Application of a novel disposable film culture system to photoautotrophic micropropagation of <Emphasis Type="Italic">Eucalyptus uro-grandis (Urophylia x grandis)</Emphasis>

Summary To overcome various disadvantages of conventional culture vessls for plant micropropagation, we previously developed the photoautotrophic micropropagation technique, with special mention for the first practical film culture system, the ‘Miracle Pack’ (MP), which was made of fluorocarbon polymer film (Neoflo^? PFA film) and supported by a polycarbonate frame. While the PFA film has superior thermal stability, high light transmittance and high gas permeability, making the MP system (MP-PFA) superior to conventional culture vessels for the micropropagation of various plant species, its high cost is a disadvantage. In this study, a possible alternative of lower-cost OTP^? film made of TPX (4-methyl-1-pentane polymer) and CPP (a polypropylene), which possesses similar characteristics to PFA film, is evaluated to develop a novel disposable film culture vesel, termed ‘Vitron’, for culturing Eucalyptus (urophylla x grandis), plantlets. The three film culture systems, MP-PFA, MP-OTP (MP with OTP film), and Vitron, were placed under CO₂ enrichment, low photosynthetic photon flux density (PPFD; 45 μmol m⁻² s⁻¹), and sugar-free medium, using phenol resin foam (Oasis^?) as a substrate. In vitro and ex vitro growth and development of Eucalyptus shoots from the four-leaf stage to the rooting stage were compared for all three culture systems. The effects of the duration and concentration of CO₂ enrichments on in vitro growth of Eucalyptus cultured in the Vitron film system were also examined. The best growth and quality of Eucalyptus plantlets was obtained for the Vitron vessel placed in 3000 ppm CO₂ enrichment for 24 hours per day at low PPFD with sugar-free liquid medium and Oasis as substate. Results of this study suggest that the novel Vitron culture system is suitable for the photoautotrophic micropropagation of Eucalyptus. These authors contributed equally to the research results.     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.     

Micropropagation of ornamental <Emphasis Type="Italic">Prunus</Emphasis> spp. and GF305 peach,a <Emphasis Type="Italic">Prunus</Emphasis> viral indicator

A micropropagation approach was developed for nine ornamental Prunus species, P. americana, P. cistena, P. glandulosa, P. serrulata ‘Kwanzan’, P. laurocerasus, P. sargentii, P. tomentosa, P. triloba, P. virginiana ‘Schubert’, commercially important in North America, and GF305 peach, commonly used for Prunus virus indexing. The micropropagation cycle based on proliferation of vegetative tissues includes establishment of tissue culture through introduction of shoot meristems in vitro, shoot proliferation, root induction and plant acclimatization steps and can be completed in 5 months. A meristem sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation in ornamental Prunus was obtained through the use of 1 mg l⁻¹ 6-benzyladenine. For GF305 peach, alteration in the sugar composition, fructose instead of sucrose, and addition of 1 mg l⁻¹ ferulic acid had a significant impact on the shoot proliferation rate and maintenance of long-term in vitro culture. Rooting and plant acclimatization conditions were improved using a two-step protocol with a 4-day root induction in indole-3-butiric acid (IBA)-containing media with consequent 3-week root elongation in IBA-free media. One-month incubation of rooted shoots in a vermiculite-based medium resulted in additional shoot and root growth and provided better acclimatization and plant recovery. The micropropagation approach can be used for maintenance of the clonal properties for Prunus spp. as well as a protocol to support meristem therapy against viral infection.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of four lupin species

The complete protocols for long-term micropropagation of some cultivars of four lupin species: Lupinus luteus, L. albus, L. angustifolius and L. mutabilis were elaborated. The shoots were regenerated in vitro via induction of axillary buds development. Plantlets were multiplicated on lowered salts MS-derived media containing BAP in diverse and generally low concentrations. Significant differences in regeneration capacity between species and cultivars were observed. The highest multiplication ratio revealed L. mutabilis and L. luteus. Regenerated shoots were rooted in vitro on low-salts MS-derived media with B5 vitamins. Media were supplemented with different auxins that affected roots formation of particular species and cultivars. Rooting ability of regenerated shoots decreased rapidly through in vitro culture. For that reason, grafting was applied as an alternative method of transfer of shoots to in vivo conditions. This method turned out to be successful for the majority of studied species and cultivars. Complete rooted or grafted plantlets were cultivated in pots with perlit in greenhouse. An erratum to this article is available at .     

Adjusting for Nonignorable Missingness When Estimating Generalized Additive Models

Generalized additive models (GAMs) have been widely used for flexible modeling of various types of outcomes. When the outcome in a GAM is subject to missing, practical analyses often assume that missingness is missing at random (MAR). This assumption can be of suspicion when the missingness is not by design. Evaluating the potential effects of alternative nonignorable missing data mechanism on the MAR inference from a GAM can be important but often challenging due to the complicatedness of alternative nonignorable models. We apply the index approach to local sensitivity (Troxel, Ma, and Heitjan 2004 (2004). Statistica Sinica 14 , 1221–1237) to evaluate the potential changes of the GAM estimates in the neighborhood of the MAR model. The approach avoids fitting any complicated nonignorable GAM. Only MAR estimates are required to calculate the resulting sensitivity index and adjust the GAM estimates to account for nonignorable missingness. Thus the proposed approach is considerably simpler to conduct, as compared with the alternative methods. The simulation study shows that the index provides valid assessment of the local sensitivity of the GAM estimates to nonignorable missingness. We then illustrate the method using a rheumatoid arthritis clinical trial data set.     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

In what situations isin vitro culture appropriate to plant conservations?

Variousin vitro techniques are available for plant propagation, including seed germination, micropropagation, meristem culture and callus culture. The role of these techniques in the conservation of endangered plants is discussed, using examples drawn from the work of the Micropropagation Unit at Kew.     

Ornamental Chrysanthemums: Improvement by Biotechnology

The in vitro tissue culture and micropropagation of chrysanthemums, important floricultural (cut-flower) and ornamental (pot and garden) plants, have been well studied. An increase in genetic transformation studies aimed at improving aesthetic and growth characteristics of the plants has been hampered by low transformation efficiencies and genotype dependence of protocols. As a result chrysanthemum regeneration studies have once again emerged as an essential complement of transformation studies. This review highlights the impact that biotechnology has had on the improvement of chrysanthemum in vitro cell, tissue and organ culture, micropropagation and transformation.     

Factors affecting the efficiency of micropropagation from lateral buds and shoot tips of <Emphasis Type="Italic">Rubus</Emphasis>

Factors affecting micropropagation efficiency of 32 selections of Rubus, including the pre-treatment and initiation culture stages, were investigated. Chilling at 4°C for 6 weeks as a pre-treatment significantly promoted in vitro initiation culture; up to 65% of initiation cultures post chilling were successful. The cytokinin 6-benzyladenine (BA) was the most effective of the three tested for promotion of multiple shoot development in culture, with an average of three to seven shoots or plantlets developed from each single node. Alternating the concentration of BA between 4.44 and 13.31 μM from sub-culture to sub-culture improved recalcitrant Rubus micropropagation, and reduced the problems associated with long-term culture in the presence of high concentrations of BA. Reduction of the strength of macro- and micro-elements in the basal medium from half to one-third, with addition of 0.49 μM indole-3-butyric acid and 0.05% activated charcoal, and placing the cultures under reduced light intensity (17 μmol m⁻² s⁻¹), was found to alleviate chlorosis and improve micropropagation with high quality Rubus plantlets. Response to in vitro culture differed greatly and consequently different methods of micropropagation were required by different genotypes. From these selections, three types of micropropagation including micro-cutting, micro-shoots and multi-shoots were observed, and their efficiency was characterized.     

Optimization of plantain (<Emphasis Type="Italic">Musa</Emphasis> AAB) micropropagation by temporary immersion system

The positive and reliable effect of temporary immersion systems on in vitroshoot proliferation was already proved for different plant genera and it is now presented as an alternative for plantain micropropagation. Some culture parameters affecting the efficiency of the twin flasks system or temporary immersion bioreactor (Escalona et al., 1999) were investigated. Three different cytokinins (benzyladenine, thidiazuron and meta-topolin) were added to the culture medium and meta-topolin at a concentration of 4.4 M was proved to be the most efficient. Successive subcultures (28 days per subculture) were performed on medium supplemented with meta-topolin, revealing a decrease in multiplication after the 6th subculture. Multiplication rate was not changed within the ranges of immersion times (4, 12 or 22 min) and frequencies (every 3, 5 or 7 h) tested. The size of the bioreactor (250, 1,000, 5,000 or 10,000 ml) and the volume of medium per inoculum (10, 20 or 30 ml) were also evaluated and appeared to have an influence on the multiplication. A proportion of 25–100 ml of headspace per inoculum and 30 ml of medium per inoculum resulted in a multiplication rate > 13 in 28 days.     

Use of sugarcane bagasse as an alternative low-cost support material during the rooting stage of apple micropropagation

Summary Studies were carried out to evaluate sugarcane bagasse as an alternative to agar for micropropagation of apple clones to reduce the cost of micropropagation and improve the quality of the propagules. Significant improvement in the in vitro rooting process, coupled with cost reduction, were obtained by the use of sugarcane bagasse as a substitute for the traditionally used agar-gelled medium. The tests were undertaken with micro-cuttings of the apple rootstock Marubakaido (Malus prunifolia Borkh.) using a rooting medium composed of half-strength Murashige and Skoog salts and vitamins, 3% (w/v) sucrose, and 0.49 μM indole-3-butyric acid. The plants grown on sugarcane bagasse yielded a 22% increase in root length, 20% increase in plant length, and 63% increase in the number of roots, compared with agar-grown micro-cuttings. Particle size of the sugarcane bagasse had a significant impact on all those parameters, and the best results were obtained with bagasse comprising particles smaller than 0.18 mm. The results demonstrated that the sugarcane bagasse could be used effectively as a substitute for agar during rooting of apple shoots.     

Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka

Summary Phytagel™ allowed the production of longer internodes, faster in vitro tuberization, and larger tubers in Solanum tuberosum L. cv. Baraka as compared to Difco Bacto-agar during both an 8-h photoperiod or in darkness. It also allowed a higher tuberization percentage in the dark. Only a 0.2% (wt/vol) Phytagel allowed optimal micropropagation and microtuberization under the photoperiod regime used. Water availability does not account for the observed differences in growth and tuberization between media containing the above gelling agents. In consequence, Phytagel appears as an advantageous alternative to agar for micropropagation and microtuberization.     

Plant regeneration from stem nodal segments of <Emphasis Type="Italic">Orthosiphon stamineus</Emphasis> benth., a medicinal plant with diuretic activity

Summary A micropropagation method for Orthosiphon stamineus, using stem nodal segments, has been developed. The highest number of regenerated shoots was obtained on Murashige and Skoog (MS) medium supplemented with 6.7 μM benzyladenine with the formation of an average of 6.1 shoots per explant over a period of 4 wk. The number of shoots increased with longer culture duration on proliferation medium. Multiple shoots which were maintained on the proliferation medium for 6 wk had the highest proliferation rate. Separation of multiple shoots and culturing in larger flasks significantly promoted the growth and formation of plantlets. All the in vitro plantlets survived when transferred to the field and showed no significant morphological differences from the mother plants.     

An introduction to predictive microbiology and the development and use of probability models withClostridium botulinum

Summary Traditionally, food microbiologists have relied on empirical studies to assess the microbiological safety of a particular food. However, these studies are time-consuming and, because only one or two inhibitory factors are usually dealt with, they are often of limited value. Today, the food industry is constantly developing new products with new formulations and alternative packaging strategies, resulting in a wide diversity of factors to be studied. It is therefore advantageous to develop mathematical models describing microbial growth which may be used to predict how changes in formulations or storage conditions may affect microbial growth. A brief overview of the basic concepts and steps of modeling procedures will be presented, along with some of the difficulties encountered therein. The safety of foods with respect toClostridium botulinum depends on the probability (P) of growth or of toxigenesis, andP has been the dependent variable in several models. The development of these probability models will be discussed.Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

Age and Physiological Condition of Donor Plants Affect in vitro Morphogenesis in Leaf Explants of Passiflora edulis f. flavicarpa

The regeneration potential of D.alata L. germplasm preserved in vitro was compared with the micropropagation of fresh material. Nodal cuttings were conserved for 9 months in different treatments based on D-571 culture medium modified, using several variable components (mannitol, benzylaminopurine and activated charcoal). Regeneration at 8 weeks, assessed by means of percentage of explant regenerating and the multiplication at 5 weeks through the shoot length and de novo bud count formation per explant were determined. The results showed high rates (100 and 98%) of explant regeneration and micropropagation from in vitro material maintained in D-571 medium with 1.5% of mannitol + 0.1 or 1 mg l^–1 of benzylaminopurine + 2 g l^–1 of activated charcoal, respectively.     

Micropropagation of <Emphasis Type="Italic">Scabiosa caucasica</Emphasis> Bieb. Cv. Caucasica blue

Summary Micropropagation of Scabiosa caucasica cv. Caucasica Blue was achieved by culturing, separating axillary and adventitious shoots, or node sectioning on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). The highest frequency of adventitious shoots regenerated from nodal or internodal explants and leaf blade (with or without petiole) appeared to occur on MS medium with 4.4 and 18 μM BA, respectively. Addition of 0.19 or 1.9 μM α-naphthaleneacetic acid to the BA-containing medium promoted callus formation and reduced shoot organogenesis. During micropropagation, shoot nodal explants derived from in vitro shoots cultured on MS medium supplemented with 4.4 μM BA yielded 8.9 shoots per explant within 40 d after culture initiation.     

Micropropagation and slow growth conservation of cardamom (<Emphasis Type="Italic">Elettaria cardamomum</Emphasis> Maton)

Cardamom (Elettaria cardamomum Maton) has great commercial value as a spice crop in India. A one-step protocol for direct regeneration of plants and in vitro conservation by slow growth method has been developed. A maximum of 6.5 shoots/culture were obtained in 2 mo or 15.1 shoots/culture in 4 mo on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) + 5 μM benzylaminopurine gelled with 0.7% agar (micropropagation medium). Rooting also occurred simultaneously on the same medium. Using one shoot tip or nodal explant, about 30,375 plants can be regenerated in a year on the micropropagation medium. In vitro conservation by slow growth method was achieved on 1/2 MS (major salts) + 5 μM BAP + 0.7% agar (conservation medium); about 70% of the cultures survived up to 18 mo at 25 ± 2°C. Successful regrowth of plants on micropropagation medium was obtained by culturing nodal explants excised from 18-mo-old conserved plants. Some 96% of the plants survived the hardening treatment and grew normally in a greenhouse. If 24 cultures are conserved on the conservation medium, it is possible to regenerate at least 750 plants by using explants derived from 70% of the surviving shoots and culturing the same in micropropagation medium for 4 mo. These plants may be used for planting or as a source of explants for the next conservation cycle. On the basis of 20 random amplified polymorphic DNA and 13 inter-simple sequence repeat primers analyses, no significant reproducible variation was detected among the in vitro-conserved plants compared with the mother plants.     

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (“Edison de Paula”, EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch’s (VS 50) and Guillard & Ryther’s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L⁻¹) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.     

A general framework for modeling population abundance data

Time-series data resulting from surveying wild animals are often described using state-space population dynamics models, in particular with Gompertz, Beverton-Holt, or Moran-Ricker latent processes. We show how hidden Markov model methodology provides a flexible framework for fitting a wide range of models to such data. This general approach makes it possible to model abundance on the natural or log scale, include multiple observations at each sampling occasion and compare alternative models using information criteria. It also easily accommodates unequal sampling time intervals, should that possibility occur, and allows testing for density dependence using the bootstrap. The paper is illustrated by replicated time series of red kangaroo abundances, and a univariate time series of ibex counts which are an order of magnitude larger. In the analyses carried out, we fit different latent process and observation models using the hidden Markov framework. Results are robust with regard to the necessary discretization of the state variable. We find no effective difference between the three latent models of the paper in terms of maximized likelihood value for the two applications presented, and also others analyzed. Simulations suggest that ecological time series are not sufficiently informative to distinguish between alternative latent processes for modeling population survey data when data do not indicate strong density dependence.     

Low-cost alternatives for the micropropagation of banana

A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial light. The micropropagation of Musa `Grande Naine' by shoot tip culture was used as model. Thirteen commercial sugars from different countries were tested. Best results were achieved using white and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin were suitable. Starches of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation rates under natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD of 65 μmol m⁻² s⁻¹ and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 μmol m⁻² s⁻¹ and photoperiods ranged from 8–16 hours. This revised version was published online in June 2006 with corrections to the Cover Date.