis L.) with leaf-section and axillary shoot-meristem explants

We have developed a new Agrobacterium-mediated transformation method for the low-frequency-regenerating pear (Pyrus communis L.) cvs. Silver bell and La France. Leaf sections derived from in vitro shoots were initially used for the transformation procedure. Under optimum transformation conditions, which included culture and selection on 30 mg/l kanamycin (Km) combined with 500 mg/l sulbenicillin, a 3.2% transformation efficiency was obtained for cv. Silver bell, but no transformants of La France were obtained because of the very low regeneration frequency. Axillary shoot meristems were then examined as potential explants for La France. Selection in 5 mg/l Km and 375 mg/l carbenicillin resulted in transformed shoots being produced at an efficiency of 4.8%, and the apparent white Km-sensitive shoots were not formed during a 2-year subculture on micropropagation medium containing 50 mg/l Km. Therefore, transformations using axillary shoot meristems may be an alternative method for pear cultivars recalcitrant to regeneration from leaf sections.     

宽皮柑橘品种的胚性愈伤组织诱导

以宽皮橘3个品种的幼嫩胚珠和8个品种成熟果实中未发育胚珠为试材,采用EME(MT 500mg/L麦芽浸出物)、MKT(EME 10mg/L KT)、MGS(EME 1mg/L GA3 40mg/L硫酸腺嘌呤)和MDB (EME 0.01mg/L 2,4-D 0.1mg/L BA)4种培养基进行胚性愈伤组织诱导,结果表明EME与MKT培养基配合使用有利于从幼嫩胚珠获得胚性愈伤组织;MGS比MDB更有利于成熟果未发育胚珠胚性愈伤组织的诱导;暗培养有利于此诱导过程。经两种途径获得的胚性愈伤组织,染色体数目稳定,均为二倍体2n=2x=18。     

An efficient mass propagation system for cassava (Manihot esculenta Crantz) based on nodal explants and axillary bud-derived meristems

Nodes from 3- to 5-week-old in vitro plants of different cassava cultivars were cultured for 2–3 days on solid Murashige and Skoog basal medium supplemented with cytokinin to induce the enlargement of axillary buds. Subculture of these buds on the same medium resulted in multiple shoot formation within 4–6 weeks. Of the four cytokinins tested (6-benzylaminopurine (BAP), thidiazuron (TDZ), zeatin, and kinetin), BAP induced shoot development most efficiently. The best results were obtained with cultivar TMS 30555, in which 63% of the explants each produced at least 25 shoots on medium with 10 mg/l BAP. In cultivars that did not produce shoots, the addition of the surfactant Pluronic F-68 (2% wt/vol) raised the percentage of explants forming at least 5 shoots from 0 to 20–60%. Axillary buds were also used to dissect meristems and test their ability to regenerate into shoots. Shoot formation from meristems of six different cultivars was observed after preculture on medium with 5 mg/l BAP followed by transfer to 10 mg/l BAP.Abbreviations MS Murashige and Skoog - BAP 6-Benzylaminopurine - TDZ Thidiazuron     

盾叶薯蓣组织培养技术的优化

以盾叶薯蓣的根状茎、茎段、叶柄、幼叶为材料,进行愈伤组织诱导、分化及再生植株形成的研究。结果表明：盾叶薯蓣不同外植体均能诱导出愈伤组织,其中茎段愈伤组织的诱导率最高;不同激素配比的培养基对愈伤组织的形成有很大的影响：以LS为基本培养基,2,4-D浓度为4．0mg／L、6-BA浓度为1．0mg／L的激素配比诱导率最高,达62．5％;以改良MS为基本培养基,2,4-D浓度为2．0mg／L、6-BA浓度为0．5mg／L的激素配比诱导率最高,达71．4％。筛选到优化的分化培养基为改良MS附加2．0mg／L的6-BA和0．5mg／L的Vc,且能直接诱导出根,并形成完整植株。     

Establishment and multiplication of red clover plants by in vitro shoot tip culture

A procedure is described for the routine establishment and multiplication of red clover, Trifolium pratense L., shoot tips which should be applicable to a wide genotypic background. The addition of CO₂ to the culture vial or use of polypropylene closures enhanced shoot multiplication at high levels of benzyladenine (BA). Horizontal orientation of crown buds resulted in more efficient multiplication. Culture of nodes from flowering stems was unsuccessful. The cytokinin BA was most effective for shoot multiplication at 2.0 mg/l with maximum shoot production by four weeks. A comparison of several genotypic sources revealed a 10-fold range in response to the multiplication medium with no differences observed among agronomic type or ploidy level. An additional study revealed that multiplication ability of a genotype can be determined after the second subculture since multiplication ability does not change during repeated subculture.     

Effect of Ploidy and Flowering Type of Red Clover Cultivars and of Isolate Origin on Severity of Clover Rot, Sclerotinia trifoliorum

Two complementary experiments were conducted in a controlled environment to elucidate the interactions between the fungus Sclerotinia trifoliorum Erikss. and red clover (Trifolium pratense L.). In one of these studies, two hardened diploid red clover cultivars (cvs) were inoculated with 20 isolates of S. trifoliorum of various geographic origins. In the other study, 20 red clover cvs, diploid or tetraploid, including late and medium‐late flowering types, were inoculated with two isolates of the fungus. Prior to inoculation, some plants were hardened by subjecting them to a low temperature and light treatment mimicking autumn conditions. Late flowering cvs were found more resistant than medium‐late ones. Isolates collected in the northern region, where late cvs are grown, were significantly more aggressive than isolates from southern locations, where medium‐late cvs are more prevalent. Such an adaptation has not previously been reported for this fungus. This is the first report concerning flowering type and resistance in red clover. Tetraploids were generally not more resistant than diploids. A hardening procedure for red clover plants was found to be a prerequisite for detecting the differences in disease resistance.     

冬凌草离体培养体系的建立及主要次生代谢产物的测定

以冬凌草叶片为外植体,研究不同浓度激素组合对冬凌草愈伤组织诱导及植株再生的影响,并对不同外植体(茎、叶)诱导愈伤、芽的分化能力及再生植株内主要次生代谢产物的含量进行了比较研究。结果表明:在MS 2.0 mg/L 6-BA 1.0 mg/L NAA培养基上诱导愈伤组织效果较好;在MS 2.0 mg/L 6-BA的培养基上诱导芽的效果较好;叶片和茎段在愈伤诱导培养基上均能产生大量的愈伤组织,但其再分化能力以茎段最好;再生苗生根培养基以0.3 mg/L IBA最好;以叶为外植体诱导的再生植株中冬凌草甲素、迷迭香酸的含量均高于以茎为外植体诱导的再生植株。     

Prolific direct plant regeneration from cotyledons of white clover

Summary A facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1^-1 6-benzylaminopurine and 0.05 mg 1^-1 -napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - MS Murashige and Skoog medium     

Variation in the development of stem nematodes, Ditylenchus dipsaci, in susceptible and resistant crop plants

Dicotyledonous plants were reliably inoculated with stem nematodes by placing drops of 1.3% carboxymethylcellulose containing the surface sterilised worms between the cotyledons or in the leaf axils of recently emerged seedlings raised in pots of cool, moist soil. Inoculating onion leaves, bean stems and potato tubers or potato leaves gave variable results and inoculating onion, tulip and narcissus bulbs and lucerne and red clover stems was usually unsuccessful. An attempt to characterise 67 stem nematode populations by the reactions of a number of different plants failed for lack of useful differential hosts. Lucerne was, however, resistant to all but lucerne populations. Multiplication of stem nematode populations varied greatly between cultivars of lucerne or red clover. Some cultivars were resistant to some populations of lucerne or red clover stem nematodes and susceptible to others. These differences could not be ascribed to differences in viability of the nematodes or to differences in success of the inoculations. They indicate the presence of different pathotypes or biotypes in different populations of a so-called ‘host race’ and indicate the need for new resistant cultivars to be tested against a range of populations before they are released for general use. Amongst lucerne cultivars tested, Vertus was resistant to some lucerne stem nematode populations and susceptible to others. The supposedly resistant lucerne cultivars Euver and Lifeuil were as susceptible as was Europe. Amongst red clover cultivars tested, Redhead, Kühn, Changins and Mt Calme were susceptible, Britta, Lucrum and Temara were the least resistant, Renova, Rittinova and Quin were intermediate and susceptible to one or more of the populations tested but Norseman and especially Sabtoron were very resistant.     

Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons

A protocol for chloroplast transformation of an elite rapeseed cultivar (Brassica napus L.) was developed based on optimized conditions for callus induction and regeneration from cotyledonary tissues. Comparison of six different media with three elite cultivars showed that B5 medium plus 3 mg/l AgNO₃ supplemented with 0.6 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-furfurylaminopurine was optimal for callus formation and maintenance without differentiation, while the medium suitable for regeneration was B5 medium supplemented with 1 mg/l 6-benzylaminopurine, 1 mg/l 6-furfurylaminopurine and 0.5 mg/l α-naphthaleneacetic acid. A rapeseed-specific chloroplast transformation vector was constructed with the trnI and trnA sequences amplified from the rapeseed chloroplast genome using two primers designed according to Arabidopsis homologs. The aadA gene was used as a selection marker regulated by the ribosome-binding site from the bacteriophage T7 gene 10L, the tobacco 16S rRNA promoter and the psbA terminator. After bombardment, cotyledonary segments were cultured for callus formation on media containing 10 mg/l spectinomycin and regeneration was carried out on medium with 20 mg/l spectinomycin. Heteroplasmic plastid transformants were isolated. An overall efficiency for the chloroplast transformation was one transplastomic plant per four bombarded plates. Southern blot analyses demonstrated proper integration of the target sequence into the rapeseed chloroplast genome via homologous recombination. The expression of the aadA gene was confirmed by Northern blot analysis. Analysis of T1 transplastomic plants revealed that the transgenes integrated into the chloroplast were inheritable with a ratio of about 8%. These results suggest that rapeseed may be a suitable crop for chloroplast transformation with cotyledons as explants under appropriate conditions.     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。     

培养因子对艾西丝南瓜芽增殖及不定根形成的影响

以艾西丝南瓜带芽茎段为外植体,研究了基本培养基、激素、糖、光照、培养基支持物等因子对芽增殖及不定根形成的影响。结果表明：艾西丝南瓜芽增殖的最佳培养条件为：MS+BA 0.5～1.0 mg/L+IAA 0.1～0.5 mg/L+食用白糖30 g/L,芽的月[KG(*9]增殖系数稳定在10左右;不定根诱导的适宜条件是：[KG)]1/2MS＋食用白糖20 g/L,生根率达86%;且自然散射光条件(1000～5000Lx)优于灯光(1000～2000Lx);以脱脂棉作生根培养基支持物效果优于琼脂,其芽增殖系数和生根率分别提高26%和7%。     

Establishment and multiplication of red clover plants by in vitro shoot tip culture

A procedure is described for the routine establishment and multiplication of red clover, Trifolium pratense L., shoot tips which should be applicable to a wide genotypic background. The addition of CO₂ to the culture vial or use of polypropylene closures enhanced shoot multiplication at high levels of benzyladenine (BA). Horizontal orientation of crown buds resulted in more efficient multiplication. Culture of nodes from flowering stems was unsuccessful. The cytokinin BA was most effective for shoot multiplication at 2.0 mg/l with maximum shoot production by four weeks. A comparison of several genotypic sources revealed a 10-fold range in response to the multiplication medium with no differences observed among agronomic type or ploidy level. An additional study revealed that multiplication ability of a genotype can be determined after the second subculture since multiplication ability does not change during repeated subculture.Contribution from the Plant Cell Culture Centre, University of Guelph, Department of Crop Science, Guelph, Ontario, Canada N1G 2W1     

Shoot regeneration from spinach hypocotyl segments by short term treatment with 5,6-dichloro-indole-3-acetic acid

Factors affecting shoot regeneration from hypocotyl segments of spinach (Spinacia oleracea L.) were investigated. When expiants were cultured on medium containing 10 mg/l IAA for 7 weeks, 3 out of 9 cultivars showed relatively high shoot regeneration response (15 – 35%). The other PGRs tested had no effect on shoot regeneration. However, the transfer of explants from auxin-containing medium to auxin-free medium 20 d after culture induced shoot formation from expiants cultured on media containing each of the auxin sources tested individually. By applying this short term auxin treatment, more than 80% shoot regeneration was obtained on medium containing 5–20 mg/l 5,6-Cl₂-IAA, compared to less than 30% with 10–20 mg/l IAA treatment.Abbreviations BAP 6-benzylaminopurine - NAA 1naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - 5,6-Cl₂-IAA 5,6dichloro-indole-3-acetic acid - PGR plant growth regulator - A-PGR auxin-like plant growth regulator     

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.)

We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum L.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Glucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, [beta]-glucuronidase (GUS), neomycin phosphotransferase, and an [alpha]-amylase inhibitor were used to transform subterranean clover. Transgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of T-DNA into the subterranean clover genome. The expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding GUS segregated as a dominant Mendelian trait.     

In vitro micropropagation of Lawsonia inermis (Lythraceae)

A successful protocol was developed for mass propagation of Lawsonia inermis Linn., an important medicinal plant. Multiple shoots were induced in apical and axillary meristems derived from mature explants of L. inermis on Murashige and Skoog (1962) medium supplemented with 0.25 mg/l 6-benzylaminopurine (BA), 0.25 mg/l Kinetin (Kn), 0.5 mg/l ascorbic acid and 3% (w/v) sucrose. The rate of multiplication was higher when the cultures were incubated under continuous light rather than the 14 hr photoperiod. Rooting was readily achieved upon transferring the microshoots onto MS basal semi-solid medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA) after ten days of culture. Micropropagated plantlets were acclimatized and successfully grown in soil.     

Combination of 6-Benzylaminopurine and Thidiazuron Promotes Highly Efficient Shoot Regeneration from Cotyledonary Node of Mature Peanut (<i>Arachis hypogaea</i> L.) Cultivars

Efficient in vitro plantlet regeneration is an important step to successfully transform genes for the improvement of agronomic traits. A combination of 6-benzylaminopurine (BAP) and thidiazuron (TDZ) plant growth regulators was applied to evaluate shoot regeneration capacity whereas α-naphthalene acetic acid (NAA) combination with 6-benzylaminopurine (BAP), and 2, 4-dichlorophenoxyacetic acid (2, 4-D) with 6-benzylaminopurine were tested to optimize root induction for two peanut cultivars. The result showed combination (BAP with TDZ) was found to be effective in promoting shoot. The highest shoot regeneration frequency (93%) was obtained on a medium supplemented with 4 mg/L BAP and 0.5 mg/L TDZ while an average regeneration frequency (87%) was achieved in a medium containing combinations of 2 mg/L BAP with 1 mg/L TDZ. The shooting rate increased for both cultivars as the concentrations of BAP increased and TDZ decreased. The highest rooting rate (93%) was obtained on a medium supplemented with 3.5 mg/L NAA with 2.5 mg/L BAP for both cultivars. The rooting rate increased as the concentration of auxin to cytokinin ratio increased. The maximum rooting rate (83%) was obtained on MS medium supplemented with 0.3 mg/L 2, 4-D with 0.2 mg/L BAP for the cultivar N3. The result indicated that BAP with NAA was much better than BAP with 2, 4-D in rooting rate. Thus, the protocol developed was genotype independent and effective for peanut tissue culture.