Efficacy of chemotherapy and thermotherapy in elimination of East African cassava mosaic virus from Tanzanian cassava landrace

Cassava mosaic disease is caused by cassava mosaic begomoviruses (CMBs) and can result in crop losses up to 100% in cassava (Manihot esculenta) in Tanzania. We investigated the efficacy of chemotherapy and thermotherapy for elimination of East African cassava mosaic virus (EACMV) of Tanzanian cassava. In vitro plantlets from EACMV‐infected plants obtained from coastal Tanzania were established in the greenhouse. Leaves were sampled from the plants and tested to confirm the presence of EACMV. Plantlets of plants positive for EACMV were initiated in Murashige and Skoog (MS) medium. On the second subculture, they were subjected into chemical treatment in the medium containing salicylic acid (0, 10, 20, 30 and 40 mg/L) and ribavirin (0, 5, 10, 15 and 20 mg/L). In the second experiment, EACMV‐infected plantlets were subjected to temperatures between 35 and 40°C with 28°C as the control. After 42 days of growth, DNA was extracted from plant leaves and PCR amplification was performed using EACMV specific primers. It was found that plant survival decreased with increasing levels of both salicylic acid and ribavirin concentrations. In general, plants treated with salicylic acid exhibited a lower plant survival % than those treated with ribavirin. However, the percentage of virus‐free plants increased with an increase in the concentration of both ribavirin and salicylic acid. The most effective concentrations were 20 mg/L of ribavirin and 30 mg/L of salicylic acid; these resulted in 85.0% and 88.9% virus‐free plantlets, respectively. With regard to thermotherapy, 35°C resulted in 79.5% virus‐free plantlets compared to 69.5% at 40°C. Based on virus elimination, ribavirin at 20 mg/L, salicylic acid 30 mg/L and thermotherapy at 35°C are recommended for production of EACMV free cassava plantlets from infected cassava landraces.     

In vitro development of plantlets from axillary buds of Acacia auriculiformis — a leguminous tree

Multiple shoots have developed from axillary buds excised from in vitro grown seedlings of Acacia auriculiformis on Gamborg's (B5) basal medium supplemented with coconut milk (5 or 10%) and benzylaminopurine (10^-6M). These shoots, if transferred individually to indole-3-acetic acid (10^-7M) or naphthaleneacetic acid (10^-6 or 10^-7M) augmented B5 medium, produced roots at their base.Abbreviations B5 Gamborg's basal medium (BM) - CM coconut milk - BA 6-benzylaminopurine - Kn kinetin - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Micropropagation by explants ofGracilaria chilensisBird, McLachlan and Oliveira

Regeneration studies were carried out on four morphotypes ofGracilaria chilensisfound on the coast of Chile (36–41°S). Vegetative explants were obtained from sections of apical and medial origin and were cultured in five media: filtered autoclaved seawater (FAS), Provasoli enriched seawater (PES), PES + indole-3-acetic acid (PIAA), PES + kinetin (PK) and PK + IAA (PKIAA). Mature female gametophyte and tetrasporophyte explants were obtained from sections of medial origin and were cultured in FAS and PES. Bud differentiation and/or callus formation were the morphogenetic responses to the wounding of the explants in all culture media. Plantlet regeneration was obtained from excised buds and calluses cultured separately.     

The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets

Large amounts of anti-inflammatory activity are present in extractsprepared from Eucomis plants. Extracts prepared from in vitroplantlets grown on a modified Murashige and Skoog medium supplementedwith 1 mg &ell^–1 NAA and 1 mg &ell^–1 BA, were tested intwo cyclooxygenase assays (COX-1 and COX-2). Ethanol extracts showedhigh levels of COX-1 and COX-2 inhibitory activity, with a COX-2/COX-1inhibition ratio of 1.1. Further experimental work aimed to determine thefactors affecting the accumulation of anti-inflammatory compounds inin vitro plantlets. High concentrations of sucrose (40 g &a,p;ell^–1) inthe culture medium significantly increased the number of shoots initiated,but had no effect on the subsequent anti-inflammatory activity. Lowconcentrations of sucrose (10 g &ell^–1) led to a significantdecrease in COX-1 inhibition. Changig the amount of nitrogen in the medium(but not the ratio of nitrate to ammonium ions) had no significant effect onthe COX-1 inhibitory activity of the extracts.     

Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163

Aims:  To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro -grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro -grown potato plantlets.
Methods and Results:  The inoculation of in vitro -grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp . using phylogenetic 16S rDNA approach . Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants.
Conclusion:  Methylobacterium sp. resides in unculturable state within tissues of in vitro -grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163.
Significance and Impact of the Study:  In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.     

怀山药种质资源的包埋玻璃化超低温保存与植株再生

通过对怀山药（Dioscorea opposita）种质资源的包埋玻璃化超低温保存与植株再生进行研究,结果表明：将低温下锻炼7天的怀山药试管苗带芽茎段放入预培养基中,低温下培养3天,然后在室温下用3%的海藻酸钠和0.5mol·L-1CaCl2包埋,包埋珠用MS＋0.2mol·L-1蔗糖＋2mol·L-1甘油＋50g·L-1二甲基亚砜在0°C下装载60分钟,用30%甘油＋15%乙二醇＋10%二甲基亚砜＋15%聚乙二醇＋0.4mol·L-1蔗糖在0°C下脱水60分钟,迅速投入液氮,24小时后立即用40°C水浴快速化冻,再用MS＋0.5mol·L-1蔗糖溶液洗涤2次,转入再生培养基中培养,可获得再生植株。再生植株成活率因基因型而异,铁棍山药、太谷山药、怀庆1号山药和B号山药的成活率分别为64.29%、49.21%、13.11%和39.81%。     

An efficient protocol for genetic transformation and shoot regeneration of turmeric (Curcuma longa L.) via particle bombardment

Turmeric (Curcuma longa L.) is an important spice crop plant that is sterile and cannot be improved by conventional breeding. An efficient method for stable transformation for turmeric, C. longa L., was developed using particle bombardment. Callus cultures initiated from shoots were bombarded with gold particles coated with plasmid pAHC25 containing the bar and gusA genes each driven by the maize ubiquitin promoter. Transformants were selected on medium containing glufosinate. Transgenic lines were established on selection medium from 50% of the bombarded calluses. Transgenic shoots regenerated from these were multiplied and stably transformed plantlets were produced. Polymerase chain reaction (PCR) and histochemical GUS assay confirmed the stable transformation. Transformed plantlets were resistant to glufosinate.     

用细胞学方法研究番木瓜组培苗的遗传稳定性

利用细胞学和形态学方法研究组织培养技术生产的番木瓜种苗的遗传稳定性。通过对番木瓜（2n=18）主栽品种蔬罗1号1～38代组培苗体细胞的染色体数的初步观察和统计,发现1～32代苗所观察的体细胞染色体全部为2n=18,大田种植后表型与亲代相比未发生明显的变异。但是在第33,36,37代发现了染色体非整倍性的细胞,田间种植后与亲代相比挂果率和抗病性下降。这一研究结果为番木瓜组培苗的继代培养和规模化生产提供了有价值的理论依据。