In vitro Studies on Pinus

Hypocotyl tissue from Pinus gerardiana was established in culture on White's basal medium supplemented with 2 % sucrose, 10% (v/v) coconut milk, 500 mg/l casein hydrolysate and 1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). Various organic and inorganic nutrients were studied in order to determine the specific nutritional requirements of the tissue in vitro. Sucrose, glucose and maltose were equally effective as fixed carbon sources. The inorganic nutrient combination of White's medium was found to be better than that of Murashige and Skoog's medium. White's modified basal medium supplemented with coconut milk, casein hydrolysate and 2,4-D was the most successful nutrient combination. Glutamine was as effective as casein hydrolysate in promoting growth of the tissue.     

Coralloid root regeneration on Macrozamia megagametophites

Abstract

Megagametophytes of Macrozamia communis were incubated in White's Basal Medium and in White's Basal Medium modified with 2,4-D and kinetin. On the medium enriched with growth substances, regeneration of coralloid roots was induced. These are morphologically identical to sporophytic coralloid roots, without any endosymbiont and displaying negative geotropism. These results confirm the fact that coralloid roots represent an inherent feature of the root system of the Cycadales rather than being the result of induction by microbial factors. Therefore it is possible to suggest that coralloid roots represent vestigial pneumatophores.     

In vitro studies on Pea (Pisum sativum L.): I. Callus formation,regeneration and rooting

Abstract

Callus production, shoot formation via organogenesis and rooting of the regenerated shoots are reported in an Egyptian variety of Pisum sativum L. Calli were initiated from hypocotyl, leaf, root and mature embryo explants when cultured on MS medium containing B5 vitamins and supplemented with 2 mg/l 2,4-D+1 mg/l kin. Among the different types of explants, hypocotyl showed best potential for callus proliferation. Hypocotyl, leaf and immature cotyledon explants were used for shoot organogenesis. The best results of shoot formation were achieved when hypocotyl explants were cultured on MS-medium supplemented with 2 mg/l BA+1 mg/l NAA. However, immature cotyledon explants showed the highest frequency of shoot formation with 1 mg/l BA. Data of in vitro rooting showed that maximum root frequency occurred on culture medium containing half strength of MS salts, 40 g/l sucrose and 2 mg/l NAA.     

Protoplast isolation,culture and regeneration from Egyptian cultures of Pea and Beans

Abstract

A protocol of protoplast isolation from Egyptian varieties of pea and bean is reported. Protoplast cultures were established from apical shoots of pea (Pisum sativum) and suspension cultures of bean (Phaseolus vulgaris). To isolate protoplasts of pea, apical shoot tissues were digested for 10 h using enzyme solution containing 1% pectinase, 0.5% cellulase, 0.5% hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. For protoplast isolation from suspension culture of bean, collected cells were incubated for 6 h in digestion solution containing 0.5% pectinase, 0.25% of each of cellulase and hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. Purified protoplasts were cultured in liquid culture medium. Microcalli were obtained after 30 days of culture. Calli colonies with a diameter of about 5 mm were developed after one month of culturing on solid B5 medium containing 2% sucrose, 2 g/l casein hydrolysate, 0.7% agar and supplemented with either 1 mg/l of each 2,4-D and kin in case of pea or 2 mg/l 2,4-D+0.5 mg/l kin in case of bean. Protoplast derived callus of pea was successfully differentiated into shoot and root, and highest frequency of shoot organogenesis was recorded on medium containing 0.5 mg/l NAA+2 mg/l BA. Protoplast derived callus of bean, on the other hand, gave rise to a high frequency of root formation when cultured on medium containing 1 mg/l NAA, but attempts to regenerate shoots from this callus was unsuccessfull.     

Tissue Cultures of Phaseolus vulgaris L.

Abstract

Callus production and plant regeneration from different explants of Phaseolus vulgaris L. cv. Giza are reported. Calli cultures were induced from leaf, hypocotyl, embryo and root explants. Rapid growth of callus was achieved by leaf explants cultured on MS salts, B5 vitamins and supplemented with 2,4— dichlorophenoxyacetic acid (2, 4—D)+0.5 mg/l kinetin (kin). Addition of casein hydrolysate at 2 g/l to maintenance medium enhanced callus growth and hindered the early appearance of necrotic parts. This report also provides a detailed method for production of multiple shoots directly from the wounded edges of immature cotyledon explant via organogenesis on 1 mg/l benzyladenine (BA) or indirectly on 0.5 mg/l naphthaleneacetic acid (NAA)+2 mg/l BA. The regeneration of bean plants through the two ways described here (direct or indirect) may be of use in genetic improvement of bean.     

A simple and efficient plant regeneration system for leaf protoplasts ofNierembergia repens by inducing single shoots on the microcolonies

A simple and efficient protocol for plant regeneration from mesophyll protoplasts ofNierembergia repens is described. The protoplasts divided in modified half-strength MS (/12 MS) medium containing benzylaminopurine (BA) and a-naphthaleneacetic acid (NAA) and formed visible colonies after 2 weeks which produced single adventitious shoots 4 weeks later. Plating efficiency (11.2%), percent colony formation (0.84%), and the number of shoot-forming colonies (368/dish) were highest in /12 MS containing 0.1 mg/l BA and 0.05 mg/l NAA. However, the percentage of colonies with shoot formation was highest (31.8%) in /12 containing 0.05 mg/l BA and 0.01 mg/l NAA. Almost all of the remaining colonies (97.5%) also regenerated shoots upon transfer onto MS medium containing 0.05 mg/l BA. The shoots with 2–3 leaves readily rooted 3–5 days after insertion in /12MS lacking plant growth regulators. Regenerated plantlets were easily established in soil 50 days after protoplast isolation. All the regenerants were normal and possessed diploid chromosome numbers.Abbreviations BA Benzylaminopurine - FDA fluorescein diacetate - MES 2-(Morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) medium - /12MS half strength MS - NAA -naphthaleneacetic acid - PGR plant growth regulator     

Nutrient Requirements of Vigna sinensis Callus Cultures

Nutrient requirements of callus tissues previously obtained from Vigna sinensis hypocotyl segments were investigated on modified White's (Miller and Skoog) and Murashige and Skoog's media containing 1 mg/1 of each of 2,4-D and kinetin, 2% sucrose and 1% agar. Yields of tissues on the latter medium were 3 times more than the former. After studying various organic and inorganic nutrients, two synthetic media have been developed for callus cultures. The above modified White's medium further supplemented with 2% sucrose, and 500 mg/1 of urea or asparagine was suitable for a compact callus with a dry-to-fresh-weight ratio around 8% and on this the tissues grew 15- to 17-fold in 5 weeks. Similarly the Murashige and Skoog's medium further enriched with 2% sucrose, 400 mg/1 of urea or a mixture of aspartic acid (750 mg/1), glutamic acid (250 mg/1), asparagine (250 mg/1) and arginine (250 mg/1), was apt for a friable callus with a dry to fresh weight ratio of 3 to 4% and it promoted a 45- to 50-fold increase in growth in 5 weeks.     

In vitro flowering and pod formation from cotyledons of groundnut (Arachis hypogaea L.)

Summary The response of groundnut cotyledons to the presence of various growth regulators in concentrations from 0.1 to 5 mg/l has been studied in detail using several genotypes of groundnut on two different media. Cotyledons with embryo axis, cultured on Blaydes' medium with cytokinins, produced shoots, in the axils of which 2–7 flower buds could be seen. The frequency of flower bud induction in general increased with increasing concentrations of cytokinins, the optimal levels being 3 mg/l of KN or 4 mg/l of BAP. Cotyledons without embryo axis, cultured on Blaydes' medium with BAP (0.5 mg/l), produced a cluster of flower buds directly, ranging in number from 8–28, without any vegetative growth. Excised embryo axes cultured on the same medium gave plantlets without flower buds. The growth regulators IAA, NAA, GA₃ and ABA failed to induce flower buds in independent treatments. However, lower concentrations of IAA and NAA in combination with cytokinins exerted a positive influence on flowering. The blooming of the flower buds was facilitated on media supplemented with low concentrations of cytokinins. Six percent of the induced flowers resulted in gynophore development and ultimately formed pods when cultured under complete dark conditions in modified MS medium supplemented with kinetin.     

Differentiation in Callus Cultures of Leaf of Two Species of Trigonella

Callusing of leaf segment of Trigonella corniculata L. and Trigonelta foenum graecum L. was observed on Murashige and Skoog's (MS) medium and White's (W) medium supplemented with naphthalene acetic acid (NAA; 0.5 mg/1) and coconut milk (15% v/v). Root, shoot and development of isolated leaf occurred in the calli of both the species on MS medium containing NAA amd coconut milk. Omission of coconut milk with subsequent addition of casein hydrolysale (CH) increased the number of differentiated organs per callus. In both the materials higher differentiation rate was observed at low irradiance than at high irradiance or in complete darkness. Initially the cells were diploid but at the 5th passage the calli became mixoploid wilh predominantly diploid cell populations. The differentiated organs were diploid.     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

In vitro Post-germination Growth and Development of Embryos of Alectra (Scrophulariaceae)

In vivo, seeds of the obligate root parasite Alectra vogelii Benth. (Scrophulariaceae) germinate only after being soaked in water for a period of time (pretreatment) followed by stimulation by certain factors exuded from a host root. Germinated seedlings do not develop beyond radicle emergence, and finally die, unless their radicles make contact with and penetrate into a host root conductive system. In vitro, germinated embryos obtained by exposing sterilized and pretreated seeds to root exudate of Vigna unguiculata were aseptically cultured on Knop's, White's and Murashige and Skoog's media. The embryos grew into seedlings with shoots and roots on a medium containing mineral salts and sucrose, but not on mineral salts alone. Seedling performance was generally not improved when the mineral salts-sucrose media were supplemented with vitamins. Shoot extension growth was better on Murashige and Skoog's mineral salts-sucrose medium than on Knop's or White's medium. However, seedling development was greatly boosted when cultivated on White's minerals salts-sucrose medium supplemented with coconut milk. Seedlings turned green on transfer to light but did not flower. The successful culture of these embryos and seedlings on a simple, chemically defined medium of mineral salts and sugar suggests that these nutrient components are the minimal external requirements for stimulation and support of normal seedling growth. These may be obtained in vivo by the parasite's tapping of the host root conductive system.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of North American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.)

North American ginseng (NAG) (Panax quinquefolius L.) is a medicinally important plant with multiple uses in the natural health product industry. As seed propagation is time-consuming because of the slow growth cycle of the plant, in vitro propagation using a bioreactor system was evaluated as an effective approach to accelerate plant production. An efficient method was developed to multiply nodal explants of NAG using liquid-culture medium and a simple temporary immersion culture vessel. The effects of plant growth regulators, phenolics, and chemical additives (activated charcoal, melatonin, polyvinylpolypyrrolidone, and ascorbic acid) were evaluated on in vitro-grown NAG plants. The highest number (12) of shoots per single node was induced in half-strength Schenk and Hildebrandt basal medium containing 2.5 mg/l kinetin, in which 81% of the cultured nodes responded. In a culture medium with 0.5 mg/l α-naphthalene acetic acid (NAA), roots were induced in 78% of the explants compared to 50% with a medium containing indole-3-acetic acid. All of the resulting plants appeared phenotypically normal, and 93% of the rooted plants were established in the greenhouse. Phenolic production increased significantly (P < 0.05) over a 4-wk culture period with a negative impact on growth and proliferation. Activated charcoal (AC; 50 mg/l) significantly reduced total phenolic content and was the most effective treatment for increasing shoot proliferation. Shoot production increased as the phenolic content of the cultures decreased. The most effective treatment for NAG development from cultured nodal explants in the bioreactor was 2.5 mg/l kinetin, 0.5 mg/l NAA, and 50 mg/l AC in liquid culture medium. This protocol may be useful in providing NAG tissues or plants for a range of ginseng-based natural health products.     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid     

GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.     

In vitro propagation of shoot buds of Carica papaya L. (caricaceae) var. Honey Dew

Shoot buds from the saplings and the fruit bearing plants of Carica papaya L.. var. Honey Dew (papaya) initially treated with Gentamycin were cultured in modified MS media, each with a different hormonal combination, for the establishment of cultures and multiplication and rooting of plants. About 43% of explants from fruit bearing plants and 69% of those from saplings remained free of contamination and retained regeneration capacity when treated in 500 mg/l Gentamycin. For the establishment of the explants a medium containing 1 mg/l GA₃ and 2 mg/l kinetin was necessary. When established buds were transferred to medium containing 1 mg/l NAA and 3 mg/l kinetin, calli were initiated at cut ends of shoot buds; multiplication started on transfer to NAA (0.1 mg/l) and BAP (0.5 mg/l) medium. Cultures have been maintained for the last twenty months without any loss in multiplication rate. Rooting was induced in medium with reduced salt concentration containing 2 mg/l IBA. Shoot elongation was induced after prolonged culture in the same rooting medium.Abbreviations MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - GA₃ Gibberellic acid - Kn Kinetin - NAA Napthaleneacetic acid - BAP 6 -Benzylaminopurine - IBA Indole-3-butyric acid - IAA Indole-3-acetic acid     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

Rapid propagation of lemongrass (Cymbopogon flexuosus (Nees) Wats.) through somatic embryogenesis in vitro

Somatic embryos induced from callus cultures of lemongrass [Cymbopogon flexuosus (Nees) Wats.] on Murashige and Skoog medium supplemented with 5 mg/l of 2,4-D, 0.1 mg/l of NAA and 0.5 mg/l of Kn developed into plantlets when plated on a medium supplemented with 3 mg/l of BA, 1 mg/l of GA₃ and 0.1 mg/l of NAA. The regeneration potential of callus was retained for more than 2 years on the nutrient medium supplemented with comparatively lower levels of growth regulators (2,4-D at 2 mg/l, NAA at 0.1 mg/l and Kn at 0.25 mg/l). Approximately 30–35 plantlets were produced after two months of culture per 100 mg of callus inoculated. Regenerants were transplanted into soil and transferred to the field for assessment of various morphological and biochemical characteristics. The results of 1 year of field trials showed that plants derived from somatic embryoids were more uniform in all the characteristics examined when compared with the field performance of plants raised through slips by standard propagation procedures. Thus, a procedure has been developed for high frequency long term plant production of lemongrass through in vitro methods.Abbreviations 2,4-D 2,4 -dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberllic acid - MS Murashige and Skoog (1962) basal medium     

Ricerche Sulla Cultura Sommersa di Cellule Singole di Piante Superiori

Abstract

Research on submerged culture of single cells of higher plants. — The author describes a method which allows to obtain submerged cultures of single cells of Phaseolus vulgaris and Nicotiana tabacum. The medium composition in macroelements in the culture on agar appears to effect to a great extent the ability of tissues to dissociate into single cells in the subsequent liquid culture. In this respect Heller's solution results to be more suitable than Gautheret's and Hildebrandt and Ri-ker's.

Cells are grown at 24 [ddot]C in 300 ml flasks containing 60 ml of broth on a rotary shaker at 220 rpm.

To prevent contaminations some antibacterial agents were added to cultures of Phaseolus vulgaris. Among these Penicillin and Neomycin were not tossic at 20 and 5 ppm concentrations respectively.

The presence of septa, which are observed also in largely vacuolate cells, seems to confirm the ability of single cells to divide.

The optimum 2,4-D concentration for growth decreases from 6 × 10^-8 to 6 × 10^-8 during successive liquid cultures, each of them being inoculated with on amount of the previous one. This fact, showing the adaptation of liquid cultures to decreasing concentrations of the growth hormone, is in agreement with previous observations in solid cultures by several authors.     

Effect of plant growth regulators,temperature and sucrose on shoot proliferation from the stem disc of Chinese jiaotou (<Emphasis Type="Italic">Allium chinense</Emphasis>) and in vitro bulblet formation

In vitro shoot proliferation from stem disc of Allium chinense, a vegetatively propagated plant, was investigated in this experiment. In the present study, shoots were formed directly on stem discs on a medium containing 1 mg/l N⁶-benzyladenine (BA) and 0.5 mg/lα-naphthaleneacetic acid (NAA). These shoots were further cultured on MS media supplemented with various levels of BA in combination with NAA, and new shoot clusters developed easily from the explants cultured despite considerable differences in the induction of shoot clusters with different levels of BA and NAA. The most productive combination of growth regulators proved to be 1.0 mg/l BA and 1.0 mg/l NAA, in which about 17 shoots were produced per cluster in 8 weeks culture. Most of the formed shoots were rooted 15 days after being cultured on MS media supplemented with 0.1–1.0 mg/l NAA. The survival rate of the plantlets under ex vitro conditions was 95% in pots filled with a peat: sand (2:1 v/v) mixture after two weeks. In vitro bulblet formation were strongly promoted by the high temperature of 30°C compared to that at 25, 20 and 15°C, and 12% (w/v) sucrose appeared to be optimal for bulblet development. Results from this study demonstrated that A. chinense could be in vitro propagated by using stem discs and in vitro bulblet formation could be achieved.     

Factors affecting shoot initiation from tuber discs of potato (Solanum tuberosum)

Discs of cortical and perimedullary tissue from tubers of potato (Solanum tuberosum L. cv. Superior) formed adventitious shoots when cultured on a medium containing Murashige and Skoog's salts, myo-inositol, 100 mg/l; folic acid, 0.5 mg/l; D-biotin, 0.05 mg/l; gibberellic acid (GA₃), 0.5 mg/l; thiamine ˙ HCl, 0.1 mg/l; glycine, 2.0 mg/l; pyridoxine ˙ HCl, 0.5 mg/l; nicotinic acid, 0.5 mg/l; sucrose, 25 g/l; casein hydrolysate, 1 g/l; Bacto agar, 9.0 g/l; and a cytokinin [N⁶-benzylaminopurine (BAP), N⁶-γ,γ-dimethylallylaminopurine (2iP), or N⁶-furfurylaminopurine (kinetin)]. Discs of pith tissue either failed to survive or produced callus but did not undergo morphogenesis. Cytokinin was essential for explant survival, while BAP at 3.0 mg/l was most efficacious in promoting shoot initiation. Auxin was not essential for shoot initiation or development; however, a low concentration (0.03 mg/l) of α-naphthaleneacetic acid (NAA) stimulated both explant survival and the number of shoots produced per disc. Indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) did not stimulate shoot initiation. GA₃ was essential for both shoot initiation and subsequent shoot development. The highest incidence of morphogenesis (over 100 shoots in 12 weeks) occurred from tuber discs cultured at 18°C constant and under a photon flux density of 60 μE m^-2s^-1. No difference in regenerative ability was found in explants taken from source tubers stored for 0 to 20 weeks at 4°C. A histological examination indicated that shoots developed from small clusters of meristematic cells which were initiated from within small protuberances on the surface of the tuber disc 3 weeks after transfer.