Bulblet Growth Research Articles

DEVELOPMENT OF PILOT SCALE PROCESS FOR MASS PRODUCTION OF LILIUM BULBLETS IN VITRO

Using a two-stage culture process separating bulblet formation and the development stage, the possibilities for a mass propagation system by bioreactor were investigated. The nutrient requirements for bulblet formation and development were varied for each stage. It was revealed that each stage could be controlled by the manipulation of nutrients and culture conditions. By using a two stage culture process, various types of bioreactors were employed for mass production of bulblets. Several thousand bulblets were produced in 5-20 liter batches of non-stirred bioreactors within 60 days. In addition, a considerable increase of bulblet weight was observed. With the passage of time, sugar and nitrogen were consumed rapidly. In particular ammonium was used more preferentially as a nitrogen source than the nitrate ion for bulblet growth.

BULB GROWTH IN LILY REGENERATED IN VITRO

High quality, in-vitro produced bulblets should grow rapidly and flower as fast as possible after transfer to soil. Fast growth occurred in large bulblets and in bulblets that formed a stem with several leaves instead of one or two leaf-bearing scales. Since stem formation occurred more often in large bulblets, we concluded that bulblet growth in vitro is an important factor for rapid growth of lily bulblets after planting. During in vitro culture, growth of the bulblets depended on sucrose concentration. Another important factor was the size of the explant: on a large explant, large bulblets regenerated. The presence of the explant was necessary during the whole regeneration period. When bulblets were excised from the explant after 5-6 weeks, and cultured onto the same medium, growth ceased. We studied changes in carbohydrate content during regeneration in vitro as influenced by explant size and sucrose concentration. We concluded that the contact area of the explant with the medium plays a key role in determining bulblet weight. Interestingly, the heaviest bulblets were regenerated on actively growing explants that accumulated starch.

Production of Commercial Lilium rubellum Baker Bulbs: Effects of Volume and Renewal of Liquid Medium on In Vitro Growth, Bulb Rot Infection during Cold Treatment, and Post-in-vitro Growth of Bulblets.

Bulblets of Lilium rubellum Baker were cultured for 16 weeks in a 20-, 30- or 40-ml liquid medium renewed 0, 1, or 3 times. The bulblets were assessed for growth and rot infection.1. Bulblets with fresh weight of more than 600 mg were obtained by using a 20- or 30-ml medium with 3 renewals, or 40 ml with one renewal.2. Bulblets frequently turned brown when they were cultured in a 30- or 40-ml medium with 3 renewals; they frequently rotted both during a cold treatment and after transplantation.3. Bulblets cultured in a 20-ml medium did not rot after a cold treatment irrespective of the number of medium renewals; they grew well after transplantation to the greenhouse.4. Bulblets cultured in a 20-ml medium with 3 renewals most frequently developed into plantlets with elongated axes 15 weeks after transplantation.These results indicate that the 20-ml medium with 3 renewals appears to be most suitable for bulblet culture of L. rubellum in a liquid medium.

組織培養におけるヤマユリ子球の分化生育に及ぼす培地当り移植リン片数と培養系統の影響

The effects of the number of inoculated bulbscales and the culture strain on differentiation and growth of newly formed bulblets of Lilium auratum Lindl. in tissue culture were investigated.The bulbscales dissected from tissue cultured bulblets were transferred successively to 300 ml flask containing 150 mE modified MS liquid medium, 5, 10, 15, 20, 25 and 30 bulbscales per flask, and cultured for 3 months. Fresh weight of bulblets increased with the decrease in the number of inoculated bulbscales.Regeneration and growth of bulblets were also affected by the culture strain. Established strain collected in Fukushima town, Hokkaido Pref. tended to form multiple bulblets and to show callus induction, but not the strains collected in Mishima city, Shizuoka Pref.

ササユリの母りん片白色カルス由来の子球の発育に及ぼすナフタレン酢酸とベンジルアデニンの影響

ササユリの母りん片白色カルス由来の子球の発育に及ぼすナフタレン酢酸とベンジルアデニンの影響

Regeneration of bulblets from twin scales ofCrinum macowanii in vitro

Tissue culture techniques were applied to study the regeneration and growth of bulblets from bulb scale segments ofCrinum macowanii Bak. (bush- or march lily)in vitro. Shoots were induced on twin scales taken from the basal plate region of flowering-size bulbs on Murashige and Skoog (MS)-medium containing 0–20 mg l−1 NAA and BA and a modified MS medium (MMS medium) containing 1.25 mg l−1 ancymidol (A-RestTM), 0.1 mg l−1 NAA and 0.1 mg l−1 kinetin (ANK). Large bulblets could only be initiated on the latter. Subsequently the bulblets of 5 mm or more in diameter were trimmed and split in half, and secondary plantlets were regenerated on MMS-medium containing ANK or MS-medium without any growth regulators which in turn grew into bulblets suitable for splitting within 12–16 weeks. A total of 700–1000 bulblets could be obtained from each initial bulb within 12 months. Anatomical studies showed that the shoots were initiated from the epidermis and hypodermis on the abaxial surface of the meristematic tissue of the basal plate of the bulb scale. This technique is useful for the multiplication and preservation of a genotype, since plantlets regenerated in this manner should be genetically uniform.

ササユリ球根のｉｎ ｖｉｔｒｏにおける増殖に及ぼす液体通気培養条件の影響

To establish a practical in vitro mass-production system using bulb scales of Lilium japonicum, several cultural methods simplifying the process to enlarge the current system were compared. In addition, the effects of cultural conditions on the differentiation and growth of bulblets in aerated liquid culture were investigated.1. Four cultural methods : (a) liquid medium culture with polyester fiber support; (b) liquid medium culture with horizontal rotation; (c) liquid medium culture with aeration; and (d) capsule culture were tested. Among them, system (c) appeared to be the most practical.2. Bulblet differentiation was observed after 3 weeks of culture. The number of bulblets increased significantly 5 to 8 weeks after culture initiation but not thereafter. No increase in diameter of bulblets was observed after the 5th week of culture, indicating that additional nutrients should be supplemented to the medium after 8 weeks of culture for subsequent growth of the bulblets.3. Bulblet differentiation in aerated liquid culture was enhanced, using the medium with 3/4 strength MS macro-elemetns and 40g• liter-1 sucrose.4. Addition of NAA to the medium promoted bulblet differentiation and its optimal concentration was 0.01 mg liter-1. Addition of kinetin or ABA did not promote bulblet differentiation.5. Bulblets differentiate efficiently when aerated with ambient air (20.9%O2), whereas higher oxygen concentration was ineffective. Effects of temperature and light conditions on bulblet development in aerated liquid culture were similar to those of agar culture.

組織培養によるササユリ子球生産の実用化

Effects of culture conditions on the differentiation and growth of bulblets from bulblet scales of Lilium japonicum in aerated liquid culture were investigated. Based on the results, a practical in vitro mass-production of the bulblets was designed using a simplified culture box.1. The agar culture period of scales to obtain a maximum number of bulblets in the following aerated liquid culture appeared to be about 3 months. When the culture period extended beyond 3 months, the number of bulblets tended to decrease. Basal segments of scales produced more bulblets and at a faster rate than did apical segments.2. Dissecting the in vitro bulbs into small segments increased the total number and hastened the propagation rate of bulblets, compared to scale segments cut from comparable sized in vitro bulbs.3. Using a polypropylene container as a culture box, segments obtained from 90100 in vitro bulbs (total fresh weight, 12.8 g) were cultured in an aerated liquid medium of 1.5 liters. About 2, 000 bulblets, with an average diam. of 4.9 mm, were produced in 16 weeks.4. To simplify the propagation process, 300 in vitro bulbs (total fresh weight, 60 g) were cut with several razor blades spaced 2 mm apart. A 10-liter container with 5 liters of liquid medium was used. In this simplified in vitro system, about 4, 500 bulblets, with an average diam. of 5.9 mm, were mass-produced in 24 weeks.

Rapid clonal propagation of Hyacinthus orientalis bulbs by shake culture

The effect of in vitro culture conditions on regeneration and growth of Hyacinthus orientalis bulbs was investigated. Bulblet regeneration was stimulated on Murashige and Skoog's solid medium containing combinations of 4.4–44 μM benzyladenine (BA) and 0.54–5.4 μM α-naphthaleneacetic acid (NAA), but their growth was slow. To stimulate bulblet growth, cultures were transferred into liquid medium and shake-cultured. The growth of bulblets which had been pre-cultured on agar medium was markedly stimulated by shake culture. Based on the results, a mass propagation scheme for Hyacinthus orientalis using shake culture has been established.

Influence of in Vitro Generation Temperature and Post-in Vitro Cold Storage Duration on Growth Response of Lilium longiflorum Bulblets

Lilium longiflorum Thunb. `Ace' bulblets generated in vitro at 25 or 30C were stored at 4C for O, 1, 2, 4, or 6 weeks after removal from culture and before planting to ascertain the effects of in vitro generation temperature and post-in vitro cold storage duration on bulblet growth responses during 36 weeks of greenhouse growth. Increasing post-in vitro storage duration decreased the number of days to first leaf emergence and percentage of plants producing shoots within 36 weeks, but increased the number of days to shoot emergence and anthesis, leaf number, and flower bud number. The length of time required for bulblet development from planting to shoot emergence was affected by storage duration more than periods from shoot emergence to visible bud and anthesis. It is feasible to produce high-quality L. longiflorum pot plants from in vitro-produced bulblets.

ヒメサユリの成球生産に関する研究試験管内で増殖した子球の生長改善

ヒメサユリの成球生産に関する研究試験管内で増殖した子球の生長改善

ウイルスフリー•ユリ球根の <i>in vitro</i> における増殖

Summary In vitro cultural conditions were investigated for promoting differentiation and growth of bulblets from segments of various parts of plantlets regenerated through shoot-tip culture of lilies (Lilium spp .). The growth and flowering of the in vitro propagated bulblets were observed in soil culture, and diagnosis of virus was also performed under electron microscope and by sap inoculation to L , formosanum seedlings. 1. Bulblet differentiation from various tissue segments and the growth of bulblets were enhanced with addition of auxin, especially 0.1 mg/l NAA, to the medium . Addition of cytokinin to the medium multiplied the differentiation of bulblet primordia (adventitious buds) remarkably, but retarded their development to bulblets. 2. Cutting bulb scales into segments increased total number of regenerated bulblets per scale. 3. High sucrose concentration of the medium (60120 g/l) and high incubation temperature (2728°C) inhibited leaf elongation from bulblets and promoted enlargement of the base of scaly leaves. 4. Although both leaf and stem-node segments regenerated bulblets , bulb scales were most suitable material for in vitro propagation of lilies because of their high regeneration rate of bulblets and simplicity of their culture process. 5. In L, longiflorum, in vitro propagated bulblets flowered without cold treatment within one year after transplanting to soil, and bulbs with marketable size for cutflower production were harvested in the first cultivation in soil . On the other hand, cold treatment to the bulblets of L, speciosum accelerated bolting in soil culture and reduced days to flowering. In the case of L , x formolongo and L, concolor, bulblets propagated by in vitro culture could flower earlier than by conventional cultivation methods in field conditions. Lilies like these two species are considered to be promising materials for practical production of bulblets through in vitro culture. 6. Diagnosis of virus in bulblets derived from shoot-tip culture of L. longiflorum indicated that these bulblets were freed from virus. These results suggested that a large number of virus-free lily bulbs can be propagated in a short time by subculturing bulb scales of in vitro bulblets derived from shoottip culture, and that the cultivation time necessary to flowering-bulb production can be reduced remarkably by use of those bulbs.

The effect of methyl jasmonate and abscisic acid on differentiation of benzyladenine-induced bulblets inMuscari bulbs

Methyl jasmonate (JA-Me) at a concentration of 0.5% in lanolin paste totally inhibited bulblets formation induced by benzyladenine in intactMuscari bulbs. Lower concentrations of JA-Me delayed development and growth of bulblets induced by benzyladenine. It seems that methyl jasmonate acts as a powerful inhibitor of cell division induced by cytokinin in used test. In comparison with methyl jasmonate, abscisic acid did not show an inhibitory effect on bulblets formation induced by benzyladenine, even in a higher concentration.

APPLICATION OF LEAF-SEGMENT CULTURES TO IN VITRO BULBLET PRODUCTION OF SIX LILIUM SPECIES

SUMMARY Three 5 mm strips excised from the basal region of young leaves of 6 Lilium species were cultured in vitro, and the rates of regeneration and the number of bulblets per explant were investigated. The bulblet productivity depended both on the kind of Lilium species and the origins of each explant. Growth regulators also affected the bulblet productivity; α-naphthaleneacetic acid (NAA) was absolutely required for the induction and growth of bulblets whereas addition of 6-benzylaminopurine (BA) was ineffective.

ヒメサユリのりん片培養における子球の形成及び生長に及ぼす要因

Lilium rubellum Baker bulb-scales excised from bulbs growing in vitro were cultured at different temperatures, in various lengths of light period, or in a medium containing different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA).Temperature affected the percentage of regenerating bulb-scales, the number of bulblets per scale, and the growth of bulblets. The optimum temperature for each of the parameters was 25°C.The percentage of regenerating bulb-scales and the number of bulblets per scale were stimulated in a lighted environment, but the fresh weight of bulblets formed in this environment was lower than the weight of those formed in darkness.NAA at concentrations of 0.05 and 0.1mg/l gave a stimulatory effect on each of the parameters, but the higher concentrations suppressed bulblet formation. BA scarcely affected any of the parameters, except in the combination of 0.1mg/l BA with 1.0mg/l NAA, which enhanced the fresh weight of the bulblets.

The effect of light and of explant orientation on the regeneration and subsequent growth of bulblets on Lilium longiflorum Thunb, bulb-scale sections cultured in vitro

Lilium longiflorum, cultivar ‘Osnat’, bulb-scale sections were cultured in the light and in the dark with explants placed with their dorsal (abaxial) side on the medium, and in the dark with their ventral (adaxial) side up or down, or in the upright or inverted position. Scale sections regenerated bulblets, roots and callus. In all cases, bulblets developed only on the ventral side of the scale. Light did not affect the regeneration capacity of the scale piece but did significantly affect bulblet growth. Bulblets developing in the light were small and bore many leaves. In the dark, a smaller number of bulblets, which were much larger but bore fewer leaves, developed. In the light, leaf dry weight was double that of bulb dry weight, whereas in the dark this proportion was reversed. In contrast to light, explant orientation greatly affected regeneration percentage. Scale sections planted with their ventral side down regenerated fewer and smaller bulbs, less root, but much more callus than those with their dorsal side down. The upright and inverted positions also reduced the number of bulblets regenerated and increased callus formation.

Growth regulator requirements for adventitious regeneration from Lilium bulb-scale tissue in vitro, in relation to duration of bulb storage and cultivar

Addition of NAA (1-naphthylacetic acid) to the nutrient medium strongly influenced the adventitious regeneration of plantlets from bulb-scale tissue of lilies in vitro. The percentage of regenerating explants, the number of plantlets per explant, and also the bulblet growth of the resulting plantlets were affected by the auxin, low concentrations being stimulatory and high concentrations inhibitory. The cytokinins BA (6-benzyladenine) and 2iP (N6-[Δ2-isopentenyl]-adenine) did not influence the adventitious regeneration, but caused aberrant growth of the regenerated plantlets. Prolonged cold storage of the mother bulbs decreased the demand for exogenous auxin for maximum adventitious regeneration. The optimum for bulblet growth was not affected. Results of experiments with cultivars ‘Pirate’, ‘Enchantment’ and ‘Connecticut King’ suggest an inverse relationship between the in vivo propagation rate by scaling and the NAA demand for optimal adventitious regeneration in vitro.

Effect of Auxins, Cytokinins, Gibberellins, Abscisic Acid and Ethephon on Regeneration and Growth of Bulblets on Excised Bulb Scale Segments of Hyacinth

AbstractThe effect of various growth substances on regeneration and growth of bulblets on excised bulb scale segments of Hyacinthus orientalis L. cv. Pink Pearl was studied. Bulblet formation was promoted by the auxins IAA and IBA but was inhibited by NAA at high concentrations. IAA hardly affected bulblet weight, whereas IBA and NAA at high concentrations decreased it. Cytokinins had hardly any effect on bulblet regeneration and bulblet weight, Gibberellins inhibited both bulblet regeneration and bulblet weight; GA4+7 acted much stronger than GA3. ABA and ethephon decreased bulblet regeneration and bulblet growth.

テッポウユリりん茎の生育反応に関する研究

The experiment was carried out to clarify the effect of temperature and light treatments on the type of leaves developing from scale bulblets. The Easter Lily cultivar “Hinomoto” was scaled at 25 or 15°C in darkness for 5 months prior to a growth production phase. On 1/15/74 the newly developed bulblets (scale bulblets) were planted in peatmoss and then grown for 75 days at constant temperatures of 25, 20, 15 or 10°C in darkness or in continuous illumination by fluorescent tubes (NEC, FL-20-BR).The development of foliage scales on the scale bulblets was greater in light than in darkness and was independent of the previous scaling or subsequent growing temperature treatments. In light, when the lily was scaled at 15°C, foliage scales developed almost equally regardless of the subsequent growing temperature treatment. When scaled at 25°C, however, the lower the growing temperature was, the greater the development of foliage scales was. Independent of growing temperatures, new bulblets with foliage scales were more numerous when scaled at 15°C than at 25°C.In darkness, bulblets formed foliage scales to a greater degree at 10°C than at any other growing temperature. These foliage scales developed particularly at the end of the experiment. At 25°C (growing temperature), scale bulblets in darkness bolted or developed stems with foliage leaves to the same degree as in light and were independent of the original scaling temperatures. At the growing temperatures of 20, 15 or 10°C, more bolted bulblets were observed when originally scaled at 25°C than at 15°C. At the growing temperatures of 20 or 15°C, bolting occurred to a greater degree in light than in darkness.These results indicate the following: 1) When new bulblets are devoloped at a higher temperature, they bolt more easily than when developed at a lower temperature and the coolly formed bulblets tend to develop foliage scales. 2) Light during bulblet growth induces the bulblet to form foliage scales and this light response is promoted by low temperature. 3) In darkness, a growing temperature of 10°C induces foliage scale development.

Regeneration of bulblets on bulb scale segments of hyacinth in vitro.

Hyacinth bulbs, cv. Pink Pearl, harvested in June were held at 25 deg C for 1-5 months, after which bulb scales were removed for tissue culture in modified basic medium. The length of the holding period had little effect on the number of bulblets that regenerated, but the average bulb weight increased as the holding period lengthened, attaining a maximum with bulbs held until 1-15 October. There were wide differences in regenerative ability and bulblet growth between bulbs of the same harvest and size. The number of bulblets that regenerated was not affected by scale age, but bulblet growth from the older scales (nos. 7-18) was much better than from the younger scales. Regeneration and growth of explants from the proximal parts of the scales was much better than from the distal parts, and 3-cm-long explants gave much better results than shorter ones. Inverting the explants in the culture medium was favourable to regeneration and growth. In further studies on the effects of temperature and light, bulb regeneration was accelerated by raising the temperature, the optimum being 21.6 deg . The greatest number of bulblets/explant, however, was obtained at 13 deg , and the highest bulb weight at 21.6 or 24.8 deg . There were no differences in regeneration and bulblet growth between scales grown in continuous light or in darkness. (Abstract retrieved from CAB Abstracts by CABI’s permission)