Bulblet Growth Research Articles

Response of Plant Growth Substances and Bio-enhancer on the Enhancement of Bulblet Growth under the In-vivo Condition of Lilium

Lilies are one of the most extensively sold cut flowers due to their quantity and variety of colours [1]. This this study aims at development of bulbs from microbulblets by the use of different bio-enhancers and growth substances to decrease the time for bulblet growth to bulb and ontogenic development. The experiment was carried out in the Department of Horticulture, Sikkim University in the year 2016-2017 to find out the effect of growth substances and bio enhancers on the enhancement of microbulblets in vivo condition of Lilium spp. This experiment was conducted using completely randomized block design (CRD) which was replicated thrice and the details of the treatments were as follows: T1-Paclobutrazol @10ppm, T2-Paclobutrazol @20ppm, T3-Paclobutrazol @ 30ppm, T4-Thiourea @10ppm, T5-Thiourea @20ppm, T6-Thiourea @30ppm, T7-Cow urine @10%, T8- Vermiwash @100%, T9- Vermiwash @50% and T10- Control. The treatments were sprayed at 15, 30 and 45 days intervals. Among all treatments used for the development of bulbs from microbulblets, the performance of the growth substance was found more effective. The treatment thiourea @30ppm (T6) and paclobutrazol 20ppm (T2) recorded maximum bulb weight (1.38g and 1.27g respectively while paclobutrazol @20ppm observed maximum bulb length (20.01 cm) and breadth (15.46 cm) after 90 days of the spray of treatment under. However, organic bio enhancers like vermiwash 100% (T7) and vermiwash 50% (T8) were found at par with the treatments of thiourea @30ppm and paclobutrazol @20ppm. The maximum root length (7.70 cm) was found in vermiwash 50% (T8) while the maximum shoot length (17.38 cm) was given by thiourea @30ppm (T6). The maximum amount of carbohydrate accumulated (76.31 mg/g) was observed in thiourea 10ppm (T4) after 60 days of the spray of treatments, which was at par with vermiwash 100% (T7) and vermiwash 50% (T8). After 90 days of the spray of treatments, the concentration of carbohydrates in the bulbs did not differ significantly where the maximum value of 78.97 mg/g was observed in cow urine @10% (T7). Maximum moisture content after 90 days of spray of treatments was observed in thiourea @10ppm (T4) (65.76%). Keeping in view of the results obtained in the study for the regeneration of microbulblets from scale and development of bulbs from microbulblets, bio enhancers like vermiwash and cow urine can be used as substitutes for the chemical growth substances like paclobutrazol and thiourea in organic farming and can be recommended to the farmers.

Molecular cloning, characterization and expression analysis of three key starch synthesis-related genes from the bulb of a rare lily germplasm, Lilium brownii var. giganteum.

Starch is the predominant compound in bulb scales, and previous studies have shown that bulblet development is closely associated with starch enrichment. However, how starch synthesis affects bulbification at the molecular level is unclear. In this study, we demonstrate that Lilium brownii var. giganteum, a wild lily with a giant bulb in nature, and L. brownii, the native species, have different starch levels and characteristics according to cytological and ultra-structural observations. We cloned the complete sequence of three key gene-encoding enzymes (LbgAGPS, LbgGBSS, andLbgSSIII) during starch synthesis by rapid amplification of 5' and 3' complementary DNA (cDNA) ends (RACE) technology. Bioinformatics analysis revealed that the proteins deduced by these genes contain the canonical conserved domains. Constructed phylogenetic trees confirmed the evolutionary relationships with proteins from other species, including monocotyledons and dicotyledons. The transcript levels of various tissues and time course samples obtained during bulblet development uncovered relatively high expression levels in bulblets and gradual increase expression accompanying bulblet growth. Moreover, a set of single nucleotide polymorphisms (SNPs) was discovered in the AGPS genes of four lily genotypes, and a purifying selection fashion was predicted according to the non-synonymous/synonymous (Ka/Ks) values. Taken together, our results suggested that key starch-synthesizing genes might play important roles in bulblet development and lead to distinctive phenotypes in bulblet size.

Functional analysis and expression patterns of members of the FLOWERING LOCUS T (FT) gene family in Lilium

Lilium is an important commercial flowering species, and there are many varieties and more than 100 species of wild Lilium. Lilium × formolongi is usually propagated from seedlings, and the flowering of these plants is driven mainly by the photoperiodic pathway. Most of the other lily plants are propagated via bulblets and need to be vernalized; these plants can be simply divided into pretransplantation types and posttransplantation types according to the time at which the floral transition occurs. We identified three Lilium FLOWERING LOCUS T (LFT) family members in 7 Lilium varieties, and for each gene, the coding sequence of the different varieties was identical. Among these genes, the LFT1 gene of Lilium was most homologous to the AtFT gene, which promotes flowering in Arabidopsis. We analyzed the expression patterns of LFT genes in Lilium × formolongi seedlings and in different Lilium varieties, and the results showed that LFT1 and LFT3 may promote floral induction. Compared with LFT3, LFT1 may have a greater effect on floral induction in Lilium, which is photoperiod sensitive, while LFT3 may play a more important role in the floral transition of lily plants, which have a high requirement for vernalization. LFT2 may be involved in the differentiation of bulblets, which was verified by tissue culture experiments, and LFT1 may have other functions involved in promoting bulblet growth. The functions of LFT genes were verified by the use of transgenic Arabidopsis thaliana plants, which showed that both the LFT1 and LFT3 genes can promote early flowering in Arabidopsis. Compared with LFT3, LFT1 promoted flowering more obviously, and thus, this gene could be an important promoter of floral induction in Lilium.

Factors affecting in vitro bulblet growth of Lilium orientalis ‘Stargazer’ and ‘Casablanca’

ISHS XXX International Horticultural Congress IHC2018: II International Symposium on Micropropagation and In Vitro Techniques Factors affecting in vitro bulblet growth of Lilium orientalis 'Stargazer' and 'Casablanca'

Factors Affecting Bulblet Growth of Lilium sp. - Tracking Ontogenic Development and Bulb Production in vitro

Poor growth of bulblet and ontogenic development are the major problems of adventitious bulb production of lilies in vitro through explant culture. Ontogenic development from the juvenile phase to the adult vegetative phase strongly correlates with bulblet growth of lily, and it is important to understand how lily bulblets grow in vitro. This study was at aimed to determining an effective in vitro culture process for lily to track bulblet growth, ontogenic development, and bulb production. In the results, bulblet performance was higher with the increase in the amount of medium; however, bulblet size was not satisfactory and was in the juvenile phase under an inducing condition. The threshold weight at which 100% ontogenic development was about 300 mg. Avoiding depletion of sucrose in the media is the key to achieving desired bulblet growth and ontogenic development in vitro and indicates growth could be sustained by subculture. The circumference of bulblets increases with its weight after subcultured in vitro. Therefore, a threshold circumference of about 3.2 cm in proportion to bulblet fresh weight of 300 - 350 mg may be an indicator of ontogenic development.
 Plant Tissue Cult. & Biotech. 30(1): 1-13, 2020 (June)

Growth of Lily Bulblets In Vitro, a Review

In micropropagation of lily, preferably bulblets should be produced: Because bulblets are compact and robust, they are much easier to handle and to plant in soil than shoots. In this review, the various factors that determine bulblet growth in vitro are discussed. Gibberellins, jasmonates (JA) and abscisic acid (ABA) are the major identified plant growth regulators (PGRs) for storage organ formation. They also play a major role in lily bulblet growth in vitro. Growth conditions such as temperature and light (quantity and quality) strongly affect lily bulblet growth in tissue culture. Moderate abiotic stresses are introduced as new tool to improve storage organ formation in vitro. The amounts of endogenous carbohydrates (starch) in the explant and exogenous carbohydrates in the medium (sucrose) influence bulblet growth in vitro. It is also discussed how compounds present in the medium or in the scale-explants are translocated to the regenerating bulblet.

Differential Effects of Paclobutrazol on the Bulblet Growth of Oriental Lily Cultured In Vitro: Growth Behavior, Carbohydrate Metabolism, and Antioxidant Capacity

Bulblet growth is crucial for global lily production, and the applications of plant growth regulators have proven effective but with poor understanding of mechanisms. The aims of this study were to identify the effects of paclobutrazol (PBZ) on tube lilies. Low PBZ concentrations (5 × 10−4 mM, LPBZ) stimulated bulblet biomass, whereas higher doses suppressed the growth of the leaves and roots. Soluble carbohydrate and starch contents increased significantly with increased PBZ dose. The activities of adenosine 5′-diphosphate glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), and soluble starch synthase (SSS) increased dramatically in response to PBZ treatments at later growth stages (60 days after transplanting) when carbon starvation occurred. In contrast, GBSS activity was enhanced throughout the whole growth period, indicating that the starch increase was attributed mainly to amylose synthesis. Carbohydrates were utilized more efficiently following LPBZ, with a relative bulblet weight of approximately 77.07%, which potentially ensured the source-sink balance. Alternatively, more carbohydrates were stored in response to high PBZ concentration. Intriguingly, PBZ usage significantly increased the activities of ascorbate peroxidase, catalase and glutathione reductase at the early stages, implicating a possible role in the elimination of ROS to maintain homeostasis. Application of LPBZ resulted in the largest bulblet, which weighed 396 mg (2.5 times that of the control) and measured 10.70 mm in diameter. The detailed characterizations of the bulblet swelling mechanism using PBZ in vitro offer suggestions of future PBZ usage in other bulbous crops.

Factors affecting bulblet growth of Lilium sp. in vitro and in vivo

Factors affecting bulblet growth of Lilium sp. in vitro and in vivo

Effects of exogenous hormones and cold storage on Asiatic lily ‘Gironde’ × ‘Renoir’ bulblet growth and development

Asiatic hybrid lily (Lilium) is an excellent cut flower because of its various types and colors. Also, because of its strong cold tolerance, Asiatic hybrid lily is a superior choice to breed new cultivars. A group of bulblets, obtained from hybridizing Lilium Asiatic 'Gironde' and Lilium Asiatic 'Renoir', was treated in vitro with nine different concentrations of exogenous hormone (abscisic acid (ABA), gibberellic acid 3 (GA3), and 6-benzylaminopurine (6-BA)) solutions, then stored at 4°C for 5 weeks. The effects of exogenous hormone levels and low temperature on further bulb growth were studied. Exogenous ABA could restrain the increase of soluble sugar during the low-temperature treatment, especially when the ABA content reached 0.2 mg L-1. Meanwhile, the exogenous hormone GA3 raised the soluble sugar content remarkably. The relative content of carbohydrates is directly related to bulb growth and development after transplanting, thus, we can draw the conclusion that exogenous hormone (GA3, ABA) treatment could have a significant impact on plant height and leaf number of bulblets after transplanting. Furthermore, a decreasing amount of starch and high accumulation of soluble sugar in outer scales of bulblets were found in the process of dormancy breaking of Asiatic hybrid lily 'Gironde' × 'Renoir'.

Study on the in vitro growth of Lily Sorbonne bulblet

In this study, the effects of light and dark condition, sucrose concentration, and plant growth regulators on the in vitro growth of bulblet from Lily Sorbonne shoot were studied. Morphological, physiological and biochemical changes in the bulblet growth were analyzed. Shoots in the light condition developed into plant while shoots in continuous darkness formed bulblet. Sucrose (90 g/L) increased the size of scale and starch accumulation of bulb. 6-Benzylaminopurine (BA 1,5 mg/L) stimulated the divison of parenchyma cell. The combination of 90 g/L sucrose and 1,5 mg/L BA in dark condition stimulated the division and size of the parenchyma cells, which increase the number and size of scales and size and weight of the bulblet. Respiratory rate, zeatin content and starch accumulation were increased in the bulblet growth. The correlation of light condition, sucrose concentration, plant hormone, respiration rate, starch accumulation, and bulblet growth were discussed. The combination of 90 g/L sucrose, 1.5 mg/L BA and 0.5 zeatin mg/L in dark strongly induced the growth of in vitro Lily bulblet, especially the number of scale.

Low humic acids promote in vitro lily bulblet enlargement by enhancing roots growth and carbohydrate metabolism.

Bulblet development is a problem in global lily bulb production and carbohydrate metabolism is a crucial factor. Micropropagation acts as an efficient substitute for faster propagation and can provide a controllable condition to explore bulb growth. The present study was conducted to investigate the effects of humic acid (HA) on bulblet swelling and the carbohydrate metabolic pathway in Lilium Oriental Hybrids 'Sorbonne' under in vitro conditions. HA greatly promoted bulblet growth at 0.2, 2.0, and 20.0 mg/L, and pronounced increases in bulblet sucrose, total soluble sugar, and starch content were observed for higher HA concentrations (≥2.0 mg/L) within 45 d after transplanting (DAT). The activities of three major starch synthetic enzymes (including adenosine 5'-diphosphate glucose pyrophosphorylase, granule-bound starch synthase, and soluble starch synthase) were enhanced dramatically after HA application especially low concentration HA (LHA), indicating a quick response of starch metabolism. However, higher doses of HA also caused excessive aboveground biomass accumulation and inhibited root growth. Accordingly, an earlier carbon starvation emerged by observing evident starch degradation. Relative bulblet weight gradually decreased with increased HA doses and thereby broke the balance between the source and sink. A low HA concentration at 0.2 mg/L performed best in both root and bulblet growth. The number of roots and root length peaked at 14.5 and 5.75 cm, respectively. The fresh bulblet weight and diameter reached 468 mg (2.9 times that under the control treatment) and 11.68 mm, respectively. Further, sucrose/starch utilization and conversion were accelerated and carbon famine was delayed as a result with an average relative bulblet weight of 80.09%. To our knowledge, this is the first HA application and mechanism research into starch metabolism in both in vitro and in vivo condition in bulbous crops.

Hormone and antioxidant responses of Lilium Oriental hybrids ‘Sorbonne’ bulblets to humic acid treatments in vitro

ABSTRACTThe lily cultivar introduction is a very long process and bulblet development a limiting element in the entire cycle. The aim of the present study was to acquire a highly synchronized model system to gain insight into the bulbing process. Subsequently, this system was implemented to quantify the efficacy of humic acid applications to evaluate the hypothesized positive effect on bulblet growth. Based on weight, bulblet production was promoted with low humic acid concentration treatment (0.2 mg/L, LHA), showing 0.47 g weight and 11.68 mm diameter, while inhibitory effects were observed with increased doses. LHA significantly decreased the gibberellic acid content, and a pronounced phytohormone balance (promotive/inhibitive) was observed, which might be beneficial for the translocation of assimilates from the shoot to sink organs (bulblets). Intriguingly, LHA increased superoxide dismutase, peroxidase, ascorbate peroxidase, catalase, and glutathione reductase activities compared with the control during the early development stages, implicating a possible role for elimination of reactive oxygen species, thereby favouring cell expansion. In conclusion, we initially reported the effects of HA on the development of bulbous plants, showing that a relatively low dose markedly increased the bulblets size via positive GA and antioxidant responses. However, the mechanism of action needs further evaluation.

大理百合的组织培养研究

采用离体组织培养的方法对大理百合的快速繁殖进行了研究，结果表明：以大理百合的鳞片为外植体进行离体培养，可获得再生植株，并初步建立了其快速繁殖体系。由试验结果可知，适宜大理百合鳞片诱导不定芽和愈伤组织的培养基为MS + 2.0 mg/L6-BA + 0.5 mg/L NAA + 3%蔗糖；而MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 3%蔗糖更有利于不定芽的诱导；不定芽增殖的培养基以MS + 1.0 mg/L 6-BA + 0.1 mg/L NAA + 3%蔗糖效果较好；不定芽形成小鳞茎的培养基以MS + 1.0 mg/LNAA + 3%蔗糖效果较好；小鳞茎膨大的培养基以1/2MS + 0.3 mg/L NAA + 0.1 mg/L IBA + 12%蔗糖效果较好。 Rapid propagation protocol of Lilium taliense Franch. by in vitro tissue culture was investigated. The results indicated that plant regeneration can be obtained from the bulb scales of L. taliense and a rapid propagation system was established efficiently. Among the media investigated, MS + 2.0 mg/L6-BA + 0.5 mg/LNAA + 3% sucrose is suitable for induction of adventitious bud and callus while MS + 0.5 mg/L6-BA + 0.1 mg/LNAA + 3% sucrose is better for induction of adventitious buds. The medium MS + 1.0 mg/L 6-BA + 0.1 mg/L NAA + 3% sucrose is proper for adventitious bud multiplication; MS + 1.0 mg/L NAA + 3% sucrose is optimum for bulblet formation from the adven-titious buds, while 1/2MS + NAA 0.3 mg/L + IBA0.1 mg/L + 12% sucrose is suitable for the growth of bulblets.

Enlarging bulblet by magnetic and chelating structures of nano-chitosan as supplementary fertilizer in Lilium

The nutrition is the effective component on the increasing size of the bulb in growth period. In this work, carboxymethyl chitosan and magnetic nano-chitosan in a size ≤ 20 nm were synthesized that can be taken up by plant roots. The purpose of this work was to evaluate carboxymethyl chitosan and magnetic nano-chitosan with chelating structures as supplementary fertilizers to increase yearling bulblet growth in Lilium. The application of magnetic nano-chitosan at a concentration of 15 mg·L-1 as compared to its lower concentrations, carboxymethyl chitosan and iron sulphate had better effect on the promoting the growth, development and protecting the shoot, and growth of bulblet. Higher growth and development of shoots and roots by high concentration of magnetic nano-chitosan could affect the enlarging bulblet. There was a positive magnetic effect on the enzyme structures and supplying the essential iron for plant. Furthermore, magnetic nano-chitosan can act as a chelating component to facilitate the uptake of elements.

Effects of growth regulators and activated charcoal on in vitro bulblet multiplication and growth in Galanthus nivalis ‘Flore Pleno’

SummaryThe in vitro multiplication of Galanthus nivalis ‘Flore Pleno’ bulblet clumps was evaluated through three 16-week sub-culture passages on media supplemented with either 1.0 mg l–1 6-benzylaminopurine (BA) and 0.1 mg l–1 naphthaleneacetic acid [NAA; the plant growth regulator (PGR) control], 0.5 mg l–1 BA and 0.05 mg l–1 NAA (PGR/2), or 0.1 mg l–1 BA and 0.01 mg l–1 NAA (PGR/10). At the end of the second sub-culture passage, clumps of bulblets from each of the three PGR treatments were also transferred to conditions to promote bulblet growth (G medium without PGRs) supplemented with 60 g l–1 sucrose and 5 g l–1 activated charcoal (AC). Lowering the PGR concentration during the initial multiplication phase reduced bulblet multiplication and overall culture growth, but did not influence bulblet size, as indicated by the diameter of the largest bulblet. Lowering the PGR concentration during the multiplication phase also reduced the multiplication of bulblets, overall tissue fresh weight, and the production of roots in tissues transferred to bulblet growth conditions. The reduced growth of tissues previously multiplied on PGR/10 medium was not, however, reflected in the growth of individual bulblets, since these were not significantly smaller than bulblets initially multiplied at the higher PGR concentrations. Bulblet sprouting and root growth on the bulblet growth medium were unaffected by prior PGR status in the multiplication phase. The results are discussed in terms of the mode of action of AC on the promotion of bulblet growth.

Micropropagation of Albuca bracteata and A. nelsonii — Indigenous ornamentals with medicinal value

In this study, two species from the genus Albuca (Hyacinthaceae) with ornamental and medicinal properties were micropropagated. Adventitious bulblets of Albuca bracteata were cut into quarters and used as explants to examine the effect of temperature (10, 15, 20, 25, 30 or 35 °C), carbohydrates (glucose, fructose or sucrose at 0, 87.5, 175, 262.5 or 350 mM) and hormones (BA, mTR, NAA, IAA, GA 3, ABA or methyl jasmonate each at 0, 0.1, 1.0 or 5.0 mg/L) on the induction and growth of bulblets. Temperatures above 35 °C completely inhibited bulb formation, while induction at all other temperatures was high. Heaviest and largest bulbs formed at 20 °C. Low concentrations (87.5 mM) of all tested carbohydrates increased bulb induction compared to media without a carbohydrate source, while higher levels decreased bulblet induction. The cytokinins mTR and BA inhibited bulb induction, diameter and mass at moderate (1.0 mg/L) and high (5.0 mg/L) concentrations. GA 3, NAA and particularly IAA promoted bulblet induction, while ABA and methyl jasmonate had no significant effect on the induction or bulblet growth. Leaf material and young inflorescences of A. nelsonii were removed, decontaminated, and dissected into seven explant types: leaves, peduncles, pedicels, whole flowers, tepals, ovaries and anthers. These were placed on MS media without hormones, or containing 0.5 mg/L mTR, 0.5 mg/L NAA or 0.5 mg/L mTR + 0.5 mg/L NAA to establish which explant type and hormone combination promoted shoot formation. Some tepal and pedicel explants were capable of shoot production on media with both mTR and NAA, but peduncle explants produced the most shoots when mTR and NAA were both present in the culture medium. Flowers, leaves, ovaries and anthers were completely unresponsive, irrespective of medium composition. These techniques will aid the further horticultural development of these plants, and can be easily adjusted for other species within the genus to promote conservation.

Eucomis zambesiaca baker: Factors affecting in vitro bulblet induction

Eucomis species having considerable horticultural potential are used in African traditional medicine to treat various ailments. The effects of environmental and physiological parameters on the initiation and growth of bulblets using leaf explants were investigated. These included the effect of temperature (10, 15, 20, 25 and 30 °C), photoperiod (8 h light, 16 h light, continuous light and continuous dark), carbohydrates (sucrose, fructose and glucose) at different concentrations and combinations as well as various plant growth regulators; gibberellic acid (GA 3), indole-3-butyric acid (IBA), napthaleneacetic acid (NAA), N 6-benzyladenine (BA), zeatin and others. Liquid shake and liquid static cultures versus solid cultures were investigated. Maximum number of bulblets per leaf explant was obtained at 20 °C, with an average of 3 bulbs per leaf explants and a bulblet mass of 57 mg. An 8 h light cycle produced 1.38 bulbs per leaf explant, at a mass of 42 mg. Fructose at 3% produced an average of 1.18 bulbs per leaf explant, 3.39 mm wide and weighing 56.6 mg. Of the plant growth regulators, 4.90 µM IBA was found to be the optimum treatment for bulblet induction, with an average bulb diameter of 4.36 mm and a mean bulblet mass of 79.07 mg. Liquid shake cultures exhibited poor growth while bulblet, leaf and root growth was improved in liquid static cultures. Successful micropropagation from leaf explants established that leaf explants can be used as an alternative explant source to bulbs. This protocol allows for the fast and economic mass propagation of Eucomis plants.

Biosynthesis of Lycorine by in Vitro Cultures of Pancratium Maritimum L. (Amaryllidaceae)

ABSTRACTSeeds of Pancratium maritimum collected from the natural populations along Bulgarian Black Sea coast were in vitro germinated in 2005 and cultures were maintained under equal conditions for three years (MS based medium with BAP and NAA; 23±2°C and 16/8 light/dark photoperiod). Directly regenerated bulblets with diameter of about 6–8 mm were cultured in parallel for two months on agar solidified and in liquid media containing 3 mg/L BAP and 0.1 mg/L NAA. Alkaloid determination by HPLC proved biosynthesis of lycorine which antiviral and antitumor properties are well known. Lycorine accumulation was up to 2.90 and 1.53 mg/g DW in the cultures grown on agar and in liquid medium, respectively, which was highly promising as first trials to produce this alkaloid in vitro. Additionally, bulblets were regenerated from callus cultures and comparison was done concerning the morphology of directly and indirectly obtained plantlets. Bulblet growth varied in accordance with the sucrose quantity in the medium as well.

히아신스(cv. Pink Pearl)의 인편 기내 배양시 효과적인 자구의 재생과 생장

The regeneration and growth of bulblets from the bulb scale segments of Hyacinthus orientalis L. cv. Pink Pearl were more efficient in IBA than IAA at the same concentrations (1.0 and 3.0 ㎎/l).. The normal (base-down) orientation of explants was more effective for bulblet regeneration and root growth than the inverted (base-up) orientation. The growth of bulblets and roots was increased higher in the perlite than the agar medium. These results suggested that the alternate culture system, first cultured in the agar medium for bulblet regeneration, and then in the perlite medium for bulblet growth, may be more useful for efficient in vitro culture of hyacinth (H. orientalils) cv. Pink Pearl.

STIMULATION OF IN VITRO BULBLET GROWTH IN GALANTHUS SPECIES WITH SUCROSE AND ACTIVATED CHARCOAL

We report here a reliable method to stimulate growth of in vitro-produced bulblets of Galanthus species to a size suitable for acclimatization. Proliferating bulblet cultures, from bulb chip explants, were induced and maintained on MS medium supplemented with 30 g/L sucrose, 1 mg/L BA and 0.1 mg/L NAA. The six treatments, prepared in plant growth regulator-free MS medium, were 30 or 60 g/L sucrose in all combinations with 0, 1 or 5 g/L activated charcoal. Fifteen replicates per treatment per plant type (G nivalis, G nivalis 'Flore Pleno' and G. elwesii) were inoculated with bulblet clumps containing 2-4 bulblets. Assessments were made after incubation for 16 weeks at 18°C with 16h photoperiods. Even in the absence of plant growth regulators the cultures continued to form new bulblets in all treatments. Bulblet growth was greatly stimulated by the addition of activated charcoal. In contrast, doubling the amount of sucrose in the medium to 60g/L had only a marginal effect on bulblet growth in the absence of charcoal. Addition of charcoal and sucrose together was found to have a synergistic effect on bulblet growth, in that the benefit of adding the higher rate of charcoal could be seen at only the higher sucrose concentration. Charcoal also induced the formation of large numbers of roots and stimulated root elongation. Bulblets had a normal anatomy in longitudinal section, and, although many of the bulbs sprouted in vitro and lateral bulb units developed in larger bulbs, none formed flowers. Thus it is expected that in vitro-produced bulbs will require either flower initiation treatment(s) or a minimum of one season in field conditions to achieve flowering size.