Efficient somatic embryogenesis and bulblet regeneration of the endangered bulbous flower <Emphasis Type="Italic">Griffinia liboniana</Emphasis>

Using flower organs as primary explants and via somatic embryogenesis, we developed an efficient protocol for bulblet regeneration from in vitro-derived seedlings (bulblets) of Griffinia liboniana. Callus induction was tested on five types of floral organ (perianth, filament, pedicel, ovary and anther) in the presence of three combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA). Filament constituted the most responsive primary explant for regenerative callus induction, and the highest frequencies of callus induction (63.0?±?1.9%) and numbers of differentiated buds (3.7?±?0.3 buds/callus) were found on Murashige and Skoog (1962) medium (MS) supplemented with 1.0 mg L^?1 2,4-D and 1.0 mg L^?1 6-BA. Starting with in vitro-derived bulblets (0.8–1.5 cm in diameter), somatic embryo (SE) formation occurred within 6 weeks, followed by 8 weeks for SE germination and development on PGR-free media. The highest percentage (78.9?±?2.2%) of embryogenesis was obtained on MS media supplemented with 0.5 mg L^?1 6-BA and 1.5 mg L^?1 2,4-D, with an average of 28.0?±?2.1 bulblets/explant. Well-rooted bulblets were successfully acclimated to ex vitro conditions. A stable ploidy level of the regenerated bulblets was confirmed by flow cytometry (FCM) analysis. This is the first report about micropropagation methods of G. liboniana and constitutes an efficient and reusable method for bulblet regeneration of this endangered species. Additionally, this protocol enables large-scale vegetative production, germplasm preservation and genetic engineering of endangered Griffinia species.     

Differentiation in Lilium Bulbscales Grown in vitro. Effect of Various Cultural Conditions

Tissue cultures of Lilium auratum Lindl. and L. speciosum Thunb., which were derived from bulbscales, all appeared to differentiate organs. The effect of cultural conditions on the differentiation of bulblets and roots was examined. The best material for bulblet formation was bulbscales of intact or in vitro produced bulblets. The optimum temperature was 20°C and optimum pH was 6. Effect of irradiance on organ formation was not obvious but leaf emergence was stimulated. Higher kinetin concentrations stimulate the formation of numerous bulbscalcs. High NAA concentrations induce roots. On the other hand kinetin inhibits the NAA effect on root formation. A high sucrose concentration stimulated organ formation, but the number of bulblets was at a constant level in the medium containing between 10 and 90 g/l of sucrose. The formation of bulblets and their growth were stimulated at increasing strength of Murashige-Skoog's (MS) medium, but the length of roots was inhibited. Inter action of strength of MS medium and sucrose concentration was examined. High concentration of both components stimulated bulb lei growth, but the second strength of MS medium containing 90 or 120 g/l sucrose stimulated callus induction and inhibited the growth of bulblets. Maximum growth took 100 days for bulblets and about 50 days for roots. The change of fresh weight/dry weight ratio during differentiation is also discussed.     

In vitro bulblet production of Lachenalia

In vitro bulblet formation and subsequent transplanting of bulblets to soil were studied in order to develop a cost-effective method for the mass production of three Lachenalia varieties. Clumps of adventitious shoots regenerated from leaf explants were used. Bulblet formation was initiated after 2 weeks when shoots were subjected to low temperature (4–15 °C). The size (age) of the adventitious shoot affected the bulblet size, and shoots shorter than 4 mm did not form bulblets. Larger bulblets formed on medium containing 6% sucrose compared to 3% sucrose. Following bulblet initiation, illumination was not necessary for the completion of bulblet formation. Bulblets went into dormancy 3–4 months after they had been initiated or when the culture medium dried out, and they were released from dormancy when the natural night temperatures started to decrease in the late summer. The survival rate of the bulblets after transplanting was directly correlated to the size of the bulblets.The most important factors influencing in vitro bulblet formation of Lachenalia were sucrose concentration, temperature and length of explant shoots. Received: 12 June 1998 / Revision received: 8 September 1998 / Accepted: 23 September 1998     

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

The 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.     

Phase change in lily bulblets regenerated in vitro

During the development of the lily ( Lilium ), three phases can be distinguished: the juvenile, the vegetative adult and the flowering phase. Juvenile bulblets sprout with one or a few leaves whereas vegetative adult bulblets sprout with a stem with elongated internodes. The transition to the vegetative adult phase was studied in lily ( Lilium  × cv. Star Gazer) bulblets regenerating on bulb scale segments in vitro. The phase change was marked by the development of a tunica-corpus structure in the apical meristem which leads to the formation of an actively growing stem primordium. This structure is absent in juvenile bulblets. Juvenile bulblets first developed competence for phase change during a culture period of at least 6 weeks at 25°C. Subsequent induction of the phase change occurred during a period of 2 weeks at lower temperature (15°C). A major factor influencing phase transition was bulblet weight. Small bulblets never formed a stem whereas large bulblets always formed a stem under inducing conditions. Large bulblets more often formed a stem than small ones but the relation between bulb growth and phase transition was not absolute. A high sucrose concentration, a large explant and a prolonged period for competence development stimulated bulb growth but also phase transition independently of growth. Lowering the concentration of MS-minerals reduced bulb growth but did not affect phase transition. Under these conditions, phase change was correlated with a low phosphorus content.     

Enhanced bud and bulblet regeneration from bulbs of<Emphasis Type="Italic"> Nerine sarniensis</Emphasis> cultured in vitro

Nerine (Nerine sarniensis) cv. Salmon Supreme in vitro-grown bulblets, 7-9 mm in diameter, were cut in half longitudinally and used for adventitious bud initiation following dissection of the roots and two-thirds of the upper part of the bulblets. The terminal apex was injured with a hot, sterile microscope dissecting needle. The highest number of buds formed (seven to nine buds per halved bulblet) on a semi-solid Murashige and Skoog (MS) basal salts medium supplemented with 3% sucrose and either 1 microM 6-benzylaminopurine (BA) and 1 microM alpha-naphthaleneacetic acid (NAA) or 0.5 microM BA and 0.1 microM NAA. Bulblet halves were cultured in the dark for 11-13 weeks with one subculture after 6 weeks. Anatomical studies indicated that the initiation of adventitious buds on the abaxial side of the inner scales of the halved bulblet was adjacent to the basal plate and started from the leaf primordium and a meristematic bulge. Buds developed directly into small bulblets after they were transferred to semi-solid MS basal salts medium supplemented with 6% sucrose, 10 microM indole-3-butyric acid (IBA) and 0.25% activated charcoal. Small bulblets cultured in liquid MS medium supplemented with additional KH(2)PO(4 )(170 mg l(-1)), 6% sucrose and 0.1 microM NAA under a 16/8-h (light/dark) photoperiod for 8 weeks grew into larger bulbs faster than those cultured on semi-solid medium. The bulbs were rooted on a semi-solid medium after 4 weeks and then transferred to the soil. As many as 18 bulblets developed and rooted from one in vitro-grown bulb after 25-27 weeks.     

Stimulating effect of spermine on bulblet formation in bulb-scale segments of Lilium longiflorum

When bulb-scale segments of Lilium longiflorum were cultured on a medium containing auxin and cytokinin, the proportion of the expiants with newly-formed bulblets was significantly increased by the application of different polyamines. The most effective polyamine was spermine, where more than 90% of segments formed an average of 5 bulblets as compared to controls where less than 50% explants formed an average of 1.5 bulblets. Application of arginine one of the precursors putrescine biosynthesis, slightly promoted bulblet formation. The putrescine-stimulated bulblet formation was strongly inhibited by simultaneous addition of an inhibitor of the spermidine synthase, cyclohexylamine. The spermidine-promoted bulblet formation, however, could not be suppressed by this inhibitor. The promotive effect of spermidine on bulblet formation was reversed by an inhibitor of the spermine synthase, N-(3-aminopropyl)cyclohexylamine, but application of this inhibitor with spermine did not show any apparent effect on the bulblet formation. Endogenous level of spermine increased in common during bulblet formation that were stimulated by exogenous polyamines. Thus, spermine seemed to be the main stimulating chemical on bulblet formation in lily bulb-scale segments.Abbreviations APCHA N-(3-aminopropyl)cyclohexylamine - Arg arginine - BA benzyladenine - CHA cyclohexylamine - MS Murashige and Skoog's - NAA naphthaleneacetic acid - Orn ornithine - Put putrescine - Spd spermidine - Spm spermine     

Effects of storage temperature and sucrose on bulblet growth, starch and protein contents in in vitro cultures of Hyacinthus orientalis

The scale segments of the bulblets of Hyacinthus orientalis L. cv. Anna Marie were examined to improve their growth and development with cold-pretreatment and sucrose. The cold-pretreated (4 °C for 4 months) segments showed higher growth and better development of the bulblets on medium without sucrose than ones stored at 20 °C. A rapid decrease in starch content of bulb pieces was found during the first 2 weeks in all cultures and thereafter the content decreased gradually. A scanning electron microscopic observation during the bulblet growth and development showed a gradual decreasing trend of the starch granules from 2 to 16 weeks of the cultures. SDS-PAGE electrophoresis revealed the presence of a characteristic polypeptide of approximately 45 kD, which is assumed to be a major storage protein in the bulblets.     

Dynamic changes in carbohydrate metabolism and endogenous hormones during <Emphasis Type="Italic">Tulipa edulis</Emphasis> stolon development into a new bulb

The stolon is the main asexual reproductive organ of Tulipa edulis (Miq.) Baker. It has a special morphology and can develop into a new bulb for propagation. In the current greenhouse experiment, the dynamic changes in carbohydrates and related enzymes, protein and endogenous hormones during T. edulis stolon development were investigated. The results showed that soluble sugar levels were basically declining, whereas starch and protein content rose continuously during stolon development. The adenosine diphosphoglucose pyrophosphorylase (AGPase) activity peaked in the initial swelling stage and stayed a relative high level in the middle swelling stage; sucrose synthase (SS), soluble starch synthase (SSS) and granule-bound starch synthase (GBSS) activities followed the same law that showed rising trends during stolon development. SS activity was significantly inversely related to sucrose content but had significantly positive relations with starch content, SSS and GBSS activities. Gibberellin (GA), indole-3-acetic acid (IAA) and zeatin riboside (ZR) peaked in the initial swelling stage and maintained high levels in the middle swelling stage; they then decreased significantly in the later swelling stage. A substantial increase was observed in abscisic acid (ABA) content until the middle swelling stage, followed by a significant reduction in the later swelling stage. The ratios of ABA to IAA, GA and ZR reached their lowest levels in the initial swelling stage. In conclusion, T. edulis stolon development is a process of new bulb morphogenesis along with the starch accumulation catalyzed by AGPase, SSS and GBSS, using the product of sucrose cleavage caused by SS. Initial low ABA content and low ratios of ABA to IAA, GA and ZR, together with the GA, IAA and ZR of high-content, soluble sugars worked more efficiently to induce new bulb formation.     

In vitro propagation from axenic explants of <Emphasis Type="Italic">Lilium oxypetalum</Emphasis> (D. Don) Baker,an endemic bulbous plant of high altitude Himalaya

In vitro propagation protocol for Lilium oxypetalum, a high altitude Himalayan endemic lily, has been developed. Effect of explant types (i.e., callus and in vitro bulblet scales) and sucrose concentration [0–6.0% (w/v)] on in vitro bulblet regeneration of L. oxypetalum was tested in previously optimized Murashige and Skoog basal medium supplemented with 2.0 μM 6-benzyladenine and 0.1 μM α-naphthaleneacetic acid. Callus explants produced significantly (P < 0.01) higher number of bulblets per explant than bulblet scale explants. Of the different concentrations of sucrose tested, 4.5% (w/v) sucrose showed significantly (P < 0.01) higher percentage regeneration (i.e., 70.8 ± 4.2 and 79.2 ± 4.2% regeneration on callus and bulblet scale explants, respectively), and produced higher number of bulblets per explant (i.e., 9.0 ± 0.4 and 5.4 ± 0.5 bulblets on callus and bulblet scale explants, respectively). Regenerated bulblets developed 2–3 leaves when subcultured for 4 weeks and were subsequently transferred ex vitro with a survival rate of 66.7% after 6 weeks. Leaves of the survived plantlets became dry after growing ex vitro for 10 weeks, amongst which 86.4% re-sprouted after remaining dormant for 5–6 weeks and produced 1.5 bulblets per explant. Findings of the present study hold promise for efficiently multiplying the target species in view of its potential economic and conservation significance.     

百合鳞茎发育过程中淀粉合成相关酶基因的克隆及表达分析

该研究通过同源克隆技术克隆腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、颗粒结合淀粉合酶(GBSS)和可溶性淀粉合酶(SSS) 3类百合淀粉合成关键酶基因,分析这三类淀粉合成关键酶基因的表达变化,测定百合鳞茎膨大发育中淀粉含量变化。结果表明:(1) AGPase具有GlgC家族蛋白PLN02241蛋白结构特征及cl11394家族蛋白ADP_Glucose_PP与NTP_transferase结构域,获登录号KP751443; GBSS与SSS具有cl10013家族蛋白Glyco_transf_5,GT1_Glycogen_synthase_DULL1_like结构域,获登录号分别为KP751444、KP751445。(2)百合鳞茎形成与膨大发育过程中,淀粉含量呈现递增趋势,鳞茎盘开始分化茎杆时其淀粉含量最高,达到44.52%。鳞茎与叶片部位的三个淀粉合成相关酶基因表达量均逐渐增加;在鳞茎膨大后茎杆分化阶段,三个淀粉合成相关酶基因表达量达到最高,AGPase、GBSS、SSS在鳞片中的表达量分别为10.79,6.92和5.12,叶片中的表达量分别为6.79,5.22和4.41,鳞片中的表达量大幅度高于叶片;淀粉合成相关酶基因的表达量变化与淀粉含量、鳞茎的膨大发育成正相关。这为鳞茎的繁殖生产提供了可通过调节淀粉合成关键酶基因表达促进百合鳞茎膨大发育的思路。     

Lilium candidum bulblet and meristem development

Lilium candidum L., commonly known as the Madonna lily, is a wild Lilium species with medicinal properties and excellent potential as an ornamental crop, but one that has been scarcely investigated. The aim of this research was to study (1) tissue culture propagation of L. candidum bulblets, (2) early bulblet development, and (3) the effect of temperature and bulblet weight on bulblet and plant growth and meristem development. An investigation of the effect of explant type and temperature on in vitro bulblet propagation showed that scales were the most efficient explants for in vitro propagation and that exposing the regenerating bulblets to 15°C for 4 wk increased bulblet weight but reduced the number of bulblets produced. For bulblets planted in soil after 12 wk of exposure to 15°C or 25°C, the fastest growth was observed in the bulblets that had been exposed to 15°C and that had a larger initial size. Histological examination showed that young in vitro-grown bulblets had a rudimentary meristem comprising few cells with no layer organization. After 12 wk of growth, all bulblets showed a layered meristem, regardless of bulblet size or exposure to 15°C. However, an increased amount of leaf primordia was detected in larger bulblets. Furthermore, the histological examination revealed that in L. candidum, as opposed to other lily species, there had been no real "phase change" in the meristem and that the phase change from juvenile to vegetative adult occurred at a much later stage in L. candidum than in other species.     

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0 × MS medium containing 4.44, 8.88, 17.76 μM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74 μM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA and 17.76 μM BAP plus 2.685 μM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0 × MS medium containing 4.9 μM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA) were transferred to 1.0 × MS medium containing 30 g/l sucrose to break negative carry over effect of this dose of BAP–NAA, where they grew 2–3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0 × MS medium containing 4.44 μM BAP plus 5.37 μM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.Abbreviations: MS medium, Murashige Skoog medium; BAP, 6-Benzylaminopurine; NAA, α-Naphthalene acetic acid; IBA, Indole-3-butyric acid     

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.     

Effects of chilling stress on the accumulation of soluble sugars and their key enzymes in <Emphasis Type="Italic">Jatropha curcas</Emphasis> seedlings

As osmolytes and signaling molecules, soluble sugars participate in the response and adaptation of plants to environmental stresses. In the present study, we measured the effect of chilling (12 °C) stress on the contents of eight soluble sugars in the leaves, cotyledons, stems, and roots of Jatropha curcas seedlings, as well as on the activities of eight rate-limiting enzymes that are critical to the metabolism of those soluble sugars. Chilling stress promoted both starch hydrolysis and soluble sugar accumulation. The soluble sugar contents of the leaves and cotyledons were affected more than that of the stems and roots. Meanwhile, the activities of the corresponding metabolic enzymes (e.g., β-amylase, uridine diphosphate glucose phosphorylase, and sucrose phosphate synthase) also increased in some organs. The gradual increase of soluble neutral alkaline invertase activity in the four studied organs suggested that sucrose catabolic production, such as glucose and fructose, was especially important in determining resistance to chilling stress and hexose signal transduction pathway. In addition, the substantial accumulation of raffinose family oligosaccharides and increase in corresponding metabolic enzyme activity suggested that galactinol and raffinose play an important role in determining the chilling resistance of J. curcas. Together, these findings establish a foundation for determining the relationship between the chilling resistance and soluble sugar accumulation of J. curcas and for investigating the mechanisms underlying sugar signaling transduction and stress responses.     

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, GA₃, 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. GA₃, 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.     

兰州百合组培鳞茎发育研究

该研究以兰州百合商品种球鳞片为外植体材料,通过组织培养诱导丛生芽萌发及高频增殖,再以丛生芽为材料诱导其发育形成小鳞茎,调节培养基对小鳞茎进行膨大发育培养,最终形成促进兰州百合组培鳞茎膨大发育的“三步”组培培养技术路线;对发育过程中形成的丛生芽、小鳞茎及膨大鳞茎进行淀粉含量测定与生长特征参数统计,分析各步培养对鳞茎形成发育过程中淀粉含量与形态变化的影响。结果表明：所建立的“三步”培养方案培育出的组培鳞茎直径、重量与鳞片数分别为1.66 cm、2.48 g和26.33片,有效地促进了鳞茎的膨大,并能诱导鳞茎主茎杆的形成发育;在培养进程中其淀粉含量呈现逐步增加的趋势,这表明与鳞茎膨大发育正相关,同时鳞茎大小、重量及鳞片数三者也表现为正相关性;当鳞茎所含鳞片数在26片以上时,其生长点易发育形成主茎杆。该文研究了兰州百合组培鳞茎的形成与膨大发育技术,所研发的“三步”培养组成的鳞茎膨大发育组培技术有效地促进了鳞茎的膨大发育,而膨大发育的鳞茎能有效地缩短田间生长周期,从时间上提高百合生产量,同时为实现兰州百合膨大的鳞茎种球规模化生产提供技术支撑。     

Somatic embryogenesis and plant regeneration of <Emphasis Type="Italic">Lilium ledebourii</Emphasis> (Baker) Boiss., an endangered species

A somatic embryogenesis (SE) protocol was established for the regeneration of Lilium ledebourii (Baker) Boiss. whole plants using new vegetative bulblet microscales and transverse thin cell layers (tTCLs) of young bulblet roots as the explant sources. Bulblets were induced from bulb scale explants cultured for at least 3 months in the dark on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar, and different concentrations of α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron. Embryo-like structures were obtained from tTCL explants of 3-month-old bulblets (excised from bulb scale explants) following culture on solid MS medium containing 3% sucrose and various concentrations of NAA and BA for 3 months in the dark. Both the explant source and the type of plant growth regulators affected the differentiation of somatic embryos. The highest percentage (65.55%) of embryogenesis was obtained from bulblet microscale tTCLs cultured on solid MS medium containing 0.54 μM NAA and 0.44 μM BA. Plants with normal shoots and roots were obtained following a 3-month culture of embryos on growth regulator-free MS medium at 25 ± 1°C under a 16/8-h light/dark photoperiod (light intensity 40 μmol m⁻² s⁻¹, cool-white fluorescent light). The plants were successfully acclimatized in the growth chamber.     

<Emphasis Type="Italic">Narcissus</Emphasis> bulblet formation <Emphasis Type="Italic">in vitro</Emphasis>: effects of carbohydrate type and osmolarity of the culture medium

Shoot clump cultures of Narcissus cultivars St. Keverne and Hawera were used to investigate the effects of culture medium carbon supply, type of carbohydrate and osmolarity on in vitro bulblet development. Increasing the medium osmolarity using mannitol or sorbitol, which did not act as substrates for growth, failed to stimulate bulblet formation with either cultivar. An exception to this was a relatively small increase in total bulblet dry weight per culture, in the cultivar Hawera only, caused by adding 30 g l ^–1 sorbitol in combination with 30 g l^–1 sucrose. Simultaneously increasing the medium osmolarity and carbon supply using the metabolisable carbohydrate sources, sucrose, glucose, fructose or an equimolar mixture of glucose and fructose stimulated bulblet production, total dry matter accumulation and partitioning into bulblets. At comparable levels of carbon supply up to 19.0 g l^–1, bulblet development of both cultivars was similar with monosaccharide and sucrose media. This indicates that substrate supply is more important for bulblet development than osmolarity of the culture medium. The cultivar Hawera also showed similar responses to monosaccharide and sucrose media supplying 37.9 g C l^–1, despite the high osmolarity of monosaccharide media (c. 650 m Osm kg^–1, equivalent to –1.6 MPa, compared to 380 m Osm kg^–1 for sucrose medium). However in St. Keverne total dry matter accumulation and dry weight per bulblet were further stimulated only by increasing the sucrose supply from 19.0 to 37.9 g C l^–1, not by increasing the monosaccharide supply. Implications of the findings for Narcissus micropropagation are discussed.     

细叶百合无性繁殖条件的选择

以栽培的2 年生细叶百合(Lilium pumilum DC.)鳞茎为扦插材料, 将鳞茎分内、中、外三层剥取其鳞片, 观察鳞片在不同温度、光照强度、基质中的扦插生小鳞茎的效果。扦插40d 时, 鳞片基本枯萎, 此时的实验结果是:①影响扦插效果的重要因子是温度和光强, 25℃高温避光生鳞茎最佳;其次是鳞片位置, 中鳞片和外鳞片好于内鳞片;基质对扦插影响不大。②每百鳞片生小鳞茎数和小鳞茎直径呈正相关。③相同条件下, 相同部位的刀切鳞茎段所产生小鳞茎数量明显高于手掰的整片鳞片, 这一现象至今未见报导。