Photoautotrophic micropropagation

Summary Sugar in the medium is considered to be an essential cause for the high production costs of plantlets in conventional, heterotrophic micropropagation. Chlorophyllous explants, shoots, and plantlets in vitro have high photosynthetic ability to develop photoautotrophy, but their photosynthetic activity is restricted largely by the low CO₂ concentration in the vessel during the photoperiod and in part by the presence of sugar in the medium. The growth of plantlets in vitro is often greater under photoautotrophic conditions than under heterotrophic conditions, provided that the in vitro environment is properly controlled for promoting photosynthesis. The advantages and disadvantages of photoautotrophic micropropagation are discussed.     

Control of microbial contamination for large-scale photoautotrophic micropropagation

Summary Photoautotrophic (sugar-free) micropropagation has been examined for many different plant species. One of the advantages of photoautotrophic micropropagation is the low risk of contamination, which facilitates the use of large culture vessels, and thus contributes to the reduction in production cost. In this article, pathogenic and nonpathogenic contaminations are discussed, and guidelines for development of large-scale, pathogen-free, photautotrophic micropropagation systems are introduced. A preliminary experiment was conducted for producing pathogen-free plantlets by large-scale photoautotrophic micropropagation. Addition of AgNO₃ in the medium was shown to suppress growth of nonpathogenic contaminants without reducing fresh and dry weight, and number of leaves of the tomato plantlets.     

Recent advancement in research on photoautotrophic micropropagation using large culture vessels with forced ventilation

Summary Successful fundamental or basic research, while being stimulated by applied studies, provides the development of new technologies for the benefit of mankind. Photoautotrophic micropropagation or micropropagation using sugar-free medium is no exception from this generalization. The concept of photoautotrophic micropropagation is derived from research that revealed the relatively high photosynthetic abilities of chlorophyllous cultures such as leafy explants and plantlets in vitro. To meet the ever-increasing demand for quality transplants, the scaling-up of photoautotrophic micropropagation systems, for commercialization, has become necessary. This article reviews the recent advancement in the development and utilization of large culture vessels for photoautotrophic micropropagation with special emphasis on the feasibility of the system for the commercial-scale propagation. The review also includes choices for supporting material, ventilation type, planting density, vessel volume, and vessel sterilization procedure, and problems and solutions to achieve uniform growth in a large culture vessel. A case study of the commercial application of a photoautotrophic micropropagation system using large culture vessles, which recently has been established in Kunming, China, is also presented in this article.     

Development and application of photoautotrophic micropropagation plant system

Research has revealed that most chlorophyllous explants/plants in vitro have the ability to grow photoautotrophically (without sugar in the culture medium), and that the low or negative net photosynthetic rate of plants in vitro is not due to poor photosynthetic ability, but to the low CO₂ concentration in the air-tight culture vessel during the photoperiod. Moreover, numerous studies have been conducted on improving the in vitro environment and investigating its effects on growth and development of cultures/plantlets on nearly 50 species since the concept of photoautotrophic micropropagation was developed more than two decades ago. These studies indicate that the photoautotrophic growth in vitro of many plant species can be significantly promoted by increasing the CO₂ concentration and light intensity in the vessel, by decreasing the relative humidity in the vessel, and by using a fibrous or porous supporting material with high air porosity instead of gelling agents such as agar. This paper reviews the development and characteristics of photoautotrophic micropropagation systems and the effects of environmental conditions on the growth and development of the plantlets. The commercial applications and the perspective of photoautotrophic micropropagation systems are discussed.     

Enhancement of somatic embryogenesis frequency by gibberellic acid in fennel

The effect of GA₃ on somatic embryogenesis from petiole fragments excised from micropropagated fennel plantlets was studied. Explants were maintained for 4 weeks on an induction medium containing, 2,4-d and kinetin and were then transferred to a medium devoid of these growth regulators to allow embryo development. The addition of autoclaved or filter-sterilized GA₃ to the induction medium or to the embryo development medium increased the number of embryogenic explants. No positive effect was observed when GA₃ was added to the micropropagation medium of the mother plantlets. Gibberellic acid also counteracted the inhibiting effect of continuous light on the number of embryogenic explants. Moreover, the embryogenic frequency of petiole explants from several genotypes previously considered as poorly reacting was highly enhanced by GA₃.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid     

Enhanced growth and quality of St. John's wort (Hypericum perforatum l.) under photoautotrophic in vitro conditions

Summary Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.     

Photoautotrophic growth of sugarcane plantlets <Emphasis Type="Italic">in vitro</Emphasis> as affected by photosynthetic photon flux and vessel air exchanges

Summary Explants of sugarcane, a C₄ plant, were cultured in vitro for 18d on Floridalite (a solid cube consisting of vermiculite and cellulose fibers) used as supporting material with sugar-free Murashige and Skoog liquid medium with double-strength KH₂PO₄, MgSO₄, FeSO₄, and Na₂-EDTA in the vessel with enhanced natural ventilation. CO₂ concentration in the culture room was kept at 1500 μmol mol⁻¹ (four times the atmospheric CO₂ concentration) during the photoperiod. A factorial experiment was designed with two levels of photosynthetic photon flux (PPF) and three levels of N (number of air exchanges of the vessel). The results were compared with those in the control treatment (photomixotrophic culture using sugar-containing agar medium under low PPF and low N). PPF and N showed significant positive effects on the growth of sugarcane plantlets in vitro. In the photoautotrophic (using sugar-free medium) treatments with relatively high PPF (200–400 μmol m⁻² s⁻¹) and high N (2–10 h⁻¹), the growth of plantlets was four to seven times greater than that in the control. Also, the culture period for multiplication and rooting was shortened from 30 d in the control to 18 d or less in the photoautotrophic, high PPF, and high N treatments. Use of porous supporting material in photoautotrophic treatments promoted rooting and plantlet growth significantly.     

Implication of peroxidase activity in development of healthy and PPV-infected micropropagated GF305 peach plants

In this work, we describe the micropropagation of PPV-infected GF305 peach plants for their use as a continuous source of PPV inoculums. We observed that PPV was maintained in each sub-culture as well as in the complete micropropagation cycle, including shoot proliferation, rooting and acclimatization to ex vitro conditions. We assayed the addition of fructose and ferulic acid to the multiplication media, to improve the quality and vigour of shoots. This increased the growth of explants and this effect was correlated with a reduction in POX activity, suggesting the possible participation of POX in the growth regulation of micropropagated GF305 shoots. In general, PPV-infected explants did not show evident Sharka symptoms under in vitro conditions, but some explants showed the typical interveinal chlorosis produced by PPV. However, all the PPV-acclimated plants developed normal Sharka symptoms. The data shows that shoot cultures can be used as a reservoir of PPV inoculums as well as to study different metabolic response of healthy and PPV-infected plantlets. In addition, this strategy could serve as a secure and constant source of both healthy and virus-infected plants for other molecular and biological studies.     

A comparative study on growth and morphology of wasabi plantlets under the influence of the micro-environment in shoot and root zones during photoautotrophic and photomixotrophic micropropagation

The growth of wasabi (Wasabia japonica Matsumura) plantlets under different micro-environments inside culture vessels in photoautotrophic micropropagation (PA) and photomixotrophic micropropagation (PM) conditions were compared. After 28 days of culture, dry weight, relative growth rate, leaf area, and leaf chlorophyll contents of plantlets in PA were greater than those in PM. The number of leaves did not differ significantly between PA and PM conditions. PA promoted root growth and development with a greater number of roots, root length, root diameter, root fresh weight, root dry weight, and root xylem vessel system. Dissolved oxygen concentration in PA culture medium sharply decreased after 7 days of culture and then recovered. In PM culture medium, no significant fluctuation of dissolved oxygen concentration was apparent. The net photosynthetic rates of plantlets in PA were much higher than those in PM and increased with culture time. In contrast, the net photosynthetic rates of wasabi plantlets in PM kept a low and constant value during the culture period. With the presence of gas exchange membranes attached to the vessel lids, the detected vapor pressure deficit was higher in PA than in PM conditions. Higher stomatal density and larger stomatal aperture on the abaxial and adaxial surfaces of the leaves in PM medium promoted leaf water loss following ex vitro conditions. Thus, PA is applicable for producing healthy wasabi transplants.     

Development of a forced ventilation micropropagation system for large-scale photoautotrophic culture and its utilization in sweet potato

Summary A forced ventilation system has been developed for large-scale photoautotrophic micropropagation of chlorophyllous plants. The major goal of the system is to provide a uniform supply of CO₂ inside a large culture vessel (volume 3480 ml) to achieve uniform growth of the plantlets. The system has been designed such that sterile nutrient solution can be supplied throughout the culture period, which is essential for long-term culture. Sweet potato (Ipomoea batatas L. Lam., cv. ‘Beniazuma’) was used as a model plant for photoautotrophic culture with stagnant and nonstagnant nutrient solution in large vessels. Growth and net photosynthetic rates of the plantlets were compared with those of the plantlets grown in a small vessel under photoautotrophic conditions (with natural ventilation) and conventional photomixotrophic conditions. The results indicated that the large vessel with the forced ventilation system was effective for improving growth and uniformity of the plantlets and the rate of net photosynthesis. The stagnant nutrient solution condition under photoautotrophic forced ventilation treatment significantly increased the fresh mass of the plantlets; however, percent dry mass was highest in the treatment with nonstagnant nutrient solution condition. The results demonstrated that the conventional photomixotrophic culture system can cause seriously inhibited growth and development.     

An efficient in vitro regeneration protocol for Tagetes minuta

Tagetes minuta is a source of secondary products which are used as pharmaceuticals, pesticides and as flavour components in the food industry. Cotyledons and hypocotyls of T. minuta were cultured on MS medium with combinations of IAA or NAA and BA. Hypocotyl-derived callus developed adventitious shoots which failed to develop further. Cotyledon-derived callus, cultured on medium with IAA, regenerated adventitious shoots which developed into plantlets on MS medium or half-strength MS with 2.85 μM IAA. Cotyledons cultured on medium with 5.71 μM IAA + 44.4 μM BA and transferred to MS medium for shoot growth yielded the highest number of shoots. Nodal segments from developing shoots were micropropagated on half-strength MS medium with 2.58 μM IAA and 95% of plantlets produced adapted successfully to greenhouse conditions. In vitro plants micropropagated from nodes had many shoots whereas plants regenerated from shoot tips had only a single main stem. This difference in morphology was retained after two months growth in a greenhouse. There were no significant differences in leaf and shoot fresh and dry weights among the regenerated plants after two months growth. After six subcultures of cotyledon-derived callus on medium with IAA and BA all explants lost their ability to regenerate except those cultured on medium with 17.23 μM IAA and 44.4 μM BA. The methods of regeneration developed will facilitate selection of T. minuta plants more tolerant of environmental stress, their micropropagation, and the in vitro production of secondary products. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoautotrophic shoot and root development for triploid melon

The aim of this investigation was to establish environmental factors which promote growth and photosynthesis of melon (Cucumis melo L.) shoot buds, in vitro, and determine if photoautotrophic shoots had superior root forming ability in photoautotrophic environments. Buds from the triploid melon clone ‘(L-14×B)×L-14’ were observed for 21 days after transfer from a multiplication MS medium with 3% sucrose and 10 μM benzyladenine (BA) to a shoot development medium with 1 μM BA at three levels of sucrose in the medium (0, 1 and 3%), and light (50, 100 and 150 PPF) and CO₂ (500, 1000 and 1500 ppm) in the culture chamber. More shoot buds were observed with 3% sucrose in the medium. Increased light and CO₂ had a positive interaction with shoot proliferation. Fresh and dry weights were greatest at 3% sucrose, 150 PPF light and 1500 ppm CO₂. Shoot buds grew more slowly in sugar-free medium, but fresh and dry weight still doubled over 21 days of culture. Net photosynthetic rates (NPR) of buds were negative after four days in treatment conditions, but became positive after transfer to fresh, sugar-free medium. Two triploid genotypes of melon were (1) grown in vitro with sugar (photomixotrophic) and without sugar (photoautotrophic), (2) rooted in sugar-free media, both in a laboratory controlled environment chamber (in vitro) and a greenhouse acclimatization unit (ex vitro), and (3) compared for subsequent nursery growth in the greenhouse unit. The genotype ‘(L-14×B)×L-14’ produced more shoots than ‘(L-14×B)×Mainstream’ in both photomixotrophic or photoautotrophic conditions. ‘(L-14×B)×L-14’ rooted as well from either photoautotrophic and photomixotrophic shoots but ‘(L-14×B)×Mainstream’ rooted less frequently from photoautotrophic shoots. Seventy-six percent of the shoots in the laboratory controlled environment chamber were able to root photoautotrophically, whereas 47% of the shoots in the greenhouse acclimatization unit were rooted. Between 77% and 88% of plantlets from all treatment combinations survived transfer to the nursery. After growth in the nursery, the sizes of plants (fresh weight, dry weight, leaf area) were the same for either genotype, from either photoautotrophic or photomixotrophic shoots. Nursery plants that had been rooted in the laboratory controlled environment chamber were larger than those rooted in the acclimatization greenhouse chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of<Emphasis Type="Italic"> Thapsia garganica</Emphasis>—a medicinal plant

The micropropagation of T. garganica, a medicinally important plant, was investigated as an option for conservation purposes as wild populations are becoming sparse. A Murashige and Skoog agar medium supplemented with 0.5 mg l(-1) alpha-naphthaleneacetic acid and 1.5 mg l(-1) benzyl-6-adenine significantly improved the production of multiple shoots directly from petiole and leaflet explants compared to other plant growth regulator (PGR) combinations. Medium free of PGRs promoted rooting at a low incidence. Acclimatisation was low--52% plantlet survival--as micropropagated plantlets were highly susceptible to fungal rot once removed from culture. Application of antifungal agents to in vitro-derived plantlets as pre- and post-acclimatisation treatments during transplanting significantly reduced ex vitro mortality.     

Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easy- and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes

Net photosynthetic rates (P _n) of easy (EK 16-3) and difficult-to-acclimatize (EK 11-1) sea oats genotypes were examined under the following culture conditions: (1) photoautotrophic [sugar-free medium, high photosynthetic photon flux (PPF), high vessel ventilation rates and CO₂ enrichment, (PA)]; (2) modified photomixotrophic [sugar-containing medium diluted with sugar-free medium over time, high PPF, and high vessel ventilation rates (PM)]; (3) modified photomixotrophic enriched [same as PM with CO₂ enrichment, (PME)]; or (4) conventional photomixotrophic [sugar-containing medium, low PPF, and low vessel ventilation rates (control)]. Regardless of genotype, plantlets cultured under PA conditions died within 2 wk, whereas under PM and PME conditions, plantlets increased their P _n. After 6 wk, P _n per gram dry weight was 1.7 times greater in EK 16-3 than EK 11-1 plantlets cultured under PME conditions. In vitro-produced leaves of EK 16-3 plantlets were elongated with expanded blades, whereas EK 11-1 produced short leaves without expanded blades, especially under control conditions. After in vitro culture, EK 16-3 PME plantlets exhibited the highest dry weights among treatments. EK 16-3 PME and EK 16-3 PM had similarly high survivability, shoot and root dry weights and leaf lengths ex vitro compared to EK 16-3 control and EK 11-1 PM and PME plantlets. Ex vitro growth, survivability and P _n per leaf area of either genotype were not affected by CO₂ enrichment under modified photomixotrophic conditions. These results suggest that growth and survivability of sea oats genotypes with different acclimatization capacities can be enhanced by optimizing culture conditions.     

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.     

Photoautotrophic growth response of in vitro cultured coffee plantlets to ventilation methods and photosynthetic photon fluxes under carbon dioxide enriched condition

Effects of two ventilation methods (forced and natural) and two photosynthetic photon fluxes (PPF, 150 and 250 μmol m⁻² s⁻¹) on the photoautotrophic growth of in vitro cultured coffee (Coffea arabusta) plantlets were investigated. Number of air exchanges was 2.7, 5.9 and 3.9 h⁻¹ for forced low rate, forced high rate and natural ventilation, respectively. Single node cuttings of in vitro cultured coffee plantlets were cultured on Florialite, a mixture of vermiculite and cellulose fibers with high air porosity, emerged in liquid half strength basal MS medium, without sucrose, vitamins and plant growth regulators. The study included 40 days in the in vitro stage and 10 days in the ex vitro stage. Mean fresh and dry weights, leaf area, shoot and root lengths and net photosynthetic rate per plantlet were significantly greater in forced high rate treatments compared with those in natural and forced low rate treatments. PPF had a distinct effect on shoot length suppression and root elongation of coffee plantlets in forced high rate treatments. The control of carbon dioxide concentration inside the culture box according to the plant demand when growing was easy with the forced ventilation method in photoautotrophic micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acclimatization of tissue cultured plantlets: from laboratory to land

The ultimate success of micropropagation on a commercial scale depends on the ability to transfer plants out of culture on a large scale, at low cost and with high survival rates. During field transfer the in vitro grown plantlets are unable to compete with soil microbes and to cope with the environmental conditions. The in vitro culture conditions result in the plantlets with altered morphology, anatomy and physiology. In order to increase growth and reduce mortality in plantlets at the acclimatisation stage, efforts are focused on the control of both physical and chemical environment and biohardening of micropropagated plantlets. This review describes the abiotic and biotic stresses and current developing methods for the acclimatization of microshoots.     

Plant regeneration of <Emphasis Type="Italic">Erigeron breviscapus</Emphasis> (vant.) Hand. Mazz. and its chromatographic fingerprint analysis for quality control

An efficient micropropagation system for Erigeron breviscapus (vant.) Hand. Mazz., an important medicinal plant for heart disease, has been developed. Shoot organogenesis occurred from E. breviscapus leaf explants inoculated on a medium supplemented with a combination of plant growth regulators. On average, 17 shoots per leaf explant were produced after 30 days when they were cultured on MS basal salts and vitamin medium containing 5 μM 6-benzylaminopurine (BAP) and 5 μM 1-naphthaleneacetic acid (NAA). All the regenerated shoots formed complete plantlets on a medium containing 2.5–10 μM indole-3-butyric acid (IBA) within 30 days, and 80.2% of the regenerated plantlets survived and grew vigorously in field conditions. Based on the variation in common peaks and the produced amount of the most important bioactive component, scutellarin, a high performance liquid chromatography (HPLC) fingerprinting system was developed for quality control of these micropropagated plants. Chemical constituents in E. breviscapus micropropagated plants varied during plant development from regeneration to maturation, the latter of which showed the most similar phytochemical profile in comparison with mother plants. The regeneration protocol and HPLC fingerprint analysis developed here provided a new approach to quality control of micropropagated plants producing secondary metabolites with significant implications for germplasm conservation.     

植物组织培养新技术:光自养微繁

系统地综述了常规植物组织培养存在的不足,如易染菌、生长周期长、生产成本高等,从而引出了光自养微繁的概念、研究现状、控制方向以及它的优势。如植株长势较好、生长周期短、生产成本低等,并对该技术做了展望。光自养微繁技术作为一种新型的组织培养方法,克服了传统组培无法克服的缺陷,必将成为今后组培生产的一种重要手段。     

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.