Micropropagation and induction of autotetraploid plants of <Emphasis Type="Italic">Chrysanthemum cinerariifolium</Emphasis> (Trev.) Vis

Rapid propagation technology was established and optimized in vitro for Chrysanthemum cinerariifolium (Trev.) Vis., an important botanical insecticide plant with a huge international market. A large number of buds could be induced directly from epicotyl and hypocotyl explants on Murashige T; Skoog F. J. Plant. Physiol. 15: 473–479; (1962) medium [Murashige and Skoog (MS) medium] supplemented with 0.3 mg l⁻¹ benzyladenine (BA) and 0.3 mg l⁻¹ α-naphthaleneacetic acid (NAA). Root induction and development could be observed within 15 d after inoculation on 1/2 MS medium supplemented with 0.2 mg l⁻¹ indole-3-acetic acid (IAA) and 0.1 mg l⁻¹ rooting powder (ABT). Furthermore, a polyploid breeding study in vitro was reported to obtain superior breeding lines with high yield and good quality. Autotetraploid lines of C. cinerariifolium were obtained by colchicine treatments and identified by root-tip chromosome determination and stoma observation. The chromosome number of the autotetraploid plantlet was 2N = 4x = 36. Obtained autotetraploid lines will be of important genetic and breeding value and be used for further selection and plant breeding.     

In vitro production of Indian citrus ringspot virus (ICRSV) free Kinnow plants employing thermotherapy coupled with shoot tip grafting

Indian citrus ringspot virus (ICRSV) is known to cause serious disease problem in Kinnow (Citrus nobilis Lour × C. deliciosa Tenora) plants. This work reports the elimination of ICRSV by using thermotherapy coupled with shoot tip grafting in vitro. Nodal segments from infected mother plants (indexed by indirect ELISA and RT-PCR) were treated both in water bath and moist hot air at different temperatures viz. 40, 45 and 50°C for 30, 60 and 120 min and cultured on MS medium containing 2-iP (1 mg/l) and malt extract (800 mg/l). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (C. jambhiri) rootstock under aseptic conditions. Water bath treatment was found to be more effective as compared to moist hot air treatment as maximum number of ICRSV free plants (36.84%) were obtained by grafting the tips (0.7 mm) taken from the nodal segments treated at 50°C in water bath for 2 h. In an alternate treatment regime, 1-year-old infected plants were kept at various temperatures viz.36, 38 and 40°C in a thermotherapy chamber. Maximum of 60% ICRSV free plants were obtained by grafting the tips (0.7 mm) from the plants placed at 40°C followed by the plants placed at 38°C (59.09%) and the least was observed in case of the plants placed at 36°C (40.74%). Only those plants/plantlets were considered virus free, which showed negative reaction both in Indirect ELISA and RT-PCR.     

种植茼蒿对蚕豆苜蓿蚜的田间控制作用

[目的] 探讨不同生育期和不同种植方式的茼蒿对蚕豆蚜虫的诱集作用,为利用茼蒿控制蚕豆蚜虫提供理论依据。[方法] 在蚕豆田四周种植不同生育期（幼苗期、现蕾期、开花期）和不同行数（1行、2行）的茼蒿,观察不同处理的蚕豆田有蚜株率和蚜害等级,各处理设在互不干扰的小区内进行。[结果] 蚕豆四周种植不同生育期茼蒿后,蚕豆上有蚜株率和蚜害等级比例存在显著差异,且与茼蒿的生育期有明显的相关性,各处理有蚜株率从低到高分别为茼蒿开花期（28.33%） < 现蕾期（41.67%） < 幼苗期（55.00%）,并均显著低于对照（63.33%）;种植不同生育期茼蒿后,各处理蚕豆蚜害等级也不同,5级蚜害在种植开花期茼蒿处理后仅为5.00%,现蕾期为23.33%,幼苗期为33.33%,对照蚕豆上蚜害最高,达40.00%。分别种植1行（33.33%）和2行（23.33%）茼蒿后,最高有蚜株率均显著低于对照（66.67%）,低蚜害等级比例明显增高,高蚜害比例明显下降,且种植2行的效果更佳。[结论] 开花期的茼蒿对蚕豆蚜虫诱集作用最强,种植2行开花期茼蒿可以有效降低蚕豆蚜虫为害。在蚕豆生产上,种植茼蒿可以作为蚕豆蚜虫生态防控的重要手段之一。     

The preparation in vitro of chrysanthemum for transplantation to soil

Plantlets of Chrysanthemum x morifolium were grown from nodal sections in cellulose plugs which were saturated with liquid rooting medium containing 1 mg l^–1 paclobutrazol in culture vessels that maintained relative humidities (RH) of 100%, 96% and 94%, respectively. After 4 weeks, plantlets were transferred to compost and exposed to 40% RH at 29°C. Marked differences in wilting were observed that were directly related to the RH of the vessel in which the plantlets had been grown. Thicker leaves, improved closure of stomata and increased thickness of the cuticle were associated with increased resistance to wilting. Reduced RH of the culture vessel was also associated with significantly higher concentrations of chlorophyll in the leaves.     

Altered expression of CmNRRa changes flowering time of Chrysanthemum morifolium

Flowering time is an important ornamental trait for chrysanthemum (Chrysanthemum morifolium, Dendranthema x grandiflorum) floricultural production. In this study, CmNRRa, an orthologous gene of OsNRRa that regulates root growth in response to nutrient stress in rice, was identified from Chrysanthemum and its role in flowering time was studied. The entire CmNRRa cDNA sequence was determined using a combinatorial PCR approach along with 5′ and 3′ RACE methods. CmNRRa expression levels in various tissues were monitored by real‐time RT‐PCR. CmNRRa was strongly expressed in flower buds and peduncles, suggesting that CmNRRa plays a regulatory role in floral development. To investigate the biological function of CmNRRa in chrysanthemums, overexpression and knockdown of CmNRRa were carried out using transgenic Chrysanthemum plants generated through Agrobacterium‐mediated transformation. CmNRRa expression levels in the transgenic plants were assayed by real‐time RT‐PCR and Northern blot analysis. The transgenic plants showed altered flowering times compared with nontransgenic plants. CmNRRa‐RNAi transgenic plants flowered 40–64 days earlier, while CmNRRa‐overexpressing plants exhibited a delayed flowering phenotype. These results revealed a negative effect of CmNRRa on flowering time modulation. Alteration of CmNRRa expression levels might be an effective means of controlling flowering time in Chrysanthemum. These results possess potential application in molecular breeding of chrysanthemums that production year‐round, and may improve commercial chrysanthemum production in the flower industry.     

Evaluation of Tip Culture, Thermotherapy and Chemotherapy for Elimination of Peanut Mottle Virus from Arachis hypogaea

Peanut (Arachis hypogaea L.) plants (cvs Florunner and Pronto) were inoculated at the two-leaf stage with peanut mottle virus (PMV) to obtain PMV-infected plants. Shoot-tips from plants grown in the glasshouse (27°C) or from plants maintained at 35°C were used for tip culture. In some experiments ribavirin was added to the culture medium at 5 mg/1, 10 mg/1, 15 mg/1 or 20 mg/1. Plants regenerated from meristems or shoot-tips taken from virus-infected plants were not virus-free. After 45 days at 35°C, foliar tissue of 93 % of Florunner and 95 % of Pronto plants tested negative for PMV by enzyme-linked immunosorbent assay (ELISA). When shoot-tips from the plant that tested negative by ELISA were used for tip culture, no virus-free plants were obtained. No virus-free plants were obtained from tips cultured on medium supplemnted with ribavirin. However, when tip culture, thermotherapy and chemotherapy were combined; 80 % of Florunner and 100 % of Pronto plants were found negative for PMV.     

3种菊花病毒/类病毒实时荧光定量RT-PCR检测体系的构建与准确性评价

菊花容易受到病毒感染而造成品质下降,目前国内对菊花病毒的检测主要根据外观表现或者定性PCR检测,无法准确判定病毒载量。为构建一种可同时用于检测菊花B病毒(Chrysanthemum virus B,CVB)、番茄不孕病毒(Tomato aspermy virus,TAV)和菊花褪绿斑驳类病毒(Chrysanthemum chloritic mottle viroid,CChMVd)的实时荧光定量RT-PCR检测方法,本研究分别以保守区域作为靶标设计相应的引物探针,通过优化扩增体系中CVB、TAV、CChMVd 3种病毒/类病毒探针浓度、引物浓度、Mg²⁺浓度、dNTPs浓度,摸索扩增程序中反转录时间、退火温度和扩增循环数,构建了一种可同时用于CVB、TAV、CChMVd的3重实时荧光定量RT-PCR检测体系,优化后的扩扩增体系中CVB、TAV和CChMVd的探针浓度分别为100 nmol/L、120 nmol/L和80 nmol/L,引物浓度分别为200 nmol/L、240 nmol/L和160 nmol/L,Mg²⁺浓度为3.0 mmol/L;dNTPs浓度200μmol/L;最适反转录时间为25 min,退火温度为60℃,循环数为40。敏感性实验结果表明,该反应体系对3种病毒/类病毒的敏感性为1.0×¹⁰3拷贝/mL,敏感性好;定量线性范围为1.0×¹⁰3拷贝/mL~1.0×¹⁰10拷贝/mL,线性范围宽;特异性好,对菊花矮化类病毒、烟草花叶病毒和黄瓜花叶病毒核酸检测结果为阴性;对1.0×¹⁰4拷贝/mL的低浓度参考品平行检测10次,定量结果lg值偏差(CV%)为4.81%,重复性好。在南京农业大学"中国菊花种质资源保存中心"基地随机选择菊花20株进行本研究试剂检测,检出6例CVB病毒株和4例TAV病毒株,其病毒载量为2.5×¹⁰4拷贝/mL~5.5×¹⁰7拷贝/mL,随机选择1株CVB病毒株定量PCR,产物进行TA克隆后经测序与NCBI Blast比对,其与MH678704.1的同源性为100%。因此,本研究建立了一种能同时检测CVB、TAV、CChMVd 3种菊花常见病毒/类病毒的灵敏、快速、可定量的检测方法。     

Virus infections reduce in vitro multiplication of ‘Malling Landmark’ raspberry

Summary Virus-infected plants are often symptomless and may be inadvertently used as explant sources in tissue culture research. Our objective was to determine the effect of virus infection on micropropagation. We studied the effects of single and multiple infections of three common raspberry viruses on the in vitro culture of ‘Malling Landmark’ red raspberry (Rubus idaeus L.). Virus-infected reaspberry plants were produced by leaf-graft inoculation from known-infected plants onto virus-free ‘Malling Landmark’. Single-virus source plants were infected with either tobacco streak ilarvirus (TSV), tomato ringspot nepovirus (TomRSV), or raspberry bushy dwarf idaeovirus (RBDV) and were free of other viruses as determined by enzyme-linked immunosorbent assay (ELISA) and bioassay. Virus-free, single, and multiple virus-infected ‘malling Landmark’ explants were initiated into culture and multiplied on Anderson's medium with 8.9 μM N⁶-benzyladenine (BA). At the end of the multiplication tests, ELISA reconfirmed virus infections. In vitro multiplication of ‘Malling Landmark’ was significantly reduced by multiple infections, and multiplication of plants infected with all three viruses (RBDV+TomRSV+TSV) was less than half that of virus free cultures. Shoot height and morphology of in vitro cultures were not influenced by virus infection. The greenhouse stock plant with the three-virus infection was stunted and yellow compared to the control and the other infected plants. Part of a thesis submitted by C.-W.V.T. in partial fulfilment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

In vitro micrografting for production of Indian citrus ringspot virus (ICRSV)-free plants of kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora)

Production of Indian citrus ringspot virus (ICRSV)-free plants from an infected plant of kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora) is reported. The shoot apices of different sizes (0.2–1.0 mm) excised from the ICRSV-infected plant were micrografted onto decapitated rootstock seedlings of rough lemon (C. jambhiri). Micrograft survival depended on the size of shoot apex and the sucrose concentration of the culture medium. Increase in scion size from 0.2 to 0.7 mm resulted in an increase in micrografting success rate from 30.55 to 51.88%. Further, micrograft survival obtained with 0.2 mm was improved from 30.55 to 38.88% by increasing sucrose concentration in the culture media from 5 to 7.5%. The micrografted plants were tested for ICRSV using ELISA and RT-PCR. All plants raised from 0.2-mm scion were found negative with both ELISA and RT-PCR whereas only 20% of the ELISA negative plants raised from 0.3-mm scion were found negative for ICRSV with RT-PCR. The outcome of this research is the successful establishment, acclimatization and virus testing of micrografted plants.     

Elimination of virus and rapid propagation of disease-free sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. cultivar NCo376) using apical meristem culture

The use of apical meristem culture for simultaneous virus elimination and shoot proliferation in sugarcane was assessed. Virus-free plants were propagated from Sugarcane mosaic virus and Sugarcane yellow leaf virus-infected material of the South African commercial cultivar, NCo376. A combination of thermotherapy by hot water treatment of stem sections (nodes) and subsequent germination of vegetative buds at 40°C and optimal meristem size were key factors for the production of virus-free plants. Only meristems of 2 mm in length or of a smaller size (but >0.5 mm) resulted in virus-free sugarcane. Shoot induction and proliferation via direct organogenesis were achieved on Murashige and Skoog nutrient medium supplemented with 0.1 mg l⁻¹ 6-benzyladenine and 0.015 mg l⁻¹ 6-furfurylaminopurine (KIN). The established protocol provides for the rapid proliferation of virus-free shoots from infected sugarcane plants and approximately 1,300 shoots were propagated from a single 2 mm meristem in 11 weeks. Plants remained virus-free when tested 12 months later.     

Production of herbicide-resistant transgenic sweet potato plants through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> method

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l⁻¹ PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing 900 mg l⁻¹ of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide Basta.     

Rates of cell division in the young prefloral shoot apex ofChrysanthemum segetum L.

Summary The rate of cell division was determined by the colchicine induced metaphase-accumulation technique in the young prefloral shoot apex of the quantitative long-day plantChrysanthemum segetum L. growing under conditions favourable to flowering (16-hour photoperiod; 124Em^–2s^–1; 22 °C). Cell cycle duration was evaluated in relation to the location of the cells in the intact apex. The cell cycle durations were 53.5 hours, 47.4 hours, and 97.7 hours in the axial, lateral and subapical central cells respectively. Compared with previous results, these data give evidence of the major role played by the early increase in cell division rate of axial cells in the new pattern of the prefloral shoot apex at its initial stage of development. By comparison with the vegetative shoot apex, the cell cycle duration was preferentially shortened in the axial zone; it was only slightly altered in the lateral zone while it was lengthened in the vacuolating subapical central cells. In the three zones within the prefloral shoot apex, the duration of mitosis was constant (3.2 to 3.3 hours) and the same as in the vegetative shoot apex.     

Micrografting of ASBVd-infected Avocado (<Emphasis Type="Italic">Persea americana</Emphasis>) plants

Shoot tips (meristem plus 2–3 leaf primordia) from in vitro-germinated avocado seedlings of 2 ASBVd-infected cultivars were micrografted in vitro onto decapitated seedlings from 2 ASBVd-free cultivars, and plants were recovered. Shoot tips consisted of two different sizes, i.e., <0.5 mm long and >0.5 mm but <1 mm long. The recovered plants were indexed for ASBVd using RT-PCR. More plants (58.8%) were recovered from scions >0.5 mm than from those that were <0.5 mm (10.3%). RT-PCR demonstrated that ASBVd replicated in all micrografts from infected sources irrespective of the scion size, while no ASBVd was detected in micrografts from plants that tested negative. ASBVd therefore cannot be eliminated by in vitro micrografting.     

The effects of dsRNA mycoviruses on growth and murine virulence of Aspergillus fumigatus

Some isolates of the opportunistic human pathogenic fungus Aspergillus fumigatus are known to be infected with mycoviruses. The dsRNA genomes of two of these mycoviruses, which include a chrysovirus and a partitivirus, have been completely sequenced and an RT-PCR assay for the viruses has been developed. Through curing virus-infected A. fumigatus isolates by cycloheximide treatment and transfecting virus-free isolates with purified virus, as checked by RT-PCR, isogenic virus-free and virus-infected lines of the fungus were generated whose phenotypes and growth have been directly compared. Mycovirus infection of A. fumigatus with either the chrysovirus or the partitivirus resulted in significant aberrant phenotypic alterations and attenuation of growth of the fungus but had no effect on susceptibility to common antifungals. Chrysovirus infection of A. fumigatus caused no significant alterations to murine pathogenicity.