Rapid Clonal Propagation Research Articles

Rapid clonal propagation of Pinellia ternata by tissue culture

Adventitious buds or protocorm-like bodies were regenerated directly from excised explants without intervening callus. Differences in the ability of regeneration were observed among different plant organs with bulbils showing the highest regenerative ability followed by leaf blade and petiole. Ability of vegetative propagation of bulbil could be maintained by alternate solid-and liquid-medium culture. Theoretically, 1.7×10(27) plantlets could be produced from a single bulbil by this technique within one year based on the production and rapid growth of protocorm-like bodies and adventitious buds. Concentration of MS salts, NAA and sucrose influenced not only root formation from the differentiated adventitious buds, but also root number and length. For root formation, the best combination was one-half strength MS salts with 3-5% sucrose and 1 mg/l NAA. The high survival rate of 96% was recorded when plantlets were transplanted into a mixture of vermiculite:loam soil:peat moss (1:2:1). Plants from in vitro culture were morphological similar to field-grown plants. The acute toxicity of crude extracts from protocorm-like bodies was about one-fourth that of extracts from tubers of field-grown plants when tested with white mice. Tissue culture has potential for clonal propagation of Pinellia ternata plants for commercial use.

Clonal propagation of Lemon Grass (Cymbopogon citratus Stapf) through shoot tip culture.

A tissue culture method for rapid clonal propagation of lemon grass (Cymbopogon citratus) is described. Shoot cultures were induced by culturing small excised shoot apices on the LS basal medium supplemented with 0.1μM NAA and 10μM BA. Multiple shoots were divided and cultured on a basal medium containing 1μM NAA for rooting. The regenerated plantlets were successfully transferred to soil.

Rapid Clonal Propagation of Papaya in Vitro from Mature Field-grown Trees

Abstract Rapid development of axillary branches on rooted cuttings from mature field-grown papaya (Carica papaya L.) trees was promoted by removal of stem apices and application to the cut surface of a mixture containing 225 mg BAP/liter of lanolin. Small axillary buds were removed from these branches and cultured, initially on solid medium, and then in liquid medium on a roller drum at 3 rpm in Drew and Smith (DS) medium containing 1 µm BAP + 1 µm NAA. After two or three subcultures on the roller drum with alternate periods on hormone-free basal medium, apically dominant shoots were produced. Growth of axillary buds subsequently occurred on DS medium containing 1 µm BAP + 0.25 µm NAA. Cultures also were established from buds of 6-month-old trees, followed by enhanced axillary shoot growth on DS medium containing 2 µm BAP + 0.5 µm NAA. Roots initiated on actively growing shoots when incubated at 27°C in a 12-hr photoperiod on a reduced mineral medium containing 10 µm IBA. Rooted shoots were established in a high-humidity (>90%) cabinet in a glasshouse and planted in the field. Field plants were characterized by a reduced juvenile stage, excessive vegetation at the lower nodes, and production of flowers and fruit close to the ground. Chemical names used: N6-benzylaminopurine (BAP); 1H-naphthaleneacetic acid (NAA); 1H-indole-3-butyric acid (IBA).

In vitro propagation of Drosera natalensis

The successful in vitro propagation of Drosera natalensis Diels, is described. Rapid clonal multiplication was achieved on modified Murashige and Skoog medium supplemented with various hormones. The production of buds on the surface of explant tissue, their subsequent subculture onto a series of defined media and the hardening off of the resultant plantlets are outlined.

Rapid clonal propagation of ginger (Zingiber officinale Roscoe) by roller tube culture.

Ginger has a low propagation rate and pathogens can be transmitted through the rhizomes to the next generation, as it is a vegetatively propagated crop. The purpose of this study was to develop a technique of efficient clonal propagation of g;nger by tissue culture. In the apical meristem culture on, agar media, treatment with high BA concentrations induced the increase of the number of buds and inhibited rooting. There was no clear relationship between the NAA concentration and the number of regenerated buds and the development of roots. Many plants with normal shoots and roots were obtained on modified MS medium with low BA concentrations, and at the maximum 6.3 plants were obtained at I mg/l BA without NAA supplementation(Table 1). In the culture of stem discs with axillary buds on agar media, the modified MS medium was more suitable than the B5 medium in relation to the rate of shooting or rooting. There was no relationship between the growth regulator concentration and the rate of shooting or rooting. However, the modified MS medium containing 3 mg/l BA and 5mg/l NAA was the most suitable medium for plant regeneration from stem discs (Table 2). In the rollel- tube culture of stem discs in liquid media, 2, 4-D induced either callus formation or tissue expansion. Multiple bud clumps were obtained in the modlfied MS medium with high BA concentrations, and the BA concentration was positively correlated with the number of buds (Table 3, Fig. 1). The mean bud number of a multiple bud clump was 44.5 and the maximum was 61. The multiple bud clumps could be subcultured and propag.ated by cutting the clumps into pieces in the liquid medium containing 10 mg/l BA. When the cut buds were cultured on agar medium with 3mg/l BA they began to grow quickly up to on intact plant, and acclimation of the regenerated plant was easy. It is concluded that multiple bud clumps obtained by roller tube culture are useful for the micropropagation of ginger.

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava ‘Banaras local’ was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 μM 6-benzyladanine (BA) alone or in combination with either 0.6 μM indole-3-acetic acid (IAA), 0.5 μM indole-3-butyric acid (IBA) or 0.3 μM gibberellic acid (GA3). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 μM) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 μM) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 μM each of IBA and α-naphtha-leneacetic acid (NAA), and 1 gl-1 activated charcoal. Regenerated plantlets were successfully established on soil.

Micropropagation through Cotyledon Culture in Camellia japonica L. and C.sinensis L.

Regeneration of plantlets from slices of cotyledons in Camellia japonica L. and C.sinensis L. has been accomplished. Differentiation originated from the surface of cotyledon without the mediation of callus formation. It was obvious from histological observations that adventitious embryos were formed directly at the surface of the swelled up part of the cotyledon. Adventitious embryos were formed always in the same way on the cotyledon slices independently of the presence or concentration of growth regulators. In C.japonica, adventitious embryos grew shoots and roots on the medium supplemented with GA3 (Gibberellin), and after several monthly subcultures many plantlets were established. Moreover these somatic embryos formed additional embryos on the hypocotyls with the subcultures and this phenomenon was perpetuated throughout the successive generations of embryos on the GA3 medium. In C.sinensis, adventitious embryos grew shoots and roots on the medium supplemented with 10.0 mg/l BA (N6-Benzylaminopurine) and 0.5 mg/l IBA (Indol-3-butyric acid), but not on the medium supplemented with GA3. It was considered that rapid mass clonal propagation through the culture of cotyledon slices could be obtained from one seed of a selected cross in the Camellia genus.

Eradication of mosaic disease and rapid clonal multiplication of bananas and plantains through meristem tip culture

Heat therapy and meristem tip culturing were used in various cultivars of banana (Musa acuminata ‘AAA’ cvs Grande Naine and Valary), and plantain (Musa acuminata x M. balbisiana ‘AAB’ cvs Maricongo, Common Dwarf and Super Plantain) for rapid clonal propagation of mosaic disease-free plants. Suckers were subjected to heat therapy at 38–40°C for 14 days prior to the culture of their meristem tips (1.5–2.0 mm long having 6–8 vertical incisions) on modified MS medium containing 1.0 mg l-1 thiamine HCl, 0.5 mg l-1 nicotinic acid, 0.5 mg l-1 pyridoxine HCl, 25 mg l-1 ascorbic acid (filter sterilized), 0.7 mg l-1 BA and 0.7 mg l-1 kinetin. This culture medium alone was effective in preventing the oxidation of phenolic compounds present in explants, and in producing up to 13 rooted plantlets from a single meristem within 10 to 12 weeks. Plants derived from heat-treated meristems of infected plants were free from the disease, as determined by visual inspection, mechanical inoculation to Cucumis sativus, and electron microscopy.

Tissue Culture Technology in the Rapid Clonal Propagation of Japanese White Birch

Tissue Culture Technology in the Rapid Clonal Propagation of Japanese White Birch

A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (Banana and plantain)

A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions is presented in this paper. Shoot-tip cultures of Musa cultivars (both banana and plantain) are induced by culturing small excised shoot apices on modified MS semisolid medium supplemented with various concentrations and combinations of auxins and cytokinins. The effects of cytokinin concentration in the medium as well as the genotypic configuration of the cultivars on the rate of shoot-bud proliferation have been tested. The established shoot-tip cultures grown on modified MS semisolid medium supplemented with IAA (0.18 mg/l) and BA (2.30 mg/l) have been successfully stored at 15°C with 1000 lux light intensity up to 13-17 months depending on the cultivar. The cultivars tested in the present investigation seem to vary in their ability to withstand minimal growth temperature.

Direct Somatic Embryoid Formation on Immature Embryos of Trifolium repens, T. pratense and Medicago sativa, and Rapid Clonal Propagation of T. repens

Direct Somatic Embryoid Formation on Immature Embryos of Trifolium repens, T. pratense and Medicago sativa, and Rapid Clonal Propagation of T. repens

Handbook of Plant Cell Culture. Volume 1. Techniques for Propagation and Breeding.

TORRE YA Handbook of Plant Cell Culture. Volume 1. Tec hniques for Propagation and Breeding. EdiLed b}' D. A. Evans, \V. A. Sharp, P. V. AmmiraLo, and Y. Yanell. 470 pages. ~lcMillan Publishing Co. 1983. As far back as 1949, Gavino Rotor, at thaL Lime a studenL at Cornell University, reported on the regeneration of whole plants from axillary buds on Phalaenopsis (Orchi- daceae) flower stem sections. As far as we can dcLermine, this was the first instance of micropropagation (i.e., the successful use of Lissue culture methods for rapid clonal propagation of plants). Approximately 10 years later, Georges Morel in France and Donald Wimber in the United States re- ported separately the development of prac- tical methods for rapid clonal propagation of Cymbidium (Orchidaceae). The signifi- cance of the e Lechniques became instantly apparent to orchid growers who adopLed them and exLended them to other genera. This and Lhe pioneering work of Toshio Murashige led Lo a veritable explosion in tissue culture research both in tem1s of basic sLuclies and as a means of rapid clonal propagation. Cell and prowplast cultures were not far behind. The result is a vast literature on the subjecL and a plethora of techniques, media, and approaches-all of them scattered through the literature. l n practical terms, this proliferation meant long searches for t he right tech- nique or unnecessary repetition of re- search. Computer searches facilitate the process, but one must still locate the papers listed by the machine and tllis can take time. Also, it may become necessary to request reprints or use interlibrary loan services. Thus, convenient sources of information are needed, especially if they take the handbook, manual, or cookbook approach and couple it wiLh relevant literature re- views. The intent of the editors of this book seems to be to upply critical literature re- view , detailed protocols of techniques, and applications for improvement of crop plants. This they have succeeded in doing, but not completely. For example, orchids and taro, plants of special interest to us, are barely mentioned in this volume despite their eco- nomic importance and the use of tissue cul- ture in their propagation . The question, then, i which other plant are also missing? But, then, thi is onlr the first volume in a series and future volumes mar have more extensive coverage of the Orchidaceae, the Araceae, and other important planes. The title of the book is not fully rep- resentative of its contents because it is more than a handbook. l t contains reviews on organogenesis, embryogenesis, biochemis- try of hormone action, and extensive lists of literature. And, it is not really limited to cell cultures, with chapters and discussions on shoot tip explams, callus masses, eval- uation of d isease resistance and coffee rust, a11d breeding for nematode resistance. Prefatory chapters by noted scientists are always imercsting to read and to that extent the contribution by F. C. Stewart is most welcome (especially since there is an orchid plant in the background of his pho- tograph). This book contains numerous tables, protocols, and recipes. From Lhis point of view it is a gold mine, but ince details are not alway given, the reader may often have to seek the primary source. Therefore, in some instances the handbook is a valuable shortcut to the literature rather than a di- rect ource. The only major problems with the handbook are the indices; they are simply not detailed enough for a book of this type. Altogether, such a handbook has long been needed and we are sure that this one will be of help to many workers in need of in- formation on the su~jects it covers. Its flaws can be corrected in the last volume which cou ld include more deLailed indices and in- formation on species and families which were omitted from the present one. - JOSCPJ I ARD!Trl AND LESLIE P. NYMAN, Developmental and Cell Biology, Univer- sity of California, Irvine 92717. The Biology and Ecology of Mangroves. Edited by Howard J. Teas. viii - 188 page . Ta k for VegetaLion Science, Serie Number 8. Di-. W. Junk Publish- ers, The Hague. 1983. $64.00. This thin tome is the first of two books planned for publication on mangroves in the Tasks for Vegetation Science Series. The second will supposedly deal with Physi-

Rapid clonal multiplication of Angelonia salicariefolia through tissue culture

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

Micropropagation of myrtle

Rapid clonal propagation of a local cultivar of myrtle ( Myrtus communis L.) was achieved in vitro using tips excised from vegetative shoots of outdoor-grown plants. Optimum shoot proliferation was obtained on a medium containing half-strength Murashige and Skoog (1962) (MS) salts plus (in mg l −1): nicotinic acid, 0.5; pyridoxine HCl, 0.5; thiamin HCl, 0.1; glycine, 2.0; inositol, 100; sucrose, 30 000; BA, 1.5; NAA, 0.1. Transfer of excised shoots to half-strength MS salts plus vitamins, and supplemented with 0.5 mg l −1 BA and either 0.4 or 0.6 mg l −1 NAA, resulted in good root development. Plants were successfully transferred to soil.

A tissue culture method for rapid clonal propagation of mature trees of Eucalyptus torelliana and Eucalyptus camaldulensis

Multiple shoots were induced from nodal segments of mature Eucalyptus torelliana F. Muell. and E. camaldulensis Dehnh trees on Murashige and Skoog's medium supplemented with Kn, BAP, Cal.Pan and Bio. Incubation in semi-solid media at 15°C with continuous illumination followed by growth in agitated liquid media was essential for shoot induction and development in primary explants of E. camaldulensis. For culture of E. gorelliana, growth in agitated liquid media alone was sufficient. Rooting could be induced in shoot cultures of E. torelliana by treatment with NAA whereas treatment with a mixture of IAA, IBA, IPA and NAA was essential for E. camaldulensis. After auxin treatment, transfer to a charcoal-containing medium was necessary. Rooted plantlets could be successfully transferred to pots and field. By this method it is estimated that about 50,000 plantlets of E. torelliana and 20,000 of E. camaldulensis can be produced, in a year, from a single nodal segment of a mature tree.

In vitro propagation of willows (Salix spp.), European mountain-ash (Sorbus aucuparia L.) and black locust (Robinia pseudoacacia L.)

Willows were rapidly propagated by repeated division of cultured rooted shoots into a larger number of nodal segments. Rapid clonal propagation of mountain-ash and black locust was achieved by induction of shoots from axillary buds and a multiple shoot culture was used for rapid multiplication. Excised shoots were rooted in an agar medium with a low concentration of auxin and rooted plantlets were transplanted to soil.

Micropropagation of Difficult-to-Propagate Almond (Prunus Amygdalus, Batsch) Cultivar

Summary An in vitro culture scheme has been developed which allows rapid clonal multiplication of shoots starting with excised shoot apexes (0.4-0.7 mm) from flushing buds of a “difficult-to-root” almond cultivar, “Ferragnes”. Up to 55% of these shoots could then be rooted in vitro for establishment in the soil. After an initial 3–4 subcultures on media allowing slow-growth, shoots were placed on Murashige and Skoog (MS) medium with 0.9% agar, 0.7 mg/l 6-benzylaminopurine (BAP) and 0.01 mg/l α-naphthaleneacetic acid (NAA). Shoot multiplication rates of six could be obtained with 20-day periods of subculture continued for at least 24 months. At any points these shoots could be elongated by placing them on identical medium, lowering the BAP (0.2 mg/l) and omitting NAA. Each shoot was then reduced to 2-3 leaf microcuttings for rooting tests. Rooting was induced quickly in the dark in about 55% of the microcuttings using the medium of Bourgin and Nitsch (BN) with only the macronutrients reduced to half, but containing 0.9% agar and 1 mg/l NAA or 1 mg/l γ-(indole-3)-butyric acid (IBA). Further proliferation of roots was successfully achieved in the liquid medium in the absence of any added auxin; sterile vermiculite served as the support. Some plants have been established in the soil.

Propagation of fern (Nephrolepis) through tissue culture

Rapid clonal propagation in three stolon bearing species of Nephrolepis was achieved by tissue culture methods. Explants from young stolons were grown on B5 (Gamborg et al. 1968) medium supplemented with indolebutyric acid where they produced several leaf primordia. These leaf primordia were isolated and grown into entire plantlets. An overall multiplication factor of 30-40 was attained within three months.

Effect of various growth regulators on growth in vitro of cherry shoot tips

An investigation was undertaken to examine the effects of nine different growth regulators on growth in vitro of shoot cultures of the semi-dwarfing cherry rootstock Colt (Prunus avium × P. pseudocerasus). The effects of each supplement on shoot extension and proliferation and also leaf and callus production were noted, and it was found that BAP has the ability to proliferate shoots, IBA nullifies this effect and that kinetin, ABA, GA3 and ethylene inhibit the growth of colt cultures. Conditions were established which resulted in a) optimum shoot growth prior to subsequent rooting or grafting; b) maximum shoot proliferation for rapid clonal multiplication and c) minimum shoot growth. This study will form the basis of an investigation into germplasm conservation of temperate fruit trees by both cryogenic storage and minimal growth techniques.

CLONING HIGHER PLANTS FROM ASEPTICALLY CULTURED TISSUES AND CELLS

Summary1. For a number of years considerable attention has been given both in the scientific and lay press to dramatic advances in the rapid clonal multiplication of plants by means of so‐called ‘modern’ tissue culture techniques. Because the words clone, cloning and tissue culture mean different things to different people, there is considerable confusion even among biologists as to what has actually been achieved.2. An historical approach has been used to put the accomplishments into perspective.3. Organ culture, tissue culture, cell or suspension culture and protoplast culture all offer varying degrees of potential for clonal multiplication but the most substantial progress has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These, in turn, can be separated or subdivided and induced to root and thus yield populations of genetically and phenotypically uniform plantlets.4. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. This method of stimulating organ development on callus is based on the now classic work of Skoog and Miller done in the I950s with the Wisconsin 38 strain of a ‘Havana’ cultivar of tobacco. Because of the growing criticism that when callus cultures are maintained for more than a few subcultures genetic or chromosomal aberrations are induced and these nullify the ultimate goal ‐ namely to get large numbers of genetically identical plants ‐ callus procedures are generally being relinquished in favour of precocious axillary branching methods.5. The number of plantlets obtainable through the procedures with precocious axillary shoots or callus culture are fewer, in theory, than the number of plantlets obtainable from a somatic embryo system starting with cells grown in suspension culture. But the fact is that cell culture techniques required to produce somatic embryos are also demanding and very much in their infancy as far as their applicability to clonal multiplication is concerned.6. Even so, slowly but steadily, advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension culture. In cases where it has proven feasible, not only may somatic embryos be generated in great quantities, but their discrete bipolar nature often obviates the need for time‐consuming, laborious procedures to assure their success in a conventional soil or greenhouse setting.7. While protoplast cultures are of very limited direct use for clonal propagation of higher plants, the procedures hold some hope as a vehicle whereby select useful genes can be introduced into plants. Parasexual hybridization, that is the production of hybrids by fusing totipotent protoplasts of different origin, offers no imminent prospects for the development of useful and novel plant types but may be applicable in the long term in very specific situations.8. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits that could accrue when investigators learn to overcome these problems far outweigh any disadvantages that they may have. In short, cloning via cultured free cells should be seen in the context of potentialities rather than limitations.