M. Elena González-Benito & César Pérez

Dpto de Biología Vegetal, Escuela Técnica Superior de Ingenieros Agrónomos

Ciudad Universitaria, 28040 Madrid, Spain


Nodal segments from in vitro cultured shoots of Centaurium rigualii were frozen by direct immersion in liquid nitrogen. Explants had been precultured for 2 days at 25ºC on MS medium containing 2% DMSO or 5% glycerol and subsequently immersed in a vitrification solution for 30 minutes. After thawing and 10 weeks culture, 15% recovery was achieved.


The threat to natural habitats of some endemic plant species creates the need for ex situ conservation. Seeds are the usual propagules for conservation due to the simplicity of storage. However, in some species seeds are produced in small quantities or the plant population is not numerous. Cryopreservation of vegetative plant material could be an alternative in these cases. Among the different techniques used for desiccation of tissues prior to immersion in liquid nitrogen (LN), chemical desiccation (vitrification) provides an easy and inexpensive technique. Vitrification is the physical process by which a concentrated aqueous solution solidifies into a metastable glass state at low temperature without crystallization (Yamada et al., 1991).

Centaurium rigualii Esteve is an endemic species from the SE Iberian Peninsula, and is in danger of extinction according to the IUCN categories (Alcaraz et al., 1987). The micropropagation and in vitro conservation techniques for this species have already been established (Iriondo, 1991). The development of cryopreservation protocols for C. rigualii would allow some of the problems of in vitro conservation (losses due to contamination, frequent subculture, risks of genetic variation) to be overcome. In the present paper, we report

preliminary studies of vitrification and subsequent cryopreservation of nodal explants of C. rigualii.

Materials and methods

Shoots of C. rigualii were propagated in vitro on MS medium (Murashige and Skoog, 1962) with 0.1 mg/l 6-benzylaminopurine (BAP) and 0.01 mg/l 1-naphthaleneacetic acid (NAA) (Iriondo, 1991). Nodal segments, 3-5 mm long, were excised from these shoots for use as explants (Plate 1).

Plate 1. Micropropagated shoot of C. rigualii, showing the nodal segment explant.

Explants were treated with a vitrification solution prior to freezing. The composition of this solution was based on the cryoprotective mixture of Sakai et al. (1990) : 30% (v/v) glycerol (GL), 15% (v/v) ethylene glycol (EG) and 15% (v/v) dimethyl sulphoxide (DMSO) in MS containing 0.4 M sucrose (SUC) (pH 5.7-5.8). Five explants were placed in a vial containing 1 ml solution and left on ice for different periods of time. Subsequently, twenty of these explants were placed on a square of filter paper (2 x 2 cm2) which was folded with the explants inside. This "envelope" was immersed in LN for 1-2 minutes. Thawing took place in 10 ml of liquid MS + 1.2 M SUC warmed to 35°C. Explants were cultured on MS + 1 mgl-1 BAP and 0.1 mgl-1 NAA and incubated at 25°C, 16 h photoperiod with 50 µmol m-2 s-1 irradiance provided by cool white fluorescent tubes.

In a second experiment explants were precultured for two days on MS medium containing 2% EG, 2% DMSO, 5% GL or 0.8 M SUC. Cultures were incubated in the dark at 5°C or 25°C. Explants were subsequently immersed in the vitrification solution, left on ice for 30 minutes and treated as described previously.

Four replicates of five nodes each were used per treatment. After 10 weeks in culture, explants were scored for shoot or callus induction.

Results and discussion

With non-frozen nodal segments, the number of explants which did not survive vitrification increased with its duration (Table 1). Similarly, shoot formation from apple shoot tips decreased after long exposures (>80 min) to vitrification solution (Niino et al., 1992).

Vitrification   % Recovery                     
(minutes)     Control       Frozen      

 0            100±0      0±0         

 15           90±5       0±0         

 30           95±4       0±0         

 60           75±4       0±0         

 90           57±15      5±4         

Table 1.- Recovery (x±SE) of nodal explants of C. rigualii in the 10th week of culture, after being immersed in the vitrification solution for different periods of time before freezing in liquid nitrogen.

After eight weeks no explants which had been frozen seemed to have survived. Only two explants which had been in the vitrification solution for 30 min and one for 90 min showed a small green area in the meristem. After two more weeks, the explants from the 30 min treatment had died while the bud had elongated in the other explant (Table 1, Plate 2a).

Pregrowth for a few days on medium containing high concentrations of SUC (0.5 M) has been used for the cryopreservation of date palm meristems (Bagniol and Engelmann, 1991), or together with DMSO (4% DMSO + 0.75 M sucrose) for mint shoot tips (Towill, 1990). In the present study SUC concentration used (0.8 M) seemed to be too high for the survival of C. rigualii nodal explants, especially when incubation was at 25°C (Table 2).

Plate 2. Recovery responses obtained after the cryopreservation of nodal explants of C. rigualii Esteve: (a) shoot elongation, (b) callus induction.

Cryoprotectant Preculture               
               temperature 5ºC          
             Control    Frozen      

2% EG        60±12      0±0         

2% DMSO      60±12      0±0         

5% GL        65±8       0±0         

0.8M SUC     45±8       0±0         

Cryoprotectant  Preculture              
                temperature 25ºC        
             Control    Frozen       

2% EG        80±10      0±0          

2% DMSO      50±9       15±8         

5% GL        60±12      15±8         

0.8M SUC     10±5       0±0          

Table 2. % recovery (x±SE) of nodal explants of C. rigualii in the 10th week of culture, after preculture on MS with different cryoprotectants for 2 days at 25° or 5°C, immersed in vitrification solution for 30 minutes and frozen in LN.

Other preculture treatments increased the recovery percentage after freezing (Table 2). Positive results were obtained when incubation took place at 25°C on 2% DMSO or 5% GL. In these cases recovery after ten weeks in culture was as callus growth (Plate 2b). After two more weeks in culture, the callus from the explants which had been on 2% DMSO turned white and did not show further growth. These results show that the absorption of cryoprotective compounds, which would occur more quickly at 25°C than at 5°C, is more important than the cold treatment. The incubation of cultures at low temperatures >0°C for a few days prior to freezing has been used in the cryopreservation protocols of shoot tips of apple and pear (Niino et al., 1992).

These preliminary results show that it is possible to achieve recovery after cryopreservation of vegetative explants of C. rigualii after vitrification. However, further research is required to improve the recovery percentage and to obtain shoot elongation instead of callus induction.


This work was supported by CICYT Project NAT89-0865


Alcaraz F., Garre M., Martinez J.M. and Peinado M. (1987) Centaurium rigualii Esteve (Gentianaceae). In: Gómez-Campo C. (Ed.) Libro Rojo de las Especies Vegetales Amenazadas de España Peninsular e Islas Baleares. 188-189. ICONA, Madrid.

Bagniol S. and Engelmann F. (1991) Effects of pregrowth and freezing conditions on the resistance of meristems of date palm (Phoenix dactylifera L. var. Bou Sthammi Noir) to freezing in liquid nitrogen. Cryo Letters 12: 279-286.

Iriondo J.M. (1991) Aplicación de las técnicas de cultivo in vitro a la conservación de Coronopus navasii Pau, Lavatera oblongifolia Boiss y Centaurium rigualii Esteve, tres especies endémicas de la Península Ibérica en peligro de extinción. PhD Thesis. Universidad Politécnica de Madrid.

Murashige T. and Skoog F. (1962) A revised medium for rapid growth bio-assays with tobacco tissue cultures. Physiol. Plant. 15 : 473-497.

Niino T., Sakai A., Yakuwa H. and Nojiri K. (1992) Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification. Plant Cell Tissue and Organ Cult. 28: 261-266.

Sakai A., Kobayashi S. and Oiyama I. (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification. Plant Cell Rep. 9: 30-33.

Towill L.E. (1990) Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Rep. 9: 178-180.

Yamada T., Sakai A., Matsumura T. and Higuchi S. (1991) Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification. Plant Sci. 78: 81-87.


A micropropagation unit has been set up at the RBG Edinburgh. It is a facility which has been long planned and at last money has been found to enable it to go ahead. A bequest was left to the garden by 'Mr. Ferguson' who had a great interest in both the botanic gardens and also in a particular group of plants the Cactaceae.

Initially the unit will be small, involving probably a maximum of two people. The main purposes will be to provide a service for the scientific work at RBG and for the living collections.

One aspect will be the propagation of rhododendrons, many of which are diseased, or are original introductions by plant collectors such as Rock and Forrest from the early 20th century and no longer respond to conventional propagation techniques.

Many plants are infected with virus (e.g. Liliaceae and Primulaceae), so cleaning these up will be among some of our first projects.

Propagation of rare and endangered species will be a feature of the unit once techniques are established, as much of the work at RBG is involved in conservation of species.

The unit will also be collaborating on current work being undertaken at Edinburgh such as the Conifer Conservation Programme, a linked scientific and horticultural project, with the aim of establishing breeding populations of rare and endangered conifers in cultivation to ensure their future conservation and possible re-introduction.

Currently we are trying to establish contact with as many people and organisations as possible to exchange information and publicise our existence; any help in this area would be greatly appreciated.

Dr Hans Sluiman, Faith Nelson

Royal Botanic Garden, Inverleith Row, Edinburgh, EH3 5LR, UK.

Tel: 031 552 7171

Fax: 031 552 0382

APUNTES SOBRE LA PROPAGACIÓN IN VITRO DE Melocactus bellavistensis Rauh et Backeb. DEL PERÚ

Jacinto Hernández Hernández*, Genaro Ruiz Campos* y Emiliano Sánchez Martínez**,

ITESM-CQ, AP 37, 76000 Querétaro, Qro,


*Laboratorio de Cultivo de Tejidos

**Programa de Cactáceas


Seeds of M. bellavistensis subsp. bellavistensis were germinated on modified Murashige and Skoog medium (MS) without growth regulators. When the seedlings reached 1 cm long, they were transferred to MS with a range of growth regulators. The largest number of shoots was obtained with 5 mg/l BA + 1mg/l NAA, but 33% were hyperhydric. Fewer shoots were formed with 2 mg/l 2iP + 0.3 mg/l NAA, but these were all normal. 90-95% rooting was achieved on MS + activated charcoal or 1 mg/l IBA. 80% of the plantlets survived weaning.


En 1990 el ITESM-CQ (Instituto Tecnológico y de Estudios Superiores de Monterrey - Campus Querétaro) inició un programa de propagación in vitro de especies de la familia Cactaceae del estado de Querétaro. Curiosamente la primera especie con la que se trabajó no fue una especie local, ni siquiera mexicana: la experimentación se inició con un Melocactus del Perú del cual recibimos semillas como obsequio. Aún cuando los resultados carecen de valor estadístico, hemos decidido publicarl0s resultados considerando que: (1) la técnica nos permite obtener plantas que se han aclimatado perfectamente, completando su ciclo de cultivo en los invernaderos; (2) existe escasez de información acerca de métodos (en particular in vitro) para la reproducción artificial de este género, el cual ha sido considerado hasta hace poco como difícil de obtener o imposible de cultivarse (Labbett, 1991). Por lo expuesto, se tituló al presente artículo como "apuntes", y de ellos esperamos que el lector obtenga datos útiles para ulteriores desarrollos de sistemas de

propagación para este género que impulsen su uso racional y su conservación.

Consideraciones taxonómicas

En base al análisis de un ejemplar adulto se determinó a la especie con la que se trabajó como Melocactus bellavistensis subsp. bellavistensis. Para la determinación se utilizó la clave para especies de Melocactus de México y Sudamérica recientemente publicada por Taylor (1991). Esta especie pertenece al grupo del Melocactus curvispinus y se distingue del Melocactus peruvianus (la otra especie presente en Perú) por sus costillas más altas y agudas. La subespecie bellevistensis se caracteriza por tener de 12 a 18 costillas (14), aréolas de 2 a 5 mm de longitud (5 mm) y con ausencia de espinas centrales.

El ejemplar examinado pertenecía al Ing. Hamlet Chírinos Urbina, oriundo del Perú y actualmente Director de Fitotecnia del ITESM-CQ; según Chírinos (pers. comm.), esta especie crece naturalmente en la localidad de Bagua (Departamento de Amazonas), en el norte del Perú.

Metodo de propagación

Las semillas se desinfectaron durante 10 minutos en una solución de hipoclorito de sodio al 1%, a la que se adicionó un champú comercial al 1% (que actúa como surfactante). Acto seguido se lavaron tres veces con agua destilada esterilizada.

Las semillas se sembraron en medio de Murashige y Skoog (1962) libre de reguladores de crecimiento, complementado con 100 mg/l de mio-inositol, 1 mg/l de tiamina, 30 g/l de sacarosa, 7 g/l de agar (Bioxon) y l.5 g/l de carbón activado. El pH se ajustó a 5.7. El medio nutritivo se esterilizó en autoclave durante 20 minutos a 1.3-1.4 kg/cm2.

El material se incubó a 25(±)2°C de temperatura, iluminación de 700 lux (luz difusa) y fotoperiodo de 16 horas luz.

Posteriormente, las plántulas obtenidas de l cm de longitud se subcultivaron en un medio basal similar al antes mencionado, pero con diferentes concentraciones de citocininas (6-bencil-aminopurina (BA), 5 mg/l y 2-isopentenil-adenina (2iP), 2 mg/l) y auxinas (ácido naftalenacético (ANA), 1 y 0.3 mg/l).

En esta fase se omitió el uso de carbón activado, incubándose en condiciones similares a la germinación. Se observó la formación de múltiples brotes en el medio con 5 mg/l de BA y 1 mg/l de ANA, sin embargo una tercera parte de ellos estaban hiperhidratados. En el medio que contenía 2 mg/l de 2iP y 0.3 mg/l de ANA se produjo una menor cantidad de brotes, pero no se detectó hiperhidratación.

Figura 1 : Cultivo in vitro de Melocactus bellavistensis

Para el enraizamiento se probaron dos medios, uno libre de reguladores de crecimiento y con 1.5 g/l de carbón activado y otro con ácido indol butírico (AIB, 1 mg/l). En este caso, la iluminación fue de 2000 lux. No se observaron diferencias entre los dos medios, ya que en ambos se obtuvo un porcentaje de enraizamiento similar (90-95%).

Las plántulas enraizadas se aclimataron a condiciones de invernadero en una cámara con temperatura media diurna de 20 a 25°C (no controlada), iluminación media de 3000 lux (rango aproximado de 1500 a 10000 lux) y humedad relativa superior al 50%. Las plántulas se trasladaron a bandejas tipo "Speedling" (bandeja hortícola con cavidades que permiten la plantación individual), con un sustrato compuesto de la mezcla comercial Sunshine Mix 3 (Sphagnum, vermiculita, dolomita y agentes humectantes), y agrolita en una proporción de 4 a 1. El porcentaje de supervivencia obtenido fue del 80%.

Figura 2 : Plántulas enraizadas antes de transplantarlos al invernadero para su adaptación.

Finalmente, las plántulas adaptadas se transladaron a un nueva cámara, en condiciones de mayor iluminación y temperatura, así como de menor humedad relativa. En este lugar, las plantas se desarrollaron hasta alcanzar diámetros de hasta 4 cm tras 18 a 24 meses desde el inicio del proceso de propagación.

Literatura Citada

Labbett, R. 1991. Melocactus from seed to cephalium. The Cactus File 1(2):2-3.

Taylor, N.P. 1991 The genus Melocactus (Cactaceae) in Central and South America. Bradleya 9:1-80.


E. Cárdenas et al., BGMN 1(6):75-76.

The concentration of BAP used in the 1 CK medium for Astrophytum capricorne should be 5 mg/l, not 3 mg/l as stated.


R. Filippini1, R. Caniato1, E.M. Cappelletti1, A. Piovan2, G. Innocenti2 and G. Cassina3

1. Dipartimento di Biologia, Via Trieste, 75, 35121 Padova

2. Dipartimento di Scienze Farmaceutiche, Via Marzolo, 5, 35131 Padova

3. Orto Botanico, Via Orto Botanico, 15, 35123 Padova



Haplophyllum patavinum, an endangered plant which faces extinction in its Italian range, was regenerated from nodal stem segments. Best shoot induction occurred on MS + 0.2mg/l K and 1mg/l 2,4-D within 5-6 weeks. Rooting occurred on MS + 0.5mg/l BAP and 1mg/l IAA. Plantlets were hardened and subsequently weaned to the glasshouse.

Abbrevations: 2,4-D, 2,4-dichloro-phenoxyacetic acid; K, kinetin; IAA, indol-3-acetic acid; BAP, 6-benzylamino-purine; MS, Murashige and Skoog medium (1962); B5, Gamborg et al. B5 medium (1968).


Recent data (Davis et al. 1986; Anon, 1991) show that the percentage of endangered species is increasing and that conservation measures are a matter of urgency (Fay, 1992).

Haplophyllum patavinum (L) G. Don fil. (Rutaceae) has a discontinuous distribution with a wide illyric range extending from Albania to Slovenia and a punctiform relict disjointed range on the Euganean Hills near Padua (Dolcher, 1957; Pignatti, 1982), where it forms endangered and highly unstable populations confined to open habitats on well-drained calcareous soils. These populations are facing slow extinction as the result of habitat modification by man's activities and the peculiar propagation features of the species. Natural propagation is mainly vegetative by shoots arising from the rhizome, leading to disappearance after deep ploughing (Cappelletti, 1957). The germination percentage of seeds is extremely low owing to embryo abortion at different

developmental stages (Cappelletti, 1929; Guzzo et al., 1991). Slightly greater amounts of viable seeds are produced by specimens transplanted to different soil, which supports the hypothesis of a sterility of mycotic origin put forward by Cappelletti (1929). However, transplanted specimens do not survive for a long time.

H. patavinum could have pharmaceutical interest as a natural source of coumarin compounds, the wide occurrence of which in Rutaceae, including several Haplophyllum spp., is well documented (Murray, 1982).

In vitro techniques have been successfully used in recent years as a non-conventional method of plant propagation and conservation (Kumary and Sadadhi, 1991; Rathore, 1991; Lin and Griffin, 1992). This paper deals with the factors affecting in vitro regeneration of H. patavinum from stem segments of in vitro raised seedlings.

Materials and Methods

Seeds were collected from plants grown in the Botanical Garden of the University of Padova and scarified by immersion in concentrated H2SO4 for 30 min. Several rinses removed the acid. After a surface sterilization in a 7% (w/v) Ca(OCl)2 solution for 20 min, seeds were rinsed three times with sterile distilled water and sown on B5 + 3% sucrose, pH 5.7, and solidified with 0.3% (w/v) Gelrite. They were kept at 25° C in a 12h photoperiod for germination. After two weeks, every seed was wetted with 20 µl K (1mg/ml). In vitro raised seedlings, 10-12 weeks old, were used as a source of explants (leaf, nodal shoot and root segment).

Explants of 0.5-1.5 cm were cultured on MS and B5 + 3% sucrose and 0.8% agar. pH of the medium was adjusted to 5.7 before autoclaving. Media were supplemented with 0.2mg/l K and 0.1 or 1mg/l 2,4-D (Tisserat, 1985). Cultures were cultured in a 12h photoperiod at 25°C. Differentiated shoot segments were sub-cultured to fresh medium after 6 weeks for further multiplication and for rooting of individual shoots. MS + 0.5mg/l BAP and 1mg/l IAA was evaluated for root induction. Cultures for root induction were kept in the dark for 12h and then under a 12h photoperiod. After two weeks, they were transferred to ½MS. Hardening was achieved by transferring intact plantlets to distilled water for 10 min and spraying with 0.5% (w/v) Benlate fungicide followed by transfer to pots containing sterile sandy soil. They were enclosed with two interlocking clear plastic polystyrene tumblers and kept in a culture room at 25°C, 12h photoperiod. They were periodically irrigated with liquid B5 without sucrose and sprayed with Benlate. Hardened plants were finally transferred to glasshouses at the Botanical Garden, Padova.

Results and Discussion

In spite of very low seed germination percentages, seeds are preferred to vegetative material as the source of propagation material because a wider genetic base can thus be maintained. In addition, germination of seeds in some species can be greatly increased by the use of in vitro methods (Fay, 1992).

Our results show that in vitro conditions and the addition of K to the seeds greatly increased percentage germination. These results prove the presence of normally developed embryos in at least some seeds from plants cultivated in edaphic conditions different from those occurring in the Euganean habitat (Cappelletti, 1957).

               2,4-D CONC                                      

               0.1mg/l                 1mg/l                   

MS + 0.2mg/l   20 explants yelded 16   20 explants yelded 63   
K              shoots [long suited     shoots [long suited     
               for rooting]            for rooting]            

B5 + 0.2mg/l   20 explants yelded 9    20 explants yelded 18   
K              shoots [short unsuited  shoots [short unsuited  
               for rooting]            for rooting]            

Table 1. Shoots production from nodal stem segments of H. patavinum when     
cultured in vitro on MS or B5, supplemented with 0.1mg/l or 1mg/l            
2,4-dichlorophenoxyacetic (2,4-D) and 0.2mg/l kinetin (K) in factorial       

Nodal shoot segments were found to be the best explants for multiple shoot induction. Leaf and root segments produced only callus but no shoots. Abundant shoots were induced from the nodal segments on MS + 0.2mg/l K and 1mg/l 2,4-D. On B5 and/or the other growth regulator concentrations, shoot formation was observed but the incidence was much lower (Table 1). About 70% of isolated shoots rooted on MS + 0.5mg/l BAP and 1mg/l IAA. The percentage root induction was low on other media. Hardening of plants before transplantation was found to be essential.

These results prove that H. patavinum can be multiplied in culture using this protocol which involves regeneration from existing meristems. This pathway of regeneration is recognized as the most genetically conservative propagation method, and it has been used extensively with rare and endangered plants (Krogstrup et al., 1990; Clemente et al., 1991; Rathore et al., 1991). Research on H. patavinum multiplication through plant regeneration from callus is also in progress, the possibility of widening the genetic base of a species by inducing somaclonal variation having been suggested as a method of generating new vigor into populations (Bramwell, 1990; Jacobsen and Dohmen, 1990).


Anon. (1991) Center for plant conservation comes to garden. Missouri. Bot. Gard. Bull. Jan.-Feb.:3-5.

Bramwell D. (1990) The role of in vitro cultivation in the conservation of endangered species. In: Hernandez Bermejo J.E., Clemente M., Heywood V., (eds.) Conservation techniques in botanic gardens. Koeltz Scientific Books. 3-15

Cappelletti C. (1929) Ann. Bot. 18:145-166.

Cappelletti C. (1957) Atti dell'Istituto Ven. Sci., Lett. Arti, CXV:93-99.

Clemente M., Contreras P., Susin J. & Pliego-Alfaro, F. (1991) HortSci. 26:420.

Chiesura Lorenzoni F., Curti L., Lorenzoni G.G., Marchiori S., Razzara S., Tornadore N. & Caniglia G. (1978) Giorn. Bot. Ital. 112: 320-321.

Davis S.D., Droop S.J.M., Gregerson P. et al. (1986) Plants in danger. What do we know? IUCN, Cambridge.

Dolcher T. (1957) Atti dell'Istituto Ven. Sci., Lett. Arti CXV :184-216.

Fay M. F. (1992) In Vitro Cell. Dev. Biol. 28P:1-4.

Gamborg O. L., Miller R. A. & Ojima K. (1968) Exp. Cell Res. 50:151-158.

Guzzo F., De Luca A., Chiesura F. & Mariani P. (1991) Giorn. Bot. Ital. 125:229.

Jacobsen H. J. & Dohmen G. (1990) Cour. Forschungs Inst. Sencken. 125:233-237.

Krogstrup P., Nørgaard J.V. & Hamann O. (1990) Bot. Gard. Microprop. News 1:8-10.

Kumary N. & Sadadhi P.P., (1992) Plant Cell Rep. 11:476-479.

Lin G.D. & Griffin W.J. (1992) Plant Cell Rep. 11:207-210.

Murashige T. and Skoog F. (1962) Physiol. Plant. 15: 473-497.

Murray R.D.H., Mendez J. and Brown S.A. (1982) The natural coumarins: occurrence, chemistry and biochemistry. Wiley.

Pignatti S. (1982) Flora d'Italia. Edagricole, Bologna.

Rathore T.S., Tandon P. & Shekhawat S. (1991) J.Plant Physiol. 139: 246-248

Tisserat B. (1985) in: Dixon R.A. (ed) Plant Cell Culture. Irl Press, Oxford Washington DC. 79-106

Additions to List of Laboratories

Here are a few additions to the "List of Laboratories", published in the Botanic Gardens Micropropagation Newsletter Vol. 1 Parts 4 & 5.


Jardim Botânico da Madeira, Caminho do Meio - Bom Sucesso, 9000 Funchal, Madeira, Portugal

Contact: F.M. Fernandes, J.P.M. Gouveia

Tel: 226035/39

Fax: 226720


Royal Botanic Garden, Edinburgh, Inverleith Row

Edinburgh, EH3 5LR, UK

Conservation, difficult to propagate plants, disease elimination

Contacts: Hans Sluiman, Faith Nelson

Tel: 031 552 7171

Fax: 031 552 0382

See BGMN 1(7):84


Forest Research Nursery, Univ. of Idaho, Moscow, Idaho 83844-1133, USA.

Rare/endangered/difficult to propagate plants

Contact: John Edson

Tel: 208 885 7007

Fax: 208 885 6226

See: BGMN 1(7):91-92


John L. Edson and David L. Wenny

Dept of Forest Resources, University of Idaho Moscow, Idaho, 83844-1133, USA


A micropropagation unit was established at the University of Idaho in 1991 to undertake propagation studies of plants native to the interior Pacific Northwest of the United States. The unit aims to help reduce endangerment to threatened restricted endemic and coastal disjunct species in the region. Current projects involve a diverse group of species from a variety of habitats.


The University of Idaho Forest Research Nursery established a tissue culture laboratory in 1987. In 1991 a fog propagation room and an acclimatization house were added to create an operational micropropagation unit.

Until recently, our micropropagation research concentrated on multiplying valuable clones for native tree and shrub improvement projects and mass propagating common native species for habitat rehabilitation. However, as rare plant inventories and endangerment assessments have become available (Moseley & Groves, 1990), we have increasingly focused on conserving threatened and endangered plants of the montane forests, the subalpine and alpine zones, and the basin rangelands of the interior Pacific Northwest of the USA.

Endemics and disjuncts

The complex geography, geology, and climate history of the interior Northwest have probably influenced the distribution of not only the restricted endemic species but also the small disjuncts isolated since pre-glacial times from larger populations on the Pacific coast (Daubenmire, 1956). The crest of the northerly trending Cascade Ranges of the Pacific coast forms the western boundary of the region. The Cascades intercept the prevailing moist westerly winds from the Pacific to create a rain shadow in the semi-arid lowlands of the Columbia Basin, where the steppe communities include a number of

rare restricted endemics. Over 350 km to the east, the Northern Rockies rise to form the region's eastern boundary. Here, subalpine communities on mountaintops of lower elevation have become ecological islands with numerous restricted endemics. Here also, in a few relatively warm and wet low-elevation valleys, are isolated plant communities with strong coastal affinities. The Snake River plain defines the region's southern border.

The species selected for propagation have satisfied one or more of the criteria recommended by Falk & Holsinger (1991) for initiating a high priority conservation effort. The selections are either naturally rare species or small coastal populations disjunct to northern Idaho. The rare species are at risk from proximity to human activity, widespread and irreversible changes in land use, and the possible threat of rapid climate change.


A highly endangered endemic

Showy stickseed, Hackelia venusta (Boraginaceae), is a biennial herb native to the low-rainfall eastern-slope forests of the Washington Cascades. The species exists at three known sites. The surface-sterilized explants consisted of nodal segments excised from the basal sections of flower stalks, embryos dissected from immature seed, and shoot tips from basal rosettes. After incubation for a week on ½MS medium (Murashige & Skoog, 1962), + 0.1 mg/l BAP, axillary shoots began to elongate. Several microshoots have rooted after transfer to hormone-free media. Although tissue blackening currently slows multiplication, we aim to plant out several thousand plantlets at four sites.

A threatened coastal disjunct

Pacific dogwood, Cornus nuttallii (Cornaceae), an understory tree with large showy white bracts, is a unique coastal disjunct to the coniferous montane forests of northern Idaho. The dogwood population has declined rapidly since 1988 with fewer than 700 individuals remaining in their former Pleistocene Rocky Mountain refugium. Seed crops have failed since 1990 and most trees exhibit disease symptoms (Lichthardt, 1991). However, representative seed collections made in 1986 and 1990 form the basis of an ongoing conservation effort (Guerrant, 1991).

From the first seed collection, we produced 400 greenhouse seedlings for vegetative propagation. Since stem cuttings failed to root and the success rate with rooted layers was low, we micropropagated shoot-tip and nodal explants on WPM medium (Lloyd & McCown, 1980) + 0.1 mg/l BAP. We maintain over 500 microshoots in a clone bank (representative of the cultivated seedlings). We have rooted some of these shoots ex vitro using 4.5% IBA in a dry talc powdered dip.

Endangerment from urban expansion

Three species of southern Idaho, Allium aasae (Alliaceae), Astragalus mulfordiae (Leguminosae), and Lepidium papilliferum (Cruciferae), are restricted endemics in the foothills of the ranges immediately to the north of the rapidly growing metropolitan city of Boise. The impending habitat loss threatens with extinction these rare but otherwise genetically stable populations. Seed and shoot-tip explants of these species are currently in culture.

Plant adaptability studies with a rare steppe species

Astragalus columbianus, once thought extinct, was rediscovered by Sauer et al. (1979) on several benches and sandy slopes in the basalt flow terrain of the Columbia basin. Seed has germinated in vitro, and propagation systems are being studied. The on-site performance of plantlets and natural germinants from the soil bank will be compared.

Pre-empting the threat of global climate change

Douglasia idahoensis (Primulaceae) is a rare, highly ornamental ground cover plant found in several subalpine communities on ridges and peaks of the Northern Rockies. The dependence of this species on elevation-specific conditions would put this plant at risk if the climate were to warm rapidly. An incubation and multiplication protocol being developed with commoner closely related taxa will be applied to D. idahoensis.


Updated inventories and endangerment assessments have identified rare flora of the American interior Northwest in need of conservation. In response, the Forest Research Nursery at the University of Idaho has initiated micropropagation projects to help conserve the species diversity of the region.


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Banko T.J. & Stefani M.A. (1991) In vitro flowering of Oxydendrum arboreum. HortSci. 26:1425.

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Benson E., Lynch P.T. & Jones J. (1992) The detection of lipid peroxidation products in cryoprotected and frozen rice cells: consequences for post-thaw survival. Plant Science. 85:107-114.

Bera T.K. & Roy S.C. (1993) Micropropagation of Tylophora indica (Burm. f.) Merr. by multiple bud formation from mature leaf explants without callus intervention. Bot. Bull. Acad. Sin. 34:83-87.

Berardi G., Infante R. & Neri D. (1993) Micropropagation of Pyrus calleryana Dcn. from seedlings. Sci. Hort. 53 :157-165.

Besse I., Verdeil J.L., Duval Y., Sotta B., Maldiney R. & Miginiac C. (1992) Oil palm (Elaeis guineensis Jacq.) clonal fidelity: Endogenous cytokinins and indoleacetic acid in embryogenic callus cultures. J. Exp. Bot 43:983- 989.

Bessembinder J.J.E., Staritsky G. & Zandvoort E.A. (1993) Long-term in vitro storage of Colocasia esculenta under minimal growth conditions. Plant Cell Tiss. Org. Cult. 33:121-127.

Beyrle H.F. & Smith S.E. (1993) Excessive carbon prevents greening of leaves in mycorrhizal seedlings of the terrestrial orchid Orchis morio. Lindleyana 8:97-99.

Bhansali R.J. (1993) Bud culture for shoot multiplication and plantlet formation of Tecomella undulata (Rohida), a woody tree of the arid zone. Trop. Sci. 33:1-8.

Bhaskaran S. & Smith R.H. (1992) Somatic embryogenesis from shoot tip and immature inflorescence of Phoenix dactylifera cv. Barhee. Plant Cell Rep. 12:22-25.

Bonga J.M. & von Aderkas P. (1992) In vitro culture of trees. Kluwer Academic Publishers.

Bouza L., Jacques M., Scotta B. & Miginiac E. (1993) The differential effect of N6-benzyl-adenine and N6-( 2-isopetenyl)-adenine on in vitro propagation of Paeonia suffruticosa Andr. is correlated with different hormone contents. Plant Cell Rep. 12:593-596.

Brand M.H. (1993) Influence of agar and ammonium nitrate on tissue nitrogen, culture growth, and vitrification. HortSci. 28:546.

-- (1993) Initiating cultures of Halesia and Malus: influence of flushing stage and benzyladenine. Plant Cell Tiss. Org. Cult. 33:129-132.

-- & Cullina W. G. (1992) Micropropagation of red and black chokeberry (Aronia spp.) HortSci. 27:81.

Brott J.J. & Read P.E. (1993) Pre-germination treatments influence germination and plant survival of Yucca glauca germinated on LS medium. HortSci. 28:165.

Buckley P.M., Dewilde T.N. & Reed B.M. (1993) Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cell Dev. Biol. 29A:60A (Abstract).

Bustamante M.A. & Munoz L. (1993) In vitro culture of garlic in response to cytokinins and auxin. In Vitro Cell Dev. Biol. 29A:60A. (Abstract).

Cai T., Babiker A.G., Ejeta G. & Butler L.G. (1993) Morphological responce of witchweed (Striga asiatica) to in vitro culture. J. Exp. Bot. 44:1377-1384.

Chanon A.M. & Lineberger R.D. (1993) The effects of thidiazuron concentration and 2,4-D exposure on shoot organogenesis in birch. HortSci. 28:263.

-- & -- (1993) Variation in birch plants derived from organogenic callus and axillary shoot proliferation. HortSci. 28:508.

Chaturvedi H.C., Sharma M. & Sharma A.K. (1993) In vitro regeneration of Dendrocalamus strictus Nees through nodal segments taken from field-grown culms. Plant Sci. 91 :97-101.

Chávez V.M., Litz R.E., Moon P.A. & Norstog K. (1992) Somatic embryogenesis from leaf callus of mature plants of the gymnosperm Ceratozamia mexicana var robusta (Miq.) Dyer (Cycadales). In Vitro Cell Dev. Biol. 28P:59-63.

Chen J.L. & Beversdorf W.D. (1992) Cryo-preservation of isolated microspores of spring rapeseed (Brassica napus L.) for in vitro embryo production. Plant Cell Tiss. Org. Cult. 31:141-149.

Cheng B., Peterson C.M. & Mitchell R.J. (1992) The role of sucrose, auxin and explant source on in vitro rooting of seedling explants of Eucalyptus sideroxylon. Plant Sci. 87:207-214.

Chytla M.E. & Preece J.E. (1993) Adventitious micropropagation of Rhododendron 'P.J.M' hybrids. HortSci. 28:508.

Compton M.E., Gray D.J. & Elmstrom G.W. (1993) A simple protocol for micropropagating diploid and tetraploid watermelon using shoot-tip explants. Plant Cell Tiss. Org. Cult. 33:211-217.

Conner A.J., Abernethy D.J. & Falloon P.G. (1992) Importance of in vitro storage root development for the successful transfer of micropropagated asparagus plants to greenhouse conditions. N.Z. J. Crop Hortic. Sci 20:477-481.

Corchete M.P., Diez J.J. & Valle T. (1993) Micropropagation of Ulmus pumila L. from mature trees. Plant Cell Rep. 12:534-536.

Cramer C.S. & Bridgen M.P. (1993) In vitro shoot proliferation of Mussaenda 'Dona Luz'. HortSci. 28:447.

Custers J.B.M. & Bergervoet J.H.W. (1992) Differences between Nerine hybrids in micropropagation potential. Sci. Hort. 52:247-256.

--, Eikelboom W., Bergervoet J.H.W. & van Eijk J.P. (1992) In ovulo embryo culture of tulip (Tulipa L.); effects of culture conditions on seedling and bulblet formation. Sci. Hort. 51:111-122.

Dalal M.A., Sharma B.B. & Rao M.S. (1992) Studies on stock plant treatment and initiation culture mode in control of oxidative browning in in vitro cultures of grapevine. Sci. Hort. 51:35-41.

Davies H., Hobbs J., Kroese H., Fiedler J. & Aitken-Christie J. (1992) Measurement of CO2 concentration in tissue culture vessels for sugar-free micropropagation and climate change studies. Acta Hort. 319:279-283.

Davis R.E. & Lee I.M. (1992) In vitro micropropagation for maintenance of mycoplasma-like oganisms in infected plant tissues. HortSci. 27:1041-1043.

Davis T.D., Sankhla D., Sankhla N., Upadhyaya A., Parsons J.M. & George S.W. (1993) Improving seed germination of Aquilegia chrysantha by temperature manipulation. HortSci. 28:542.

--, Sankhla N., George S.W. & Parsons J.M. (1993) Micropropagation of 'German Red' carnation (Dianthus caryophyllus) from nodal explants. HortSci. 28:542.

Debergh P.C., de Coster G. & Steurbaut W. (1993) Carbendazim as an alternative plant growth regulator in tissue culture systems. In Vitro Cell Dev. Biol. 29P:89-91.

Dellevigne L.A., Adelbera J.W. & Vergano P. (1993) Optimal growth, rapid acclimatization and shipping of Cattleya orchid in single use sealed membrane vessels. HortSci. 28:263.

Demeulemeester M.A.C., Vandenbussch B. & De Proft M.P. (1993) Regeneration of chicory plants from cryopreserved in vitro shoot tips. Cryo. Letters 14:57-64.

Dewan A., Nanda K. & Gupta S.C. (1992) In vitro micropropagation of Acacia nilotica subsp. indica Brenan via cotyledonary nodes. Plant Cell Rep. 12:18-21.

Drew R.A. (1992) Improved techniques for in vitro propagation and germplasm storage of papaya. HortSci. 27:1122-1124.

--, McComb J.A. & Considine J.A. (1993) Rhizogenesis and root growth of Carica papaya L. in vitro in relation to auxin sensitive phases and use of riboflavin. Plant Cell Tiss. Org. Cult. 33:1-7.

Druart P. & De Wulf O. (1993) Activated charcoal catalyses sucrose hydrolysis during autoclaving. Plant Cell Tiss. Org. Cult. 32:97-99.

Dumet D., Englemann F. & Chabrillange N. (1993) Cryopreservation of oil palm (Elaeis guineensis Jacq.) somatic embryos involving a desiccation step. Plant Cell Rep. 12:352-355.

Ebert A., Taylor F. & Blake J. (1993) Changes of 6-benzylaminopurine and 2,4-dichlorophenoxy-acetic acid concentrations in plant tissue culture media in the presence of activated charcoal. Plant Cell Tiss. Org. Cult. 33:157-162.

Eliasson M.K. & Beyl C.A. (1993) In vitro rooting and acclimatization of Prunus serotina using paclobutrazol. HortSci. 28:545.

Elliott G.C., Smith M.A. & Bridgen M.P. (1993) Growth response of Alstroemeria 'Parigo Pink' to phosphate supply in vitro. Plant Cell Tiss. Org. Cult. 32:199-204.

Evans D.A., Sharp W.R. & Medina-Filho H.P. (1984) Somaclonal and gametoclonal variation. Amer. J. Bot. 71:759-774.

Falque M., Compan D. & Galzy R. (1991) A method for the study of CO2 exchanges in in vitro cultured Vitis rupestris plantlets. Plant Cell Tiss. Org. Cult. 27:175-181.

Fay M.F. (1992) Weaning micropropagated plantlets. Bot. Gard. Cons. News 1:41-43.

Fenning T.M., Gartland K.M.A. & Brasier C.M. (1993) Micropropagation and generation of English Elm, Ulmus procera Salisbury. J. Exp. Bot. 44:1211-1217.

Fett-Neto A.G., Teixeira S.L., Da Silva E.A.M. & Sant'Anna R. (1992) Biochemical and morphological changes during in vitro rhizogenesis in cutting of Sequoia sempervirens (D.Don) Endl. J. Plant Physiol. 140:720-728.

Finch R.P., Baset A., Slamet I.H. & Cocking E.C. (1992) In vitro shoot culture of wild Oryzae and other grass species. Plant Cell Tiss. Org. Cult. 30:31-39.

Finer J.J., Saxton R.W., Norris B.L., Steele J.A. & Rahnema S. (1991) Growth and disinfestation of six different bacteria in embryogenic suspension cultures of cotton. Plant Cell Rep. 10:380-383.

Fournioux J.C. & Bessis R. (1993) Use of carbon dioxide enrichment to obtain adult morphology of grapevine in vitro. Plant Cell Tiss. Org. Cult. 33:51-57.

Galzy R. & Compan D. (1992) Remarks on mixotrophic and autotrophic carbon nutrition in Vitis plantlets cultures in vitro . Plant Cell Tiss. Org. Cult. 31:239-244.

Halos S.C. & Go N.E. (1993) Micropropagation of Pinus caribaea Morelet. Plant Cell Tiss. Org. Cult. 32:47-53.

Hamill S.D., Sharrock S.L. & Smith M.K. (1993) Comparison of decontamination methods used in initiation of banana tissue culture from field-collected suckers. Plant Cell Tiss. Org. Cult. 33:343-346.

Hammatt N. & Grant N.J. (1993) Apparent rejuvenation of mature wild cherry (Prunus avium L.) during micropropagation. J. Plant Physiol. 141:341-346

Han K. & Stephens L.C. (1992) Carbohydrate and nitrogen sources affect respectively in vitro germination of immature ovules and early seedling growth of Impatiens platypetala Lindl. Plant Cell Tiss. Org. Cult. 31:211-214.

Hdider C. & Desjardins Y. (1993) Prevention of shoot vitrification of strawberry micropropagated shoots proliferated on liquid media by new antivitrifying agents. Can. J. Plant. Sci. 73:231-235.

Herrington E. & McPherson J.C. (1993) Light quality growth promotion of Spiraea nipponica: the influence of a low photon fluence rate and transfer time to a higher fluence rate. Plant Cell Tiss. Org. Cult. 32:161-167.

Holloway P.S. & Boyd D.J. (1993) Spore viability and germination of endangered Aleutian shield-fern, Polystichum aleuticum. HortSci. 28:182.

Hosoki T. & Tahara, Y. (1993) In vitro propagation of Salvia leucantha Cav. HortSci. 28:226.

Hossain M.A, Hossain T., Roy S.K. & Hossain S.N. (1992) Morphogenic responses in explants of Leucaena leucocephala (Lam.) de Wit following different phytohormones. Plant Tiss. Cult. 2 :31-34.

Hossain M.A. & Yeoman M.M. (1992). A modified culture technique for graft formation in explanted internodes in vitro: its development and advantages. Plant Tiss. Cult. 2(2):115-127

Hossain T., Hossain M., Roy S.K. & Hossain S.N. (1992) Effects of BAP-casein hydrolysate on in vitro multiplication of Leucaena leucocephala from different explants. Plant Tiss. Cult. 2:97-101.

Hou Q., Hu C.J, Varga W.A & Carman J.G. (1993). In vitro culture of Sego lily (Calochortus nuttallii). In Vitro Cell. Dev. Biol. 29A:90A. (Abstract).

Huetteman C.A. & Preece J.E. (1993) Simple improvements for increasing efficiency of research micropropagation laboratories. HortSci. 28:509.

-- & -- (1993) Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss. Org. Cult. 33:105-119

IBPGR (1992) In vitro research. International Board for Plant Genetic Resources. Annual Report 1991. 42-47.

Islam R., Zaman A., Joarder O.I. & Barman A.C. (1993) In vitro propagation as an aid for cloning of Morus laevigata Wall. Plant Cell Tiss. Org. Cult. 33:339-341.

Jeddi M.R. & Ashrafi P.D 1993 In vitro propagation of tea. In Vitro Cell. Dev. Biol. 29A:93A. (Abstract).

Jones R. & Geneve R. (1993) Micropropagation of gas plant (Dictamnus albus). HortSci. 28:546.

Kaeppler S.M. & Phillips R.L. (1993) DNA methylation and tissue culture-induced variation in plants. In Vitro Cell Dev. Biol. 29P:125-130.

Kane M.E. & Philman N.L. (1993) Micropropagation of wetland plants: Pontederia cordata. In Vitro Cell. Dev. Biol. 29A:60A. (Abstract).

Kano A., Fukazawa Y., Aono M. & Ohkawa K. (1992) [Effects of age of cuttings, propagation media, and cutting methods on rooting of Stephanotis floribunda Brongn.]. J. Japan. Soc. Hort. Sci. 61:619-624.

Kantharajah A.S., Richards G.D. & Dodd W.A. (1993) Roots as a source of explants for the successful micropropagation of carambola (Averrhoa carambola L.). Scientia Hort. 51:169-177.

Kapoor M.L. & Chauhan J.M.S. (1992) In vitro clonal propagation of mature Eucalyptus F1 hybrids (E. torelliana F.V. Muell x E. citriodora Hook). Silvae Genetica 41(6):305-307.

Kataeva N.V. & Popowich E.A. (1993) Maturation and rejuvenation of Coriandrum sativum shoot clones during micropropagation. Plant Cell Tiss. Org. Cult. 34:141-148.

Kendall E.J., Kartha K.K., Qureshi J.A. & Chermak P. (1993) Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment. Plant Cell Rep. 12:89-94.

Kitto S. & McMillian T. (1993) Micropropagation of Pachysandra procumbens. HortSci. 28:258.

Klimaszewska K., Ward C. & Cheliak W.M. (1992) Cryopreservation and plant regeneration from embryogenic cultures of Larch (Larix eurolepis) and Black Spruce (Picea mariana). J. of Exp. Bot.. 43:73-79.

Kohmura H., Sakai A., Chokyu S. & Yakuwa T. (1992) Cryopreservation of in vitro-cultured multiple bud clusters of asparagus (Asparagus officinalis L. cv. Hiroshimagreen (2n=30) by the techniques of vitrification. Plant Cell Rep. 11:433-437.

Kubota C. & Kozai T. (1993) Effects of temperature on the growth and chlorophyll contents of Brassica oleracea L. seedlings stored in darkness and light. In Vitro Cell. Dev. Biol. 29A:61A. (Abstract).

Kukda G. & Purohit S.D. (1993) In vitro propagation of an adult toy wood tree Wrightia tinctoria. In Vitro Cell Dev. Biol. 29A:91A. (Abstract).

Laforge F., Lussier C., Desjardins Y. & Gosselin A. (1991) Effects of light intensity and CO2 enrichment during in vitro rooting on subsequent growth of plantlets of strawberry, raspberry and asparagus in acclimatization. Scientia Hort. 47:259-269

Lal N. & Ahuja P.S. (1993) Assessment of liquid culture procedures for in vitro propagation of Rheum emodi. Plant Cell Tiss. Org. Cult. 34:223-226.

Leifert C., Pryce S., Lumsden P.J. & Waites W.M. (1992) Effects of medium acidity on growth and rooting of different plant species growing in vitro. Plant Cell Tiss. Org. Cult. 30:171-179.

Leverone L. & Pence V.C. (1993) Recovery of moss and liverwort gametophytes in vitro following desiccation and cryopreservation. In Vitro Cell Dev. Biol. 29A:88A. (Abstract).

Levy D., Seabrook J.E.A. & Coleman S. (1993) Enhancement of tuberization of auxillary shoot buds of potato (Solanum tuberosum L.) cultivars cultured in vitro. J. Exp. Bot. 44 :381-386.

Lu C. (1993) The use of thidiazuron in tissue culture. In Vitro Cell Dev. Biol. 29P:92-96.

Lucchesini M., Mensuali-Sodi A. & Vitagliano C (1993) Micropropagation of Tamarix gallica from nodal explants of mature trees. Plant Cell Tiss. Org. Cult. 35:195-197.

Lulsdorf M.M., Tautorus T.E., Kikcio S.I., Bethune T.D. & Dunstan D.I. (1993) Germination of encapsulated embryos of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.). Plant Cell Rep. 12:385-389.

Macdermott J., Bell L.M. & Trigiano R.N. (1993) Plant regeneration of two Echinacea species from leaves. HortSci. 28:543.

MacKay W.A. (1993) Micropropagation of Mexican Redbud Cercis canadensis var. mexicana. In Vitro Cell. Dev. Biol. 29A:88A. (Abstract).

Marin J.A., Jones O.P. & Hadlow W.C.C. (1993) Micropropagation of columnar apple trees. J. Hort. Sci. 68:289-297.

Marin M.L., Gogorcena Y., Ortiz J. & Duran-Vila N. (1993) Recovery of whole plants of sweet orange from somatic embryos subjected to freezing thawing treatments. Plant Cell Tiss. Org. Cult. 34:27-33.

Martin S.P., Bobisud C.A. & Sekioka T.T. (1993) Micropropagation of white taro (Colocasia esculenta var. esculenta cv. Pikokea, Pololu, Nihopuu, and Haukea). HortSci. 28:543.

Mather J. (1991) Enhanced somatic embryogenesis in Selinum candolii DC. under a mineral oil overlay. Plant Cell Tiss. Org. Cult. 27:23-26.

McKently A.H. & Adams J.B. (1993) Micropropagation of rare plants from the endangered Florida scrub habitat. In Vitro Cell Dev. Biol. 29A:88A. (Abstract).

Mencuccini M. & Rugini E. (1993) In vitro shoot regeneration from olive cultivar tissues. Plant Cell Tiss. Org. Cult. 32 :283-288.

Mensuali-Soda A., Panizza M., Serra G. & Tognoni F. (1993) Involvement of activated charcoal in the modulation of abiotic and biotic ethylene levels in tissue cultures. Scientia Hort. 54:49-57

Mercer H. & Kerbauy G.B. (1992) In vitro multiplication of Vriesea fosteriana. Plant Cell Tiss. Org. Cult. 30 :247-249.

Miguens F.C., Louro R.P. & Machado R.D. (1993) A scanning electron microscope study of normal and vitrified leaves from Datura insignis plantlets cultured in vitro. Plant Cell Tiss. Org. Cult. 32:109-113.

Mukunthakumar S. & Mather J. (1992) Artificial seed production in the male bamboo Dendrocalamus strictus L. Plant Sci. 87:109-113.

Nadgauda R.S., Nagarwala N.N., Parasharami V.A. & Mascarenhas A.F. (1993) Bud break and multiple shoot formation from tissues of mature trees of Pinus caribaea and Pinus kesiya. In Vitro Cell. Dev. Biol. 29P:131-134.

Nagakubo T., Nagasawa A. & Ohkawa H. (1993) Micropropagation of garlic through in vitro bulblet formation. Plant Cell Tiss. Org. Cult. 32:175-183.

Nakano M. & Masahiro M. (1993) Antibiotics stimulate somatic embryogenesis without plant growth regulators in several Dianthus cultivars. J. Plant Physiol. 141:721-725.

Nandwini D. & Ramawat K.G. (1992) High frequency plantlet regeneration from seedling explants of Prosopis tamarugo. Plant Cell Tiss. Org. Cult. 29:173-178.

Nawab A., Skirvin R., Splittstoesser W.E. & George W.L. (1991) Germination and regeneration of plants from old cucumber seed. HortSci. 26:917-918.

Nicholson R. (1993) Rooting Torreya taxifolia, an endangered conifer of the Florida panhandle. Botanic Gardens Conservation News 2:35-37.

Niino T. & Sakai A. (1992) Cryopreservation of alginate-coated in vitro-grown shoot tips of apple, pear and mulberry. Plant Sci. 87:199-206.

Nour K.A. & Thorpe T.A. (1993) In vitro shoot multiplication of Eastern White Cedar (Thuja occidentalis). In Vitro Cell Dev. Biol. 29P:65-71.

Nyochembeng L.M. & Zok S. (1993) Micropropagation of cocoyam (Xanthosoma sagittifolium (L.) Schott). HortSci. 28 :545.

O'Riordain F. (ed.) (1992) COST 87. Plant in vitro culture. Report of the 1991 Activities. Commission of the European Communities. 160pp.

Panizza M., Mensuali-Sodi A. & Tognoni F. (1993) Role of ethylene in axillary shoot proliferation of lavandin interaction with benzyladenine and polyamines. J. Exp. Bot. 44:387-394.

Paulet F., Engelmann F. & Glaszmann J. (1993) Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation / dehydration. Plant Cell Rep. 12:525-529.

Pedroso M.C. & Pais M.S. (1992) Minituber production from immature seed suspension culture of Orchis papiwww.cea. In Vitro Cell Dev. Biol. 28P:183-186.

Peiwen X., Ruijie S., Yuanjun Y. & Huisheng S. (1993) Rapid multiplication of virus-free garlics. In Vitro Cell. Dev. Biol. 29A:91A. (Abstract).

Pellegrineschi A. & Tepfer D. (1993) Micropropagation and plant regeneration in Sesbania rostrata. Plant Sci. 88 :113-119.

Philip V.J., Joseph D., Triggs G.S. & Dickinson N.M. (1992) Micropropagation of black pepper (Piper nigrum Linn.) through shoot tip cultures. Plant Cell Rep. 12:41-44.

Pinto J.E., Arello E.F. & Pinto C.A. (1993) Propagation of Kielmayera coriacea. The effect of sucrose and inorganic nutrition on plant regeneration. In Vitro Cell. Dev. Biol. 29A:67A. (Abstract).

Porter K. & Kuehnle A.R. (1993) Using antiviral compounds against cymbidium mosaic virus in orchid tissue culture. HortSci. 28:447. (Abstract).

Pritchard H.W., Wood J.A. & Manger K.R. (1993) Influence of temperature on seed germination and nutritional requirements from embryo growth in Arum maculatum L. New Phytol. 123:801-809.

Pupilli F., Damiani F., Nenz E. & Arcioni S. (1992) In vitro propagation of Medicago and Lotus species by node culture. In Vitro Cell Div. Biol. 28P:167-171.

Raemakers C.J.J.M., Bessembinder J.J.E., Staritsky G., Jacobsen E. & Visser R.G.F. (1993) Introduction, germination and shoot development of somatic embryos in cassava. Plant Cell Tiss. Org. Cult. 33:151-156.

Rahman S.M., Hossain M., Biswas B.K., Joarder O.I. & Islam R. (1993) Micropropagation of Caesalpinia pulcherrima through nodal bud culture of mature tree. Plant Cell Tiss. Org. Cult. 32:363-365.

Ramulu A. (1993) In vitro micropropagation of certain biomass producing plants. In Vitro Cell. Dev. Biol. 29A:79A. (Abstract).

Reddy P.J. (1993) Micropropagation of bananas through shoot tip culture. In Vitro Cell. Dev. Biol. 29A:91A. (Abstract).

Reed B.M. (1993) Rooting response of Pyrus germplasm in vitro. In Vitro Cell. Dev. Biol. 29A:88A. (Abstract).

Remphrey W.R., Palmer C.E. & Blouw M.J. (1993) In vitro branching in relation to repeated subculture in two cultivars of Potentilla fruticosa. Plant Cell Tiss. Org. Cult. 32 :235-240

Righetti B. & Facini O. (1992) Headspace gas composition in four Prunus avium cultivars with differing photosynthetic capabilities. In Vitro Cell Dev. Biol. 28P:179-182.

Robacker C.D. & Change C.J. (1992) Shoot-tip culture of muscadine grape to eliminate Pierce's disease bacteria. HortSci. 27:449-450.

Rodaway S. (1993) Substituted nitroguanidines provide cytokinin activity during in vitro cultivation of plant tissues. Plant Cell Rep. 12:273-277.

Rogers S.M.D. & Banister S. (1992) Micropropagation of Notholaena 'Sun-Tuff' fern. HortSci. 27:1224-1225.

Rogers S.M.D. (1993) Culture phenotype affects on regeneration capacity in monocot Haworthia comptoniana. In Vitro Cell Dev. Biol. 29P:9-12.

Rossetto M. & Dixon K.W. (1993) Corrigin grevillea in Western Australia. Re-Introduction News 6:8-9.

--, -- & Bunn E. (1992) Aeration: a simple method to control vitrification and improve in vitro culture of rare Australian plants. In Vitro Cell Dev. Biol. 28P:192-196.

Rossi F., Baraldi R., Facini O. & Lercari B. (1993) Photomorphogenic effects on in vitro rooting of Prunus rootstock GF 655-2. Plant Cell Tiss. Org. Cult. 32:145-151.

Rout G.R. & Das P. (1993) Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb. Plant Cell Rep. 12:513-516.

Rubluo A., Chávez, V., Martínez A.P. & Martínez-Vázquez O. (1993) Strategies for the recovery of endangered orchids and cacti through in vitro cultivation. Biol. Conserv. 63 :163-169.

Rugini E., Jacoboni A. & Luppino M. (1993) Role of basal shoot darkening and exogenous putrescine treatments on in vitro rooting and on endogenous polyamine change in difficult-to-root woody species. Sci. Hort. 53:63-72.

Sabapathy A. & Nair H. (1992) In vitro propagation of taro, with spermine, arginine, and ornithine. I. Plantlet regeneration from primary shoot apices and axillary buds. Plant Cell Rep. 11:290-294.

Sahoo Y., Pattnaik S.K. & Chand P.K (1993) Micropropagation of mulberry using alginate encapsulated shoot buds. In Vitro Cell Dev. Biol. 29A:90A. (Abstract).

Sallanon H., Tort M. & Coudret A. (1993) The ultrastructure of micropropagated and greenhouse rose plant stomata. Plant Cell Tiss. Org. Cult. 32:227-233.

Samyn G.L.J. (1993) In vitro propagation of Ponytail Palm: producing multiple-shoot plants. HortSci. 28(3):225.

Santamaria J.M., Davies W.J. & Atkinson C.J. (1993) Stomata of micropropagated Delphinium plants respond to ABA, CO2, light and water potential, but fail to close fully. J. Exp. Bot. 44:99-107

Sarker R.H. &. Roy A.R. (1993) In vitro propagation of local orchids from Bangladesh. In Vitro Cell. Dev. Biol. 29A:90A. (Abstract).

Sato S., Hagimori M. & Iwai S. (1993) Recovering vitrified carnation (Dianthus caryophyllus L.) shoot using Bacto-Peptone and its subfractions. Plant Cell Rep. 12:370-374.

Saxena S. & Bhojwani S.S. (1993) In vitro clonal multiplication of 4-year-old plants of the bamboo, Dendrocalamus longispathus Kurz. In Vitro Cell. Dev. Biol. 29P:135-142.

Sen J., Islam M.S., Roy S.K. & Hadiuzzaman S. (1992) Micropropagation of juvenile and adult Gmelina arborea. Plant Tiss. Cult. 2:89-95.

Sen S., McKinley C.R. & Newton J. (1993) Mass propagation and field evaluation of Afghan pine (Pinus eldarica Medw). In Vitro Cell. Dev. Biol. 29A:89A. (Abstract).

Shenoy V.B. & Vasil I.K. (1992) Biochemical and molecular analysis of plants derived from embryogenic tissue cultures of napier grass (Pennisetum purpureum K. Schum). Theor. Appl. Genet. 83:947-955.

Shibli R.A. (1991) In vivo acclimatization of Chrysanthemum morifolium Ramat after in vitro water stress. Plant Tis. Cult. 1:97-100.

Shimron-Abarbanell D. & Breiman A. (1991) Comprehensive molecular characterization of tissue-culture-derived Hordeum marinum plants. Theor Appl. Genet. 83:71-80.

Shrikhande M., Thengane S.R. & Mascarenhas A.F. (1993) Somatic embryogenesis and plant regeneration in Azadirachta indica A. Juss. In Vitro Cell Dev. Biol. 29P:38-42.

Sinha R.K. & Mallick R. (1993) Regeneration and multiplication of shoot in Albizia falcataria. Plant Cell Tiss. Org Cult. 32:259-261.

Slabbert M.M., de Bruyn M.H., Ferreira D.I. & Pretorius J. (1993) Regeneration of bulblets from twin scales of Crinum macowanii in vitro. Plant Cell Tiss. Org. Cult. 33 :133-141.

Standberg J.O. (1993) Meristem culture of Ophiopogon japonicus and production of embryo-like structures. Plant Cell Tiss. Org. Cult. 32:277-282.

Stimart D.P. & Harbage J.F. (1993) Growth of rooted 'Gala' apple microcuttings ex vitro as influenced by initial adventitious root count. HortSci 28:664-666.

Suzuki S., Fujino H., Tatsuo Y., Yamazaki N. & Yoshizaki M. (1992) Rapid propagation of Stephania cephalantha Hayata by tissue culture. Japan. J. Breed. 42:769-777.

Tanny G.B., Watad A.A., Kocba M. & Gaba V. (1993) Synthetic membranes for use in plant tissue culture. In Vitro Cell. Dev. Biol. 29A:61A. (Abstract).

Tawfik A.A., Read P.E. & Cuppett S.L. (1992) Stimulation of growth and monoterpene production of sage (Salvia officinalis ) by benzyladenine in vitro. PGRSA Quarterly 20 :200-206.

Taylor P.W.J. & Dukie S. (1993) Development of an in vitro culture technique for conservation of Saccharum spp. hybrid germplasm. Plant Cell Tiss. Org. Cult. 34:217-222.

Tefera H. & Chapman G.P. (1992) In vitro normal and variant development of t'ef (Eragrostis tef) spikelets. Plant Cell Tiss. Org. Cult. 31:233-237

Touchell D.H., Dixon K.W. & Tan B. (1992) Cryopreservation of shoot-tips of Grevillea scapigera (Proteaceae): a rare and endangered plant from Western Australia. Aust. J. Bot. 40 :305-10.

Toussaint A., Kummert J., Maroquin C., Lebrun A. & Roggermans J. (1993) Use of Virazole® to eradicate odontoglossum ringspot virus from in vitro cultures of Cymbidium Sw. Plant Cell Tiss. Org. Cult. 32:303-309.

Tynan J.L., Conner A.J., Macknight R.C. & Poulter R.T.M. (1993) Miconazole: an effective antifungal agent for plant tissue culture. Plant Cell Tiss. Org. Cult. 32:293-301.

Upadhyaya A., Davis T.D., Sankhla D. & Sankhla N. (1992) Micropropagation of Lupinus texensis from cotyledonary node explants. 27 :1222-1223.

Upfold S.J. van Staden J. & Edwards T.J. (1992) In vitro propagation of Rhodohypoxis baurii. HortSci. 27 :1230.

Vaquero F., Robles C. Ruiz M.L. (1993) A method for long-term micropropagation of Phaseolus coccineus L. Plant Cell Rep. 12:395-398.

Vieitez A.M., Pintos F., San-José M.C. & Ballester A. (1993). In vitro shoot proliferation determined by explant orientation of juvenile and mature Quercus rubra L. Tree Phytol. 12:107-117.

Vyapari S., Khatamian H. & Albrecht M.L. (1993) In vitro regeneration in Asclepias tuberosa L. HortSci. 28 :508.

Waldenmaier S. & Bunemann G. (1993) In vitro Kultur von Syringa vulgaris-Hybriden 1. Etablierung, Vermehrungsmethoden und Bewurzelung. (In vitro culture of Syringa vulgaris hybrids 1. Establishment, propagation methods, and rooting.) Gartenbauwissenschaft 58:2-10.

Wilson D.A., Weigel R.C., Wheeler R.M. & Sager J.C. (1993) Light spectrum quality effects on the growth of potato (Solanum tuberosum L.) nodal cuttings in vitro. In Vitro Cell Dev. Biol. 29P:5-8.

Woods S.H., Phillips G.C., Woods J.E. & Collins G.B. (1992) Somatic embryogenesis and plant regeneration from zygotic embryo explants in Mexican weeping bamboo, Otatea acuminata aztecorum . Plant Cell Rep. 11:257-261.

Wozniewski T., Blaschek W. & Franz G. (1991) In vitro propagation of Lilium testaceum and structural investigation of the storage ß-1,4- glucomannan. Plant Cell Rep. 10 :457-460.

Wysonkinska H. (1993) Micropropagation of Penstemon serrulatus and iridoid formation in regenerated plants. Plant Cell Tiss. Org. Cult. 33:181-186.

Xiaoling Y. & Reed B.M (1993) A micropropagation system for two new hazelnut rootstocks. In Vitro Cell Dev. Biol. 29A:89A. (Abstract).

Xiaoling Y. & Reed B.M. (1993) Improved shoot multiplication of mature hazelnut (Corylus avellana L.) in vitro using glucose as a carbon source. Plant Cell Rep. 12:256-259.

Yang G. & Read P.E. (1993) In vitro cultivation of Vanhoutte's spiraea explants from 'secondary cultures' and dormant stems forced in solutions containing plant growth regulators. Plant Cell Tiss. Org. Cult. 33:255-30.

Yasseen Y.M. & Davenport T.L. (1993) Fast micropropagation of tuna (Opuntia ficus-indica Mill.) and plant establishment in soil. HortSci. 28:509.

-- & -- (1993) Micropropagation of endangered succulent plants Aloe juvenna, Aloe volkensii and

Botanic Gardens Micropropagation News: edited by Dr. M F Fay,               
Micropropagation Unit                                                       
 : produced by Technical Section,                                           
     Royal Botanic Gardens Kew, Surrey, TW9 3AB, UK.                        

Stapelia semota. HortSci. 28:508.

--, --, Splittstoesser W.E. & Skirvin R.M. (1993) In vitro production of set from onion (Allium cepa L.) inflorescence. HortSci. 28:270.

--, --, -- & -- (1993) Shoot multiplication and bulb formation from garlic and shallot in vitro. In Vitro Cell. Dev. Biol. 29A:61A. (Abstract).

--, --, -- & -- (1993) In vitro propagation of kurrat (Allium ampeloprasum, var. kurrat) and leek (Allium ampeloprasum var. porrum). HortSci. 28:508.

Yeo D.Y. &. Reed B.M. (1993) Micropropagation of two Pyrus rootstocks. In Vitro Cell. Dev. Biol. 29A:89A. (Abstract).

Yuanjun Y., Ruijie S & Peiwen X. (1993). Applying research on micro-multiplication of vegetable in China. In Vitro Cell. Dev. Biol. 29A:90A. (Abstract).

Yue D., Desjardins Y.M., Lamarre M. & Gosselin A. (1992) Photosynthesis and transpiration of in vitro cultured asparagus plantlets. Scientia Hort. 49:9-16.

--, Gosselin A. & Desjardins Y. (1993) Re-examination of the photosynthetic capacity of in vitro cultured strawberry plantlets. J. Amer. Soc. Hort. Sci. 118:419.

Zaman A., Islam R., Hossain M., Joarder O.I., Ahad A. & Barman A.C. (1992) Clonal propagation through in vitro shoot proliferation of nodal explants of seven mulberry genotypes. Plant Tiss. Cult. 2:71-74.

Zhang L.X., Chang W.C., Wei Y.J., Liu L. & Wang Y.P. (1993) Cryopreservation of ginseng pollen. HortSci. 28:742-743.

Zhiming Z. (1993) Ex situ conservation of wild plants in Beijing Botanical Garden, China. Botanic Gardens Conservation News 2:17-21.

Zhong Z., Smith H.G. & Thomas T.H. (1993) Micropropagation of wild beet (Beta maritima) from inflorescence pieces. Plant Growth Regulation 12:53-57.

Zhuang S. & Steen D.A. (1993) Factors controling callus induction and propagation in the Australian stinging tree (Dendrocnide moroides). In Vitro Cell Dev. Biol. 29A:89A. (Abstract). © Copyright The Royal Botanic Gardens, Kew