Novel Agrobacterium-mediated Transformation Techniques

(From the January, 2007 issue of Agricell Report)

R.-G. Xue and coworkers at Laiyang Agricultural College describe the development of a “new and simple method” for obtaining high-frequency genetic transformation of soybean (Glycine max) (Biotechnology Letters 28(19):1551-1557, 2006). The technique involves (1) germinating soybean seeds for one day, (2) mechanically wounding the cotyledonary node cells of half seeds with a multi-needle consisting of thin 30 fibers, (3) inoculating the wounded half seeds with Agrobacterium tumefaciens cells harboring a recombinant DNA containing the bar and sgfp genes conferring phosphinothricin (PPT)-resistance and green fluorescent protein (GFP) activity, respectively, and (4) selecting the inoculated explants on medium containing 5 or 3 mg phosphinothricin/ liter.

The authors report that use of the multineedle technique resulted in a soybean transformation efficiency as high as 12%. Polymerase chain reaction and genomic Southern blot analysis confirmed stable integration of the transgenes into the genome of the phosphinothricin-resistant plants. GFP analysis revealed that the transgenes were highly expressed in the plantlets. Adult plants were resistant to 100 mg phosphinothricin/liter applied on the leaves, demonstrating their herbicide-resistance.

At Iowa State University, M.M. Paz, K. Wang and colleagues have developed an improved cotyledonary node method, using alternative explants, for Agrobacterium tumefaciens-mediated soybean transformation (Plant Cell Reports 25(3):206-213, 2006). The alternative cotyledonary explants are derived from mature soybean seeds following overnight imbibition. Transformation efficiencies using these “half-seed” explants range from approximately 2-9% with an overall efficiency of 3.8% based on the number of transformed events that have been confirmed in the T1 generation by phenotypic assay using the herbicide Liberty® (active ingredient glufosinate) and by Southern analysis. This efficiency is 1.5 times higher than the former method, using cotyledonary nodes derived from 5- to 7-day-old seedlings, which had been used in the authors’ laboratory.

Paz et al. state that “Significantly, the half-seed system is simple and does not require deliberate wounding of explants, which is a critical and technically demanding step in the cotyledonary node method.”

Shanxi University scientists J. Wang, Y. Sun and Y. Li report a novel plant genetic transformation method for maize (Zea mays) (Biotechnology and Applied Biochemistry 46(1):51-55, 2007). Using a scalpel, meristems of germinating seeds are wounded and cocultured with an Agrobacterium tumefaciens strain harboring a Ti plasmid. Seedlings resulting from the treatment are screened by hygromycin selection and fertile transgenic T0 and T1 plants are obtained. The results of PCR amplification, PCR–Southern and Southern blot analysis showed that the foreign gene was introduced into the maize plants. Approximately 29% of T0 seedlings examined were found to be transgenic, although the overall transformation was only 0.6% when total treated seeds were taken into account.

According to Wang et al., “The method circumvented tedious and prolonged tissue-culture steps, is simple and can be readily integrated into conventional plant breeding.” programs.

For further information: R.-G. Xue, Department of Life & Science, Laiyang Agricultural College, Qingdao, 266109, China. E-mail:

K. Wang, Department of Agronomy, Iowa State University, Ames, IA 50011-1010, U.S.A. E-mail:

S. Yi, The Agri-Biotechnology Research Centre of Shanxi Province, 030031 Taiyuan, P. R. of China (

Cyanobacterial Products Enhance Lily Bulblet Regeneration and Survival

(From the December, 2006 issue of Agricell Report)

University of Buenos Aires scientist M.C. Zaccaro and associates describe the morphogenetic and antioxidant effects produced by intra and extracellular substances from the cyanobacterium Scytonema hofmanni during in vitro multiplication of Lilium alexandrae (Electronic J. Biotechnol. 9(3), 2006,

In vitro-grown S. hofmanni biomass was separated from its culture medium, homogenized, and extracted with sterile distilled water. Either the medium (containing extracellular substances) or the extract (containing intracellular substances) was added to Murashige-Skoog (MS) medium used to culture microscale explants of L. alexandrae. Bulblet morphogenesis was compared with that on medium containing NAA and on MS alone.

The University of Buenos Aires scientists found that intra or extracellular cyanobacterial products (1) increased bulblet production 83% and 78% that produced by NAA respectively, (2) increased bulblet diameters as compared with NAA, and c) increased bulblet survival. According to the authors, increased bulblet survival was due to the promotion of antioxidant activity measured as catalase, ascorbate peroxidase, and glutathione reductase activity.

Zaccaro et al state that intra or extracellular cyanobacterial products can replace NAA, “not only during the regeneration phase but also during the storage of the viable bulblets cultivated in vitro.”

For further information: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales FCEN, Universidad de Buenos Aires, Intendente Güiraldes 2620 Pab. II 4ş P Lab 2, Ciudad Universitaria C1428EHA, Buenos Aires, Argentina, Fax: 0054 1145763384. E-mail:

Protocols for Micropropagation without Autoclaving

(From the November, 2006 issue of Agricell Report)

At last month’s IAPTC&B Congress, in Beijing, S.L. Teixeira and colleagues of the Universidade Estadual do Norte Fluminense described the development of a new protocol that uses sodium hypochlorite to replace the autoclaving procedure for establishing axenic in vitro banana (Musa sp.) cultures (Agricell Report 47(3):17-18, 2006). The technique uses sodium hypochlorite not only to surface disinfest explants, but also to “sterilize” culture vessels and prevent microbial growth in the culture media during incubation of the cultures.

Teixeira et al. have also used the sodium hypochlorite sterilization technique for micropropagation of pineapple (Ananas comosus) (Plant Cell, Tissue and Organ Culture 86(3):375-378, 2006). They report that total active chlorine concentrations equal to or higher than 0.0003% provided complete sterilization of the culture medium and culture vessels. Contamination occurred only in autoclaved medium control cultures and in cultures grown with lower chlorine concentrations in the medium. The authors suggest that endogenous contaminants carried into the medium are able to grow in the chlorine-free medium of the autoclaved control cultures but are unable to grow in the medium containing chlorine.

In contrast to the authors’ previously reported studies with banana, in which temporary inhibition of growth resulted from culture in the chlorine-containing medium, pineapple shoots more than doubled both their biomass and number of new shoots when cultured on 0.0003% or 0.0005% total active chlorine-containing medium as compared with growth on autoclaved medium. Teixeira et al. have also demonstrated the beneficial effect of chlorine sterilized media on Eucalyptus pellita shoot elongation, on the number of strawberry (Fragaria sp) shoots, and on the number and length of Pfaffia glomerata shoots, as compared with autoclaved media, and have maintained Sequoia sempervirens, bananas, orchids, bromeliads, potatoes, Eucalyptus, Pfaffia and other species as stock cultures using this protocol without sustaining any visible damage

The authors conclude from their study that “nutrient media for plant tissue culture can be sterilized without autoclaving by adopting a new protocol in which all the utensils used for preparing and packing the medium are sterilized with sodium hypochlorite, and a low concentration of the same sterilizing agent is added directly to the medium.”

Direct application of chlorine disinfectants as a means of disinfesting culture medium and vessels was described at the 2006 International Horticultural Congress, held 13-19 August 2006 in Seoul, Korea by T. Yanagawa and associates of the Kyoto University of Education. Yanagawa et al. reported that sterile culture medium could be prepared without autoclaving by immediately incorporating chlorine disinfectants into the medium. Residual chlorine in the medium was able to maintain medium sterility even when the culture vessels were not autoclaved. In addition, preparation and inoculation of explants could be successfully performed under nonsterile conditions by spraying the surface of the medium and of whole explants with chlorine disinfectants

The authors applied these techniques to shoot tips of Cymbidium, chrysanthemum and carnation, protocorm-like body sections of orchids, flower-stalk sections and leaf sections of Phalaenopsis, shoot sections of chrysanthemum and another ornamentals, bulb-scale sections of bulbous ornamentals, and plantlets and callus of ornamentals. The concentrations of incorporated and sprayed disinfectants that were used suppressed in vitro contamination but appeared to be toxic to neither explants nor plantlets.

According to Yanagawa et al., “This simple micropropagation could be applied to not only propagation methods for growers but also studies for students in school and horticultural practices without soil for people of middle and advanced age.”

In another paper presented at the 2006 International Horticultural Congress, held 13-19 August 2006 in Seoul, Korea, a paper by Kyoto University scientist Y. Mizuta and coworkers described the sterilization of plant tissue culture media without autoclaving by boiling and adding calcium hypochlorite, sucrose monolaurate, and nisin (an antibacterial polypeptide produced by lactic acid bacteria) or penicillin G to the medium. Before boiling, nisin (5 mg/liter) or penicillin G (2 mg/liter) and the sucrose ester (0.5g/liter) are added to the media. After boiling, available chlorine at a concentration of 1 mg /liter is added to the media twice at intervals of three minutes or more.

With the use of this method, Mizuta et al. found that one or fewer microbial colonies was formed in 107-108 liter of media made with Kyoto University tap water. When nisin and penicillin were not added to the media, however, complex media containing homogenates of potato, sweet potato, yam or oatmeal could not be sterilized. Simple media, however, such as those composed only of sucrose, gellan gum and inorganic elements, could be sterilized by only boiling and addition of chlorine twice at a concentration of 1 mg/liter. Boiling alone resulted in the formation of 10-1000 microbial colonies in one liter of media

The protocol developed by Mizuta et al. has been successfully used for micropropagation of carnation and potato without the need for autoclave sterilization.

For further information: M. T. Teixeira, Center of Sciences and Agricultural Technologies, Universidade Estadual do Norte Fluminense, 28013-600 Campos dos Goytacazes, Rio de Janeiro, Brazil. E-mail:

T. Yanagawa, Laboratory of Horticultural Science, Kyoto University of Education, Kyoto, 612-8522, Japan. E-mail:

Y. Mizuta, Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan. E-mail:

Ceramic Powders Enhance In Vitro Growth and Metabolite Production

(From the August, 2005 issue of Agricell Report)

According to Y.J. Kim, B. Hwang and J.C. Ahn of Chonnam National University and Seonam University, far infrared-radiating ferrite ceramics absorb electromagnetic waves of all ranges and turn such absorbed energy into infrared radiation. This radiation can improve metabolism or promote growth of plants and animals.

When Kim et al. investigated the effects of two soft ferrite ceramic powders on growth of and secondary metabolite production by several medicinal plants cultured in vitro, they found that addition of the powders directly to the culture medium stimulated the growth of Japanese chaff flower (Achyranthes japonica) callus by 65 %, A. japonica plantlets by 75%, adventitious roots of black henbane (Hyoscyamus niger) by 150%, and balloon flower (Platycodon grandiflorum) hairy roots by 50% (Korean J. Medicinal Crop Sci. 12(2):118-122, 2004). The ceramic powders significantly increased the growth of H. niger adventitious shoots even when the powders were added to the medium encased in translucent glass tubes, presumably acting through radiation alone. The powders had no significant effect, however on Centella asiatica callus and plantlets, Scopolia parviflora hairy roots, or white henbane (Hyoscyamus albus) adventitious roots, even when added directly to the culture medium. The powders were also found to enhance the gross production of tropane alkaloids in H. niger adventitious shoots by thirty-five percent and polyacetylene in Platycodon grandiflorum hairy roots by thirty percent.

The authors suggest that, since the growth and secondary metabolite stimulation by ceramic powders is observed even without direct contact between the powders and the culture media, the effects are not due to adsorption of a substance but rather to a physical factor such as far-infrared radiation or magnetism.

For further information: J.C. Ahn, Dept. of Biol., Seonam University, Namwon 590-170, Rep. of Korea. E-mail:

Comparison of Micropropagation Costs by Photoautotrophic & Photomixotrophic Systems

(From the November, 2004 issue of Agricell Report)

Y. Xiao of the Kunming Institute of Environmental Science and T. Kozai of Chiba University have compared plantlet growth and production costs of micropropagating calla lily (Zantedeschia elliottiana) and China fir (Cunninghamia lanceolata) using large-scale commercial photoautotrophic and conventional photomixtotrophic micropropagation systems (HortScience 39(6):1387-1391, 2004; In Vitro Cell. Dev. Biol-Plant 40(5):450-458, 2004). The photoautotrophic (sugar-free) system consisted of 5 large (120 liter) culture vessels with forced ventilation providing CO2-enriched air. The photomixtotrophic system consisted of conventional small culture vessels with culture medium containing sugar

Xiao and Kozai found that, for calla lily, shoot length, leaf area, fresh and dry weight per plantlet on day 15 were 1.8, 1.8, 1.7, and 2.0 times greater respectively in the photoautotrophic as compared with the photomixtotrophic system. Growth on day 15 in the photoautotrophic system was similar to or greater than that on day 30 in the conventional system. The photoautotrophic system shortened the time for multiplication and rooting by half as compared with the photomixtotrophic system. The percent survival of calla lily plantlets during ex vitro acclimatization was 95% in the photoautotrophic system and 60% in the conventional system. The calla lily monthly production capacity in the photoautotrophic system was about 3 times that in the photomixtotrophic system.

For China fir, stem length, shoot number, leaf area, fresh and dry weight per plantlet on day 30 were 1.7, 2.1, 5.3, and 2.5 times greater respectively in the photoautotrophic as compared with the conventional system. The percentage of rooted plantlets in vitro were 91-93% in the photoautotrophic system and 65% in the photomixtotrophic system. The percent survival of China fir plantlets during ex vitro acclimatization was 95-97% in the photoautotrophic system and 16% in the conventional system.

In calculating the production costs of calla lily using both types of micropropagation systems, the authors found that the production cost per ex vitro acclimatized calla lily plantlet obtained with the photoautotrophic system was approximately 40% less than that of a plantlet produced by the photomixtotrophic system. Although the production cost was lower, however, the sales price of ex vitro acclimatized calla lily plantlets obtained with the photoautotrophic system was 25% higher than that of those produced by the conventional system, due to the higher quality of the plantlets obtained with the photoautotrophic system.

Xiao and Kozai conclude that “the photoautotrophic micropropagation system has advantages over the conventional micropropagation system for commercial production of calla lily plantlets and China fir plantlets with respect to production costs and sales price.” They add, “This system should be useful for commercial production of micropropagated plantlets of other plant species.”

For further information: T. Kozai, Fac. of Hort., Chiba University, Matsudo, Chiba 271-8510, Japan. E-mail:

Novel Nurse Culture System Regenerates Plants from Cabbage Protoplasts

(From the August, 2004 issue of Agricell Report)

L.-P. Chen and collaborators at Zhejiang University, and the Tokyo University of Agriculture and Technology have developed a simple and novel nurse culture method for efficient regeneration of plants from recalcitrant protoplasts of red cabbage (Brassica oleracea) within three months, based on coculturing the protoplasts with tuber mustard (Brassica juncea var. tumida) protoplasts (Plant Cell Tissue Organ Cult. 77:133-138, 2004).

When red cabbage hypocotyl-derived protoplasts were cultured in modified Murashige-Skoog media containing various combinations of plant growth regulators, they failed to develop beyond colonies of 6-10 cells. They then gradually turned brown and died. In contrast, when the red cabbage protoplasts were cocultured with tuber mustard protoplasts at a 1:1 ratio, they continued to divide and formed micro calli. In experiments with three varieties of red cabbage, coculture with tuber mustard protoplasts resulted in plantlet regeneration rates ranging from 33% to 56% from the microcalli. More than 120 protoplast-derived red cabbage plants appeared morphologically normal after transfer to greenhouse conditions. All of the plants had the normal red cabbage chromosome number (2n=18) as contrasted with that of tuber mustard (2n=36), indicating that somatic hybrids resulting from fusion between protoplasts of the two species did not occur during the coculture process.

Chen et al. state, “Experiments aimed at producing regenerants from other recalcitrant plants are in progress by using the present nurse culture system.”

For further information: L.-P. Chen, Dept. of Hort., Coll. of Agric. & Biotechnol., Zhejiang University, Hangzhou 310029, P.R. of China. Fax: 86 571 86971431. E-mail:

Water Submersion Pretreatment Enhances Shoot Regeneration

(From the March, 2004 issue of Agricell Report)

At Ankara University, M. Yildiz and M. Özgen have found that temporary immersion of flax (Linum usitatissimim) hypocotyl explants in water prior to in vitro culture results in improved regeneration of shoots (Plant Cell Tissue Organ Cult. 77:111-115, 2004).

Yildiz and Özgen excised 5 mm long hypocotyl segments from 7-day-old flax seedlings. Some of the segments were submerged in sterile distilled water for 20 minutes before being explanted onto MS regeneration medium supplemented with BAP (1 mg/liter) and NAA (0.02 mg/liter) while other were directly placed on the same medium without prior submersion in water. After six weeks of culture the explants that had been pretreated by water submersion were found to have produced significantly higher shoot wet and dry weights, shoot regeneration percentages, shoot numbers per hypocotyl, shoot lengths, and total shoot numbers per Petri dish. In addition, subsequent rooting and plantlet establishment were significantly better in the shoots derived from submersion-treated explants.

According to the authors, the results presented in this study indicate that water pretreatment of explants before culture initiation can increase the success of in vitro studies and that the procedure presented in this study can easily be used for other crops to obtain high frequency shoot regeneration in vitro.

For further information: M. Özgen, Dept. of Field Crops, Fac. of Agric., Ankara University, 06110 Diskapi, Ankara, Turkey. Fax: 90 312 3182666. E-mail:

Transgenic Plant Monitor — A Free Database for Management and Analysis of Plant Tissue Culture and Transformation Information

(From the April, 2003 issue of Agricell Report)

Although the use of database systems for logging and managing complex data sets is common in industrial plant biotechnology laboratories, it is not yet common in public sector laboratories. R. Scott, E. Mutasa-Göttgens and collaborators at the University of Nottingham and TresCalas S.A. describe the Transgenic Plant Monitor (TPM) — a ‘laboratory manager-type’ database, structured to allow efficient recording, monitoring and analysis of data produced by plant tissue culture and transformation experiments, which is available to nonindustrial laboratories (Molecular Breeding 11:121-125, 2003). Whereas, in most nonindustrial laboratories, large data sets are normally stored manually or as computerized records on spreadsheets, the TPM database provides plant biotechnology researchers with the power and flexibility of a computer database. It allows the rapid retrieval of information associated with specific transgenic events, the generation of accurate reports on demand, and allows all team members to access comprehensive data summaries as required.

The authors describe TPM as “an invaluable tool when establishing new transformation systems in the laboratory.” They state, “TPM will not only facilitate the introduction of plant transformation recording fully into the electronic age, but will also become more important as plant research continues to expand into integrated collaborative research involving several independent laboratories.” Mutasa-Göttgens et al. add that “the use of databases in public sector research is likely to be desirable and, possibly, obligatory, as statutory control...on the production of transgenic organisms becomes more closely regulated.”

Copies of the TPM database are available free of charge by e-mail to E. Mutasa-Göttgens at

For further information: E. Mutasa-Göttgens, IACR-Broom’s Barn, Higham, Bury St. Edmunds, IP28 6NP, U.K. Fax: 44 1284 811109. E-mail:

Biological Hardening of In Vitro-propagated Plantlets

(From the September, 2002 issue of Agricell Report)

Tissue culture-propagated plants often require extensive hardening treatments to prevent high mortality after transfer to ex vitro conditions. A. Pandey and colleagues at the G.B. Pant Institute of Himalayan Environment and Development hypothesize that, in addition to physiological and anatomical deficiencies resulting from in vitro propagation of plants, which make transfer to field conditions difficult, the aseptic conditions of micropropagation produce plantlets that have not developed resistance to major and minor microbial pathogens. Infection with such pathogens is one of the factors that prevent establishment of tissue culture-produced plantlets in soil. In order to harden micropropagated plantlets against such microorganisms, Pandey et al. have proposed the concept of biological hardening — the use of antagonistic biological agents to inoculate micropropagated plantlets for better establishment, survival and growth after transfer to ex vitro conditions (Biological Hardening: A Promising Technology for Tissue Culture Industry, in Role of Plant Tissue Culture in Biodiversity, Conservation and Economic Development, S.K. Nandi, L.M.S. Palni and A. Kumar, Eds., pp. 565-577, Gyanodaya Prakashan, India, 2002).

Using rhizoplane and rhizosphere analysis, Pandey et al. found that the major biotic factor responsible for mortality of micropropagated tea (Camellia sinensis) plants after transfer to soil was fungal attack, particularly infection by Fusarium oxysporum. In order to harden in vitro-cultured tea plants against fungal attack, the authors inoculated the plantlets with bacteria isolated from the rhizosphere of young as well as established tea bushes, and previously screened for their antifungal activity against a broad range of tea-associated fungi. These included Bacillus subtilis, Bacillus sp., and two strains of Pseudomonas corrugata. In soil samples taken one year after inoculation Pandey et al. found that, whereas the soil surrounding uninoculated control plants had small bacterial populations and large fungal populations, the soil surrounding inoculated plant roots contained large bacterial populations and few or no fungi. Whereas survival of control plants was 36-52%, the inoculated plants survived at rates of 88-100%, depending on the season. The overall growth of inoculated plants was also superior to that of control plants. Interestingly, the Pseudomonas strains were effective as biological hardeners even though they did not inhibit fungi in Petri dish experiments.

In addition to the experiment with tea plants, a similar study was done on ex vitro establishment of in vitro-grown plantlets of the alpine medicinal herb Picrorhiza kurrooa using B. subtilis and P. corrugata as biological hardeners. In this study the authors found that, two months after transfer, only 37.5% of uninoculated control plantlets survived while the survival rate of inoculated plantlets was 85.0 - 92.5%.

According to the authors, “Inoculation with selected bacteria at the time of transplantation provided the first line of defense to tissue culture-raised plants...” They state, “For popularizing this promising technology for use in tissue culture programs, a systematic approach is required for the selection of a right microbial candidate (or a microbial consortium) which may involve the following steps: (1) isolation and characterization of microbes from various soils/rhizospheres, (2) Petri dish-based studies of the representative microbial cultures for various physiological, biochemical and antagonistic properties, (3) bioassays to examine the effects of the promising microbial inoculants and (4) once a microbial culture is established for field applications, the culture should be developed as a carrier-based inoculant.”

For further information: A. Pandey, G.B. Pant Inst. of Himalayan Environment and Devel., Kosi-Katarmal, Almora-263 643, Uttaranchal, India. Fax: 0091 5962 31360/31507. E-mail:

Micropropagation with 3-Methyleneoxindole

(From the June, 2002 issue of Agricell Report)

At Natural Science Research, V.K. Tuli has explored the use of 3-Methyleneoxindole (MO), a metabolite of IAA, for its usefulness in micropropagating tobacco (Nicotiana tabacum), Hosta plantaginea, Kalanchoe blossfeldiana and Ficus benjamina (In Vitro Cell. Dev. Biol.-Plant 38:66-72, 2002). Tuli found that MO is ten times more active than IAA in supporting the multiplication and rooting stages of micropropagation in all these species.

Tuli recommends the use of MO as an auxin for commercial micropropagation on several grounds: (1) in terms of cost and time, MO is much more economical to use than either IAA or NAA because lower MO concentrations and shorter time periods are required to achieve desired auxin effects; (2) environmental issues arising from the use of synthetic auxins such as NAA or 2,4-D do not arise with MO; and (3) plants can tolerate high levels of MO.

For further information:
V.K. Tuli, Natural Science Research, Dept. of Plant Sci., 1210 E. 223rd St., Bldg. 322, Carson, CA 90745, Fax: 310 522 3840

Mechanical Scraper Improves Bioreactor Cell Suspension Culture

(From the May, 2002 issue of Agricell Report)

Cultivation of plant cells in a bioreactor tends to result in a buildup of foam at the interface between the culture medium and the headspace. This causes cells to be deposited in a ring of scum on the side of the culture vessel, above the liquid level. The resulting ring of dead cells that accumulates during the incubation period reduces the efficiency of secondary metabolite production.

At the University of Illinois, J.E. Meyer, M.-F. Pépin and M.A.L. Smith have developed a novel method to control the formation of scum ring deposits in a bioreactor used for the production of anthocyanins by ohelo (Vaccinium pahalae) cells (Journal of Biotechnology 93:45-57, 2002). The technique involves the use of a mechanical scraper consisting of round ceramic magnets placed both on the inside and outside surfaces of the culture vessel. By moving the outer magnet, the inner magnet can be moved to manually wipe down and remove the ring of cells that accumulates during culture.

Meyer et al. report that, although the use of the magnetic mechanical scraper does not completely eliminate foam and meringue buildup, its use twice a day keeps productive plant cell aggregates in suspension without the accumulation of a scum ring or debris on the side of the culture vessel.

For further information: M.A.L. Smith, Dept. of Natural Resources & Environ. Sci., Univ. of Illinois, 1201 S. Dorner Dr., Urbana, IL 61801, U.S.A. Fax: 217 244 3469. E-mail:

Is it Always Necessary to Disinfest Explants for In Vitro Culture?

(From the February, 2002 issue of Agricell Report)

In order to reduce or eliminate microbial contamination, an almost universally accepted step in preparing plant material for in vitro culture involves chemically disinfesting explants or explant sources before transferring the plant material to sterile in vitro conditions. The results of experiments by Hong Kong Institute of Biotechnology scientists W.-L. Teng, T. Sin and M.-C. Teng, however, challenge the necessity and even the advisability of disinfesting explants or explant material sources under some conditions (Plant Cell Tissue Organ Cult. 68(3):233-239, 2002).

Using freshly dug ginseng (Panax ginseng and P. quinquefolia) roots as explant sources, Teng et al. carefully cleaned the roots, removed tiny root branches, then broke the roots into segments, exposing the interior portions. Explants, obtained by aseptically removing the internal root tissues, were transferred to sterile culture medium and subcultured monthly. Control explants were prepared in the same manner except that the roots were surface disinfested for 40-60 minutes in NaOCl solution prior to removal of the explant material.

The authors found that explants from roots subjected to the disinfestation procedure were much more difficult to establish in vitro than were those without previous disinfestation and the cumulative contamination in the cultures from disinfested explants after 8 months of subculture was 85% as contrasted with 2-3% contamination in non-disinfested explants. In addition to affecting culture initiation, disinfestation was found to adversely affect callus growth. Whereas growth of callus from disinfested explants took 3-4 weeks to begin, non-disinfested explants showed callus growth within a week and their growth was better than that from disinfested explants. Disinfestation also had a carry-over effect on somatic embryogenesis and organogenesis. Calli from disinfested explants required 5 months to regenerate adventitious roots and 10 months to produce somatic embryos. In contrast, calli from non-disinfested explants regenerated adventitious roots in 2 months and somatic embryos in 6 months. In disinfested explants, contamination generally began in the vascular tissue. In non-disinfested explants, contamination randomly took place from pith, vascular tissue and cortex.

Teng et al. conclude that in vitro cultures of ginseng can be initiated from freshly dug roots without the need for surface disinfestation and that a dramatically higher success rate can be obtained from such cultures, along with less time spent in explant preparation. They hypothesize that stress, resulting from disinfestation procedures, weakens explants, which in turn creates a condition that allows internal contaminants to grow out more easily than their counterparts in non-stressed plants.

For further information: W.-L. Teng, Hong Kong Institute of Biotechnology Ltd., 2 Biotechnology Ave., 12 Miles Tai Po Rd., Shatin, N.T., Hong Kong. Fax: 852 26036820. E-mail:

Tubular Skylights Allow Large Reduction in Micropropagation Costs

(From the September, 2001 issue of Agricell Report)

Although natural lighting is currently being used commercially to reduce micropropagation costs, especially in tropical countries, the use of glass windows for natural lighting results in generation of excessive heat, requiring the use of energy to cool naturally lit growth rooms. At the FAO/IAEA laboratories in Austria, A. Kodym, S. Hollenthoner and F.J. Zapata-Arias have found that tubular skylights allow the use of daylight for micropropagation of bananas (Musa sp.) with almost no heat accumulation, at a fraction of the cost of micropropagation in conventional, artificially lit growth rooms (In Vitro Cell. Dev. Biol.-Plant 37(2):237-242, 2001). Tubular skylights, which redirect daylight through highly reflective tubing into interior locations, can be installed on any roof. A diffuser at the ceiling level spreads the light evenly.

Kodym et al. installed tubular skylights (Solatubes) in a micropropagation growth room containing neither windows nor air conditioning and compared micropropagation rates and plant morphology in this room, between the months of May and September, with those in a standard, artificially lit growth room. The FAO/IAEA investigators found that the micropropagation rate of plantlets cultured under tubular skylights was the same or higher than under artificial light, but required no use of electricity, resulting in a saving of US$6.00 per square meter (1400 explants) per week. Each skylight, which cost US$600, was able to illuminate an area of 3-5 square meters. Although outside temperatures were as high as 36°, temperatures in the skylight-lit room never exceeded 28°C. While the naturally illuminated plantlets showed some etiolation, they were well developed, their acclimatization rates averaged 95%, and within two months their leaf numbers and chlorophyll levels were no different from those of artificially illuminated plantlets.

According to the authors, “No expertise is required to install or to maintain Solatubes. They are offered as do-it-yourself kits and their weatherproof sealed system is reported by the manufacturer to lock out dust, moisture and insects and ensure a durable and basically maintenance-free lifetime (,,, Based on their study of various growth room setups, Kodym et al. suggest installing two tubular skylights with a center distance of 1.5 meters to illuminate 2 shelves with 3 boards each (0.4 X 3.0 m) arranged in a step-like rack. The lower and possibly the middle board should be mounted in a way that allows them to be pushed back when someone enters the room, allowing uniform light distribution but easy access to all cultures. The authors also suggest coating the walls of the growth room with highly reflective paint.

For further information: F.J. Zapata-Arias, Plant Breeding Unit., FAO/IAEA Agric. & Biotechnol. Lab., A-2444 Seibersdorf, Austria. E-mail: F.

Viscous Additive Improves Micropropagation in Liquid Medium

(From the August, 2001 issue of Agricell Report)

At Nagoya University, E. Nagamori, T. Kobayashi and colleagues have developed a new approach to culturing plant cells in liquid medium. In order to reduce hydrodynamic stresses in liquid medium tissue cultures, Nagamori et al. incorporated the viscous additives sodium alginate or carboxymethyl cellulose (CMC) into the medium used for culturing embryogenic carrot (Daucus carota) explants (J. Biosci. Bioeng. 91(3)283-287, 2001). They found that, with increased levels of additive, the viscosity as well as the number of regenerated plantlets and the fresh weight of the plantlets increased. Optimal results were achieved with sodium alginate at a concentration of 0.1% or CMC at 0.4%. This corresponded to a viscosity of 3 mPa·s. However, since sodium alginate was found to cause solidification of the medium when added at a level of 0.2%, the studies focused on the use of CMC. When added at a level of 0.4%, CMC resulted in the regeneration of approximately 1.5 times more plantlets with 2.5 times higher fresh weight than cultures grown without the additives. In the viscous medium, many plants more than 10 mm in length were obtained after 10 days of regeneration whereas most plantlets in the control culture were about 5 mm long. Enhanced regeneration could be induced by a single culture in viscous medium but did not persist in nonviscous medium. Production of plantlets rapidly disappeared after a return of the explants to conventional medium.

In addition to enhancing plantlet regeneration, addition of CMC was found to greatly extend the period of time during which cultures remain capable of producing plantlets. In repeated batch culture with viscous medium, regeneration of plantlets continued at a high level for 18 batches, over a period of 250 days, with no significant decrease. In contrast, In control nonviscous cultures, the ability to regenerate plants almost disappeared by the sixth batch (84 days). The regeneration ability of these cultures could be partially restored, however, by a single culture in viscous medium.

It thus appears, from the work of Nagamori et al., that by reducing shear stress in liquid medium, addition of a viscous agent such as CMC can (1) increase the number and size of plantlets regenerated from embryogenic calli and (2) can greatly extend the time during which the calli remain capable of producing plantlets. The authors point out that, in addition to improving the feasibility of using bioreactors for micropropagation, the shear-stress reduction provided by viscous agents could enhance the formation of aggregates in cell suspension cultures, thus increasing production of secondary metabolites. They state, “...we consider that our novel method using viscous medium should be applicable to all types of plant cell cultures.”

For further information: T. Kobayashi, Dept. of Biotechnol., Grad. Sch. of Eng., Nagoya Univ., Chikusa-ku, Nagoya 464-8603, Japan. Fax: 81 52 789 3213. E-mail:,

Temporary-immersion Micropropagation of Phalaenopsis Orchids

(From the July, 2001 issue of Agricell Report)

At Chungbuk National University, P.S. Young, H.N. Murthy and P.K. Yoeup have compared four different types of bioreactor systems for mass propagation of Phalaenopsis orchids from leaf-derived protocorm-like body (PLB) explants (Plant Cell Tissue Organ Cult. 63:67-72, 2000). The systems included (1) continuous immersion in an air lift column, (2) continuous immersion in an air lift-balloon bioreactor, (3) temporary immersion with an activated charcoal filter attachment, and (4) temporary immersion without a charcoal filter attachment. All of the systems used a modified Hyponex medium. In the continuous immersion systems, PLB explants were immersed in aerated liquid medium for the entire culture period. In the temporary immersion bioreactors, PLB explants were placed on a plastic net installed in the culture vessel. The explants were immersed in liquid medium for 5 minutes every 2 hours.

Young et al. found that, although all of the bioreactor systems were suitable for proliferation of PLBs, temporary immersion with an activated charcoal filter attachment was the best proliferation system. Inclusion of the activated charcoal filter resulted in removal of phenolics, thus enhancing PLB growth. This technique produced an average of seventeen PLBs from each explant in 2 liters of medium after 8 weeks of incubation. When solid Murashige-Skoog, Vacin and Went, Knudson C, Lindemann, and Hyponex media were compared for converting the bioreactor-propagated PLBs into plantlets, the authors found that Hyponex medium yielded the best results. Eighty-three percent of PLBs converted to plantlets on this medium, with very high fresh weights and rooting percentages. During plantlet regeneration, frequent subculturing onto medium containing 0.5% activated charcoal enhanced plantlet growth. The regenerated plantlets were successfully transplanted to pots containing peatmoss and perlite.

Young et al. state, “This is the first report of multiplication of protocorm-like bodies of orchid species using a bioreactor system. This procedure can be conveniently applied for mass multiplication of other commercial orchid species with few modifications. This system/methodology will reduce the labor, space, cost of micropropagation, and also overcome the problems of hyperhydricity since protocorms are used for multiplication in liquid medium and, subsequently, plantlets are regenerated on solid medium.”

For further information: P.K. Yoeup, Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-762, Republic of Korea. E-mail:

Reducing Cost of Micropropagation Using Starch-Gellan Gum Mixture

(From the June, 2001 issue of Agricell Report)

In order to reduce the cost of hop (Humulus lupulus) micropropagation, I. Smýkalová, H. Lipavská and associates at the Czech University of Agriculture, Charles University, and the Hop Research Institute Company have tested a mixture of starch and gellan gum (Gelrite) as a low-cost solidifying agent (Biologia Plantarum 44(1):7-12, 2001). When Smýkalová et al. compared the yield of regenerants from internodal or nodal explants cultured on Murashige-Skoog medium supplemented with various concentrations of either glucose or maltose and solidified with either agar, starch or a mixture of potato starch and gellan gum, they obtained optimal results using the mixture of potato starch and gellan gum with glucose at a concentration of 15 grams per liter. Use of starch alone lead to hyperhydricity of the culture but the hyperhydricity symptoms were repressed by addition of gellan gum. Although opaqueness of the culture medium made it impossible to observe rooting during incubation of the cultures, evaluation at the time of harvest showed that the potato starch-gellan gum mixture had no adverse effects on root development.

According to the authors, “the use of this medium results in lower cost of micropropagation of healthy hop cultures without exhibition of vitrification.”

For further information: H. Lipavská, Department of Plant Physiology, Faculty of Science, Charles University, Vinicná 5, CZ-128 44 Prague 2, Czech Republic. Fax: 420 2 21953306. E-mail:

Pectic Oligosaccharide: A Low-cost Substitute for Plant Hormones

(From the February, 2001 issue of Agricell Report)

During plant cell elongation, enzymes degrade and modify the structure of cell wall polysaccharides liberating oligosaccharides, which, depending on their structure and concentration, regulate growth and development. The pectic oligosaccharide Pectimorf, produced at Cuba’s Institut Nacional de Tejidos Vegetales (INCA) by enzymatic degradation of citrus fruit rinds, has been reported to be an inexpensive substitute for traditional exogenous plant hormones for micropropagating a number of different crop plants, often with superior results as compared with traditional plant growth regulators (REDBIO '98 — Third Latin American Meeting on Plant Biotechnology, Agricell Report 31(1):3, 1998).

S. Montes and collaborators at INCA and the Agrarian University of Havana have examined the use of Pectimorf as a substitute for traditional exogenous plant hormones in micropropagating the ornamental plant Anthurium cubense via indirect organogenesis from in vitro-cultured seeds (Cultivos Tropicales 21(3):29-32, 2000). The authors cultured leaves with petiole sections on a modified Murashige-Skoog medium containing BAP, kinetin and 2,4-D under darkness until the first morphogenic responses were observed. The explants were then incubated under light at 1500-200 lux with a 16 hour photoperiod. The resulting white callus with adventitious buds was subcultured on a medium containing 4.7 microM Pectimorf, but no growth regulators. A regeneration rate of more than 19 buds per explant was obtained on this medium. More than 90% of the resulting micropropagules survived acclimatization. According to the authors, the plants regenerated with Pectimorf were morphologically superior to, and were more vigorous than the donor plants.

For further information: S. Montes, INCA, Gaveta Postal 1, San José de las Lajas, La Habana, Cuba. E-mail:

Low Shear Stress Pre-culture Induces Tolerance of Subsequent High Shear Stress Culture Conditions

(From the January, 2001 issue of Agricell Report)

At Osaka University, Y. Hitaka, M. Taya and colleagues have observed that when shake-flask cultures of red beet (Beta vulgaris) hairy roots are cultured for 50 hours under low shear stress conditions, no lateral roots are generated but the root tips remain undamaged (Biochemical Engineering Journal 6:1-6, 2000). At higher shear stresses, the root tissues are damaged. Based on this finding, the authors cultured red beet hairy roots in a single-column bioreactor to determine whether pre-culture under low shear stress conditions can improve the survivability of the hairy roots under subsequent high shear stress conditions — conditions required to obtain good growth in a bioreactor.

The Osaka University investigators found that, when red beet hairy roots are cultured in a single column bioreactor under low stress conditions for 50 hours, they become tolerant of, and remain viable at subsequent shear stresses 2.4 times as high as hairy roots that have not been pre-cultured under low stress conditions.

Optimal protocols for growing red beet hairy roots in the single column bioreactor were found to be a first stage culture for 50 hours at 0.05 N/m2 of shear stress, to induce shear stress tolerance, followed by a second stage culture for 110 hours at 1.0 N/m2, to produce an adequate supply of oxygen and nutrients for root growth. Under the conditions of the second stage culture, hairy roots that have initially undergone the first stage ultimately reach a concentration of over 7 kg dry cells/m2 whereas those that have not undergone the first stage show no growth at all. It thus appears that, by pre-culturing them under low shear stress conditions, hairy roots can be made tolerant of the high shear stress conditions required for fast growth in a bioreactor.

For further information: M. Taya, Department of Chemical Science and Engineering, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan. Fax: 81 6 6850 6254. E-mail:

Vermiculite and Paper Pulp as Supporting Materials for Micropropagation

(From the September, 2000 issue of Agricell Report)

F. Afreen-Zobayed and collaborators at Chiba University and Nisshinbo Industries Inc. have compared the use of agar with the use of a mixture of vermiculite and paper pulp for photoautotrophic micropropagation of sweet potato plantlets (Plant Science 157(2):225-231, 2000). Afreen-Zobayed et al. found that plantlets grown on vermiculite mixed with 30% paper pulp show the best growth. The root and shoot fresh weight of these plantlets are 2.7 times greater than those grown on agar, and the leaf, stem and root dry weights are at least twice those of plantlets grown on agar. The net photosynthetic rate per plantlet is also highest in plantlets grown on the vermiculite-paper pulp mixture. Growth is significantly better in plantlets grown on the vermiculite-paper pulp mixture than on vermiculite alone, but is better on vermiculite alone than on agar. After transfer to ex vitro conditions, plantlets grown on mixtures containing various proportions of vermiculite and paper pulp show survival percentages ranging from 90% to 100% whereas the survival percentage of those cultured on agar is only 73%.

For further information: F. Afreen-Zobayed, Laboratory of Environmental Control Engineering, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan. Fax: 81 47 363 1286.

Patent: Process for Packaging In Vitro Plant Tissues for Transport

(From the June, 2000 issue of Agricell Report)

Nestec S.A. scientists J.-P. Ducos, B. Florin and V. Petiard have patented a simple and rapid process for packaging plant tissues cultured in vitro (U.S. Patent No. 6,054,319). In a sterile container, the plant tissues are placed between the surfaces of two solid supports impregnated with liquid nutrient medium. The plant tissues are kept firmly attached to at least one of the surfaces of the supports by capillary action. At least one of the other surfaces of the supports is also kept firmly attached to one of the sides of the container by capillary action.

The inventors state that plant tissues cultured in vitro survive perfectly under these packaging conditions during a transport time ranging from 1 to 100 days. They add that the process is easy to implement, the risks of contamination are reduced, the tissues do not risk being completely immersed in nutrient medium, the tissues are protected from shock, and the tissues are very easily cultured after preservation because they simply have to be placed in a semisolid agar or liquid medium.

Technique for Temporary, Reversible Permeabilization of Plant Cells

(From the May, 2000 issue of Agricell Report)

A technique that would produce temporary, reversible permeabilization of plant cells without reducing cell viability or subsequent growth would have a number of useful applications including continuous or repeated recovery of secondary metabolites from cultured plant cells.

In the process of developing new vitrification solutions for cryopreserving plant cells, P. Tandon, M. Ishikawa, A. Komamine and H. Fukuda have found that some of these solutions permeabilize plant cells making them leaky (Plant Science 140:63-69, 1999; “Incorporation of antibodies into tobacco cells treated with solutions similar to vitrification solutions," In: Cryopreservation of Tropical Plant Germplasm, F. Engelmann and H. Takagi, Eds., pp. 352-354, IPGRI, 2000). Furthermore, the vitrification solution-induced permeabilization appears to be temporary and reversible. Tandon et al. report that treatment of tobacco (Nicotiana tabacum) cells with a fine balance of glycerol, sucrose, ethylene glycol and DMSO (20:5:20:5 w/v%) results in optimal cellular uptake of high molecular weight fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (FITC-IgG) from the culture medium. The presence of CaCl2 (10 mM) is also essential for optimal uptake of FITC-IgG and cell survival. The authors report that, following treatment and subsequent washing, the morphology of the treated cells remains intact with no loss of viability, lysis or destruction of subcellular organization. Uptake of FITC-IgG is evidenced by fluorescence in the peripheral and nuclear regions of the cells as well as in cytoplasmic strands. After 21 days of culture, regrowth of the permeabilized cells is 75-80% that of untreated control cells.

According to Tandon et al., the permeabilization produced by vitrification solutions “...may involve permeabilization of the cell wall and plasmalemma resulting in pore formation in the membrane, followed by uptake of FITC-IgG through hypertonic and/or hypotonic shock. While glycerol and sucrose...may provide needed osmotic stress, ethylene glycol and DMSO bring about permeation of the cell wall and plasma membranes.” The authors suggest that similar cell permeabilization may be achievable with other vitrification solutions.

For further information:
M. Ishikawa, Dept. of Genetic Resources, NIAR, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. Fax: 81 0298 38 7408. E-mail:

Control of Hyperhydricity Using Fish Protein Hydrolysates

(From the March, 2000 issue of Agricell Report)

Fish protein hydrolysates have been shown to contain considerable amounts of proline and its precursor glutamic acid. In previously unpublished results, University of Massachusetts researchers Y. Eguchi, K. Shetty and colleagues have observed that proline contributes to the prevention of hyperhydricity (vitrification) in oregano (Oreganum vulgare) tissue cultures. Based on these observations, Eguchi et al. tested a number of different protein hydrolysates for their ability to prevent hyperhydricity and improve acclimatization of oregano plants propagated in vitro (Journal of Herbs, Spices and Medicinal Plants 6(4):29-38, 1999).

Eguchi et al. addded 1000 mg/liter of dry powder hydrolysates of Atlantic cod or herring, yeast extract, bacto-peptone or casein hydrolysate to standard Murashige-Skoog culture medium supplemented with BAP. In vitro-cultured oregano shoots, regenerated from axillary bud explants, were transferred to the media and cultured for 30 days.

The authors report that only supplements of fish protein hydrolysate partially prevented hyperhydricity. The best results were obtained with medium containing herring hydrolysate. More than 60% of plants recovered from this medium were free of vitrification. These plants were dark green, vigorous, and had shorter internodes and branches with enhanced mechanical rigidity as compared with the other treatments and controls. According to Ehuchi et al., “These studies indicate that fish protein hydrolysates have the potential for reducing vitrification in plant tissue cultures.”

For further information:
K. Shetty, Dept. of Food Sci., Univ. of Massachusetts, Amherst, MA 01003, U.S.A. E-mail:

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