A simple procedure of Gossypium meristem shoot tip culture

In order to develop transgenic plants via the biolistic gun method regenerable embryogenic tissues are required. Meristem shoot tips of 19 cultivars of cotton were cultured on several media formulations and assessed for shoot and root development. The best shoot development was observed on media containing 0.46 mM kinetin while rooting was observed on media containing 2.68 mM NAA and 0.46 mM kinetin. No intervarietal variability was observed. A complete protocol was developed from meristem tip culture to field transfer. This methodology is simple and replaces the existing protocols for meristem tip culture of cotton. This revised version was published online in June 2006 with corrections to the Cover Date.     

Eradication of cassava mosaic disease from Nigerian cassava clones by meristem-tip culture

Methods previously established for the propagation of cassava plants free from cassava mosaic disease have been applied to Nigerian clones. Meristem tips from diseased plants subjected to heat treatment for not less than 30 days at 35°–38°C were cultured on modified Murashige-Skoog medium. Concentration ranges of benzyladenine in combination with α-naphthalene acetic acid and gibberellic acid were investigated and, at optimal levels, 36% of the meristems regenerated. Regenerants, with callus and shoots only, were rooted with 80% efficiency by sub-culturing following a dip in a hormone rooting powder. All plants raised from heat-treated meristems were free of the disease as judged by visual inspection of the leaves, rooted explants and assay for the suspected pathogenic agent of the disease.     

Elimination of sugarcane grassy shoot disease through apical meristem culture

Elimination of sugarcane grassy shoot disease (SGSD) through apical meristem culture technique for producing clean planting material of sugarcane has been attempted in the present study. The results showed that meristems length of 2 and 3 mm were free from the SGSD pathogen at higher frequency than larger meristem length of 4 mm. However, the frequency of survival of explants during initiation of shoot cultures was higher in larger meristems (60%) in comparison to smaller ones (40%). The micropropagated plantlets raised from meristem culture were confirmed for disease-free by nested polymerase chain reaction (PCR) analysis at monthly interval up to 6 months. This is the first report on the elimination of SGSD phytoplasma through meristem culture in India.     

Micropropagation of Abelmoschus esculentus L. (Moench.) for disease free plantlets through meristem culture

Protocol was established for mass in vitro propagation of okra using meristem culture. Meristems (0.3–0.5 mm in size) were isolated from shoot tips of three-week old in vitro grown seedlings. Isolated meristems were established rapidly in MS liquid medium containing 1.0 mg/l of BAP. For shoot development from primarily established meristem, semisolid MS medium having the same concentration of BAP was found to be the most effective. Rapid shoot multiplication of mericlone was achieved from node cutting cultured in 1.0 mg/l plus 0.5 mg/l GA₃, and a maximum of nine shoots were found from each node. Effective root development from the developed plantlets was successful in 1.0 mg/l IBA. More than 75% of the micropropagated mericlones plantlets were successfully acclimatised in soil up to maturity and found to be healthy.     

The elimination of clover diseases by shoot tip culture

Shoot tip culture was used to eliminate white clover mosaic virus (WCMV) and red clover necrotic mosaic virus (RCNMV) from red clover, and clover phyllody disease (CP) and clover red leaf disease (CRL) from white clover. Shoot tips up to 2.4 mm (in some cases 3 mm) could regenerate plants free from the pathogens, but the efficiency of elimination, at least for WCMV and CRL, tended to decrease with increasing shoot tip size. The efficiency of plant regeneration from shoot tips generally improved with increasing tip size.     

Rapid clonal multiplication of Angelonia salicariefolia through tissue culture

Nodal explants of Angelonia salicariefolia were cultured on MS basal medium and induced to form shoots when supplemented with either Kn (1.0 mg/l) or BAP (1.0 mg/l). Rooted shoots were formed in response to Kn+NAA (1.0 mg/l+0.5 mg/l). Subcultures of the shoots of these cultures grown on the same medium supplemented with 0.5 mg/l of NAA, IAA or IBA, together with lowered concentrations of inorganic salts, induced root formation in 20–30 days. Up to 18×10³ plants were produced from one plant in less than a month. Successful transfer of regenerants into soil has been accomplished.     

Elimination of cymbidium mosaic virus and odontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy

Most commercially cultivated orchid plants are generally infected with cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV). Two methods were used in order to generate virus-free plants: meristem culture and thin section culture with chemotherapy. Meristems (0.10 mm to 1.00 mm) were excised from infected axillary shoots of an infected monopodial orchid hybrid (Mokara Char Kuan ‘Pink’) and cultured in modified Vacin and Went medium. Only larger meristem explants survived and the regenerated plantlets remained virus-infected. In contrast, high percentages of virus-free plantlets were obtained from thin section cultures of infected plantlets and protocorm-like bodies with ribavirin treatment. Interestingly, regenerants from thin section cultures without ribavirin treatment were also found to be free from CyMV and ORSV. All plantlets were tested by enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR).     

Elimination of ornithogalum mosaic virus in the Ornithogalum cv. Rogel through meristem tip culture and chemotherapy

Virus-indexed Ornithogalum ev. Rojel plants were produced by eliminating Ornithogalum mosaic virus (OMV) through meristem-tip culture. The best plantlet regeneration was obtained from meristems derived from adventitious buds which developed on leaf explants taken from mother plants at the flowering stage. Acyclovir had no effect as an anti-viral compound on plantlet regeneration or virus elimination. Adenine arabinoside retarded plantlet development at concentrations of 10 mg l^-1 and higher, while 5.0 mg l^-1 suppressed the virus concentration beneath a detectable level in young plants. All the mature plants, however, tested positively for the presence of OMV.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - OMV Ornithogalum mosaic virus     

杜衡离体繁殖的研究

以马兜铃科植物杜衡为研究材料,试图通过茎尖培养来获得愈伤组织,继而诱导愈伤组织分化形成杜衡小植株。试验证明：杜衡茎尖在MS基本培养基附加6-BA 0.6mg/l和NAA0.1mg/;这一激素组合上可诱导茎尖基部形成愈伤组织;将愈伤组织分割转接到附加6-BA0.2mg/l和NAA0.01mg/l 的MS基本幅度基上可使愈伤组织分化形成小芽;分化形成的芽置于1/8-1/4MS(大量元素减少,其余成分不变)附加6-BA0.1mg/l和GA1mg/l的培养基上,可促使芽的正常生长;新生芽转接在1/4MS附加IBA0.5mg/l培养基上促使其生根。     

杭白菊茎尖组织培养及试管苗繁殖技术研究

采用茎尖组织培养技术,建立了杭白菊中大洋菊(Chrysanthemum morifolium Ramat.)的无菌试管苗体系.通过基本培养基和激素配比实验,筛选出杭白菊试管苗快速繁殖的最佳培养基组成.结果表明:最适宜的外植体为直径0.3 mm的茎尖;诱导丛生芽的最适培养基为:MS 6-BA 0.1 mg*L-1 IAA 0.02 mg*L-1;诱导试管苗生根的最适培养基为:1/2MS IAA 0.7 mg*L-1.用电子显微镜进行病毒检测后,筛选出2个脱病毒株系,脱病毒试管苗可作为今后提供优质种苗的种源.     

玉米离体根尖的多层滤纸床液体静止培养方法

设计建立了适于玉米根尖离体培养的多层滤纸床液体静止培养方法,培养的适宜体系为：1／4MS大量元素改良＋1／2MS微量元素＋IBA0.1-0.3mg/L,黑暗培养。该方法避免了传统液体培养通气状况不良的问题,玉米根的生长速度可达到1－2cm/d,分支和生长正常。该方法在控制条件下快速繁殖根系,成本低廉,简便易行,是根系发育和生理研究的理想实验体系。     

Rejuvenation of a 100-year-old Sequoiadendron giganteum through in vitro meristem culture. I. Organogenic and morphological arguments

True-to-type cloning of mature trees, especially when they do not sprout from their base, remains problematic. Special attention is focused on the shoot apical meristem, since it is an obvious choice for vegetative propagation. In Sequoiadendron giganteum a meristem removed during budbreak from a 100-year-old tree regenerated a truly rejuvenated line that exhibited the same juvenile characters as the juvenile clone used as control, especially in regard to morphological traits and organogenic capacity, and as manifested by the ability to produce adventitious roots in vitro. This rejuvenation has been maintained for 3 years in both in vitro and ex vitro conditions. This result is discussed in terms of inhibitory correlative systems acting within the donor tree in situ, especially as concerns miniaturization of the explant.     

Molecular characterisation of Onion yellow dwarf virus (OYDV) infecting garlic (Allium sativum L.) in Israel: Thermotherapy inhibits virus elimination by meristem tip culture

Garlic (cv. Shani) was tested using single step RT‐PCR and digoxygenin (DIG) labelled dot‐blot for a number of viruses. Following sequence analysis it was shown that at least three different polymorphs of the potyvirus Onion yellow dwarf virus (OYDV) infect the same plant simultaneously, together with the potyvirus Leek yellow stripe virus (LYSV), the carlavirus Garlic common latent virus (GCLV) and a multitude of allexiviruses (Shallot virus X (ShVX) related viruses]. Several garlic plants free of all the viruses tested were obtained through meristem‐tip culture. Plants infected with single viruses or with different combinations of viruses were similarly obtained. Meristem‐tip culture was confirmed as a satisfactory method of virus eradication, while thermotherapy treatment given to mother plantlets before meristem excision was found to specifically antagonise OYDV eradication. This work uses molecular methods for the first time to examine the effectiveness of meristem‐tip culture for the eradication of multiple viruses from garlic.     

Loss of CMD2‐mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis

Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer‐preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)‐mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild‐type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2‐type varieties TME 3 and TME 7, but the CMD1‐type cultivar TMS 30572 and the CMD3‐type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2‐mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field‐level resistance in CMD2‐type cultivars presently grown by farmers in East Africa, where CMD pressure is high.     

Reduced glutathione promotes callus growth and shoot development in a shoot tip culture of apple root stock M26

Reduced glutathione (GSH) was applied to prevent browning in shoot tip explants of apple (Malus pumila Mill.). Development was compared between shoot tips treated either by (a) dipping into 0.1 mm GSH solution prior to culture (dip treatment), (b) dipping into the GSH solution and transferring to a medium containing 0.1 mm GSH (dip-and-add treatment), or (c) without dipping or culturing with GSH (control). In the dip treatment, 100% of shoot tips developed into normal shoots after 120 days, while the results with the dip-and-add treatment and control were 50 and 40%, respectively. The results show that application of antioxidant GSH in the initial phase of culture promoted the normal development of shoot tips. Received: 10 October 1997 / Revision received: 12 January 1998 / Accepted: 24 January 1998     

Micropropagation of black pepper (Piper nigrum Linn.) through shoot tip cultures

Summary The morphogenetic potential of shoot tip explants of black pepper (Piper nigrum) was investigated and an effective multiple-shoot propagation method is described. Various combinations of media, growth regulators and sterilization treatments were compared. Problems with establishment in tissue culture sometimes occurred, probably caused by endogenous pathogens associated with tissue exudates. The best establishment and proliferation of shoot tip explants was obtained on MS medium containing 1.5 mg l^–1 BAP alone; subsequent growth and development of lateral branches was best on media containing 1.5 mg l^–1 BAP plus 3.0 mg l^–1 IBA. Adenine sulphate inhibited the number of explants showing regeneration but increased the number of shoot buds per regenerating explant. Shoots were rooted on a 50% strength medium containing 1mg l^–1 NAA.Abbreviations AdSO₄ adenine hemisulphate - BAP 6-benzylaminopurine - IBA indole-3-butyric acid - NAA napthaleneacetic acid     

Effect of silver nitrate on multiple shoot formation of Virginia-type peanut through shoot tip culture

Summary A protocol for micropropagation of Virginia-type peanut plants, an ancient crop of the New World, is reported. This study was conducted to explore the effect of silver nitrate (AgNO₃), alone or in combination with growth regulators, on multiple shoot formation from shoot tip culture. Incorporation of AgNO₃ into the medium, without growth regulators, induced regeneration of the explants (which did not develop at all in the AgNO₃-free medium), and stimulated the emergence of axillary shoots. When AgNO₃ was added in combination with cytokinins and α-naphthaleneacetic acid (NAA), maximum average shoot number per regenerating explant was recorded (6.3) in Murashige and Skoog (MS) medium containing 33 μM 6-benzyladenine, 5.3 μM NAA, and 23.54 μM AgNO₃. Moreover, AgNO₃ showed a positive and marked effect on both shoot elongation and the reduction of callus proliferation from the basal ends of shoot tips. Following a period of elongation, the shoots were rooted in hormone-free Ms medium, showing no residual effects due to the long-term culture in AgNO₃-containing media. Acclimatization was easily obtained after plantlets were transferred to pots under greenhouse conditions, with 90% survival.     

RAPD analysis of a variant of banana (Musa sp.) cv. grande naine and its propagation via shoot tip culture

Summary A morphological variant obtained from in vitro corm-derived plants of banana (Musa sp.) cv. Grande Naine (AAA) was evaluated up to harvest and the genetic basis of variation was confirmed by the random amplified polymorphic DNA (RAPD). The corms formed during the multiplication phase of shoot tip-derived cultures of the cv. Grande Naine grown on Murashige and Skoog (MS) medium enriched with 13.3 μM N ⁶-benzyladenine (BA) developed numerous morphological variants after transfer to MS medium with 6.66 μM BA. The variant designated as CUDBT-B1, with distinct morphological features, was further evaluated. The morphological features of CUDBT-B1 were variegated leaf, pseudostem, bracts, ovary of the male flower and fruits, reduced height, decreased lamina length and breadth, and early flowering. These features were also manifested in the second-cycle progeny of CUDBT-B1. RAPD assay showed a marker DNA band of 1650 bp, and differential band intensity between the CUDBT-B1 and normal clone. CUDBT-B1 was multiplied using shoot tip culture, and the shoots were rooted on half-strength MS medium supplemented with 2.69 μM α-naphthaleneacetic acid. All plantlets showed variegated leaves under field conditions.     

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