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     

Genetic variability in callus formation and regeneration of garlic (Allium sativum L.)

Twenty garlic Allium sativum (L.) genotypes were analysed for genetic variation in their ability to form callus (in one medium) and regenerate shoots (in four different media). Genotypes showed significant differences in the analysis of variance of all the traits tested. The accession Printanor showed the best general behaviour, with 83% callus-producing explants, 44.7% organogenic explants, and 15.35 shoots/g of callus. The best regeneration medium was MBO, without growth regulators. Shoot production capacity was examined with the additive main effects and multiplicative interaction model that proved to be a powerful tool for analysis and easy comprehension of the strong genotype×medium interactions frequently observed in in vitro culture systems. Received: 5 February 1998 / Revision received: 11 May 1998 / Accepted: 1 June 1998     

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。     

Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass (Tripsacum dactyloides L.)

Eastern gamagrass, (Tripsacum dactyloides L.) is a perennial, warm-season grass that is being developed as a forage plant. Shoots were derived from callus initiated from immature embryos and immature inflorescences of diploid (2n=2x=36) gynomonoecious eastern gamagrass. These shoots were induced to microtiller in the presence of 3 mg/l benzyladenine. Amiprophosmethyl (10, 15, or 20 μm) was applied to 27 microtillers for 3–5 days to induce chromosome doubling. All 14 surviving plants were tetraploid, (2n=4x=72), as determined by flow cytometry or chromosome counts. These plants were morphologically normal and produced seed. Test crosses were made with a known diploid. Flow cytometry and chromosome counts showed that the progeny were triploid, proving that the induced tetraploids reproduce sexually. Received: 12 February 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998     

Micropropagation of garlic through in vitro bulblet formation

We developed a novel micropropagation method for garlic (Allium sativum L.) by the combination of initial shoot-tip culture, shoot multiplication and in vitro bulblet formation. Garlic shoot-tips were cultured on LS medium containing 1 M indole-3-acetic acid (IAA) and 1 M 6-benzyladenine (BA) to regenerate proliferative shoots. These shoot-tips produced multiple shoots when transferred to modified LS medium containing 5 M 1-naphthaleneacetic acid (NAA) and 10 M BA, and cultured at 20°C under 12-h light conditions. Higher ratios of KNO₃/NH₄Cl in the media promoted multiple shoot formation, together with suppressing vitrification of these shoots. The proliferated shoots of early maturing cultivars produced bulblets by culture on LS growth regulator-free medium at 25°C under 16-h light. On the other hand, the late maturing cultivar, Howaito-roppen, formed bulblets after a low temperature treatment of the proliferated shoots for 6 months followed by culture on LS medium containing 6 to 12% sucrose for two months. The dormancy of the bulblets of cv. Howaito-roppen was broken by successive treatments at a high (35°C), a middle (20°C), and then a low (5°C) temperature.Abbreviations IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine - LS Linsmaier and Skoog macro- and microelements     

广藿香不同外植体离体培养的研究

以广藿香叶片、带节茎、不带节茎及根尖为材料进行离体培养,对影响离体再生的因素进行了研究。结果表明:BA有利于广藿香外植体出芽,浓度以0.1～0.5mg/L效果较好;不同外植体的出芽能力有较大差异,其中以叶片和带节茎出芽能力较强,出芽率均达100%;外植体在培养基上的接种方式对出芽也有一定影响,带节茎以形态学下端垂直插入,可以缩短出芽时间及增加单个外植体出芽的数量。无根苗生根以MT+IBA0.2mg/L培养基为好,苗的生长较为健壮。     

DEVELOPMENTAL CYCLES IN SHOOT GROWTH OF MALE CYCAS CIRCINALIS

Cycles of development in several male shoots of Cycas circinalis were studied by counting sequences of foliage leaves, scale leaves, and cones by observation of either their scar patterns or vascular remains. Although male cones are always produced subsequent to a cycle of foliage leaves and are fairly regularly spaced there is no obvious correlation between their presence and the previous cycles of leaves. Ratios of foliage leaves to scale leaves in each cycle are variable. The primary diameter of the axis is obconical, but a conical stem form results from secondary growth. The result of concentric development of xylem and phloem is positively correlated with stem diameter rather than stem age.     

Heteroblasty and preformation in mayapple, Podophyllum Peltatum (Berberidaceae): developmental flexibility and morphological constraint

Developmental preformation can constrain growth responses of shoots to current conditions, but there is potential for flexibility in development preceding formation of the preformed organs. Mayapple (Podophyllum peltatum) is strongly heteroblastic, producing rhizome scales, bud scales, and either a single vegetative foliage leaf or two foliage leaves on a sexual shoot. To understand how and when preformation constrains growth responses, we compare (1) how leaf homologs of the renewal shoot differ in development, (2) whether there are differences in shoot development that occur in advance of morphological determination of shoot type, and (3) whether there are points of developmental flexibility in renewal shoot growth prior to preformation of the foliage and floral organs. We use scanning electron microscopy and histology to show that the three vegetative leaves (both types of scale leaves and the vegetative foliage leaf) are similar in the initial establishment of an encircling and overarching leaf base. Differences among them are found in the timing of differentiation of the leaf base and in the relative timing and degree of growth of the lamina and petiole. In contrast, foliage leaves on sexual shoots show less expression of the leaf base and precocious growth of the lamina and petiole. Prior to shoot type determination, there are no morphological differences in the sequence or position of leaf homologs that predict final shoot type. In this colony, leaves at positions 12 and 13, on average, appear to be identical in development until they are between 700 and 800 μm in length, when it becomes possible to distinguish leaves that will become vegetative foliage leaves from additional bud scale leaves on vegetative or sexual shoots. We suggest that late developmental determination of leaves at positions 12 and 13 reflects ontogenetic sensitivity to a transition to flowering. Thus, in mayapple, heteroblasty appears to facilitate developmental flexibility prior to the point where shoot growth becomes constrained by preformation of determined aerial structures.     

二甲戊灵对离体大蒜染色体的加倍研究

在MS分化培养基中分别添加不同浓度的秋水仙素和二甲戊灵,以 "改良蒜" 的蒜瓣基部为外植体诱导愈伤组织染色体变异并再生植株,观察根尖细胞染色体数和叶片下表皮保卫细胞特征检测诱变效果.结果表明:大蒜染色体数变异受诱变剂浓度和处理持续时间的影响,诱变剂高浓度和短时间处理均有利于大蒜愈伤组织的分化;添加0.1%秋水仙素处理5 d,愈伤组织存活率为80.7%,分化率为78.1%,四倍体的诱导率为4%;添加 100 μmol/L二甲戊灵处理5 d,愈伤组织存活率为100%,四倍体的诱导率为6%.对根尖细胞染色体和叶片下表皮气孔数目和大小观察显示,诱变株具有四倍体的基本特征.说明二甲戊灵能在离体条件下有效诱导大蒜产生四倍体,可替代秋水仙素对大蒜染色体的加倍作用.     

Micropropagation of Dendrocalamus asper by shoot proliferation using seeds

An efficient and reproducible procedure for the large-scale propagation of Dendrocalamus asper is described. High-frequency direct shoot proliferation was induced in aseptic seed cultures of D. asper on modified Murashige and Skoog's (1962) medium supplemented with 1.0–10.0 mg/l benzyladenine (BA). Multiple shoots (1–25) were formed within 5 weeks of seed culture without root formation. The shoot-forming capacity of seeds was influenced by the BA concentration in the medium. Proliferating shoot cultures were established by repeatedly subculturing shoots in propagules of 3 shoots each. A multiplication rate of 15–16 fold was achieved on MS medium +3.0 mg/l BA. Roots were formed on excised propagules of 3–5 shoots when transferred to MS medium containing 10.0 mg/l indole-3-butyric acid (IBA) or 3.0 mg/l 1-naphthaleneacetic acid (NAA). Plantlets were hardened, acclimatized and established in soil, where they exhibited normal growth. Received: 10 April 1998 / Revision received: 18 November 1998 / Accepted: 12 December 1998     

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.     

Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer

 By using highly regenerative calluses, we developed a stable transformation system in garlic (Allium sativum L.). The temperature and number of days of co-cultivation with Agrobacterium tumefaciens was shown to be an important factor in transient expression of the uid A gene. After a culture period of 5 months in selection medium containing hygromycin, 20 shoots were induced from ca. 1000 calluses, among which 15 plants expressed β-glucuronidase activity upon staining with X-Gluc. Shoots developed into transgenic garlic after 1 month. Integration of the uid A gene was confirmed by Southern blot analysis for genomic DNA of transgenic garlic plants. Received: 25 October 1999 / Revision received: 16 February 2000 / Accepted: 22 February 2000     

Leaf Development, Metamorphic Heteroblasty and Heterophylly in Berberis s. l. (Berberidaceae)

Shoot development of temperate and tropical members of Berberis s. l. was examined in order to assess: (1) the homology of the spines along the long shoots and the foliage leaves that form on the short shoots; (2) the occurrence of heterophylly and/or heteroblasty in the genus; and (3) the structural correspondence between cataphylls, spines, and foliage leaves. The 1-5-armed spines have been interpreted as modified compound leaves lacking stipules, as a modified lamina (central spine) with stipules (lateral spines), or less often, as transformed branches, or as epidermal outgrowths. On the other hand, the foliage leaves of the short shoots have been interpreted as leaflets of palmately compound leaves. Our results indicate that there are three distinct leaf types per node: (1) Leaves modified in spines spirally arranged in long shoots; (2) foliage, expanded leaves densely arranged in short shoots; and (3) cataphylls protecting axillary buds. The spines are leaf homologs with a clear distinction between the leaf base with stipules, and a laminar portion modified into the 1-5-armed spine; the lateral spines are not stipules as they arise from the marginal meristem of the laminar portion, and not from the leaf base. The foliage leaves also have stipules flanking the leaf base. Both spiny leaves and foliage leaves develop an articulation between the base and the laminar portion. Cataphylls of the short shoots of Berberis s. str. and those of the reproductive short shoots of Mahonia correspond to the entire leaf base, but those of the renewal (vegetative) shoots of Mahonia are spiny and have an odd vestigial pinnately compound lamina. Heterochrony due to ontogenetic truncation caused by the formation of the terminal inflorescence at the apex of the short shoots could be responsible for the lack of petiole/lamina differentiation in the foliage leaves. The spiny long-shoot/foliose short-shoot system of branching in Berberis s. str. appears to be genetically and phylogenetically fixed and not environment-dependent. This represents a clear example of metamorphic heteroblasty sensu Zotz et al. (Botanical Review 77:109–151, 2011) with further occurrence of heterophylly along the short shoots.     

Etiolation of `Royal Gala' apple (Malus×domestica Borkh.) shoots promotes high-frequency shoot organogenesis and enhanced ,-glucuronidase expression from stem internodes

Internodal explants from etiolated `Royal Gala' apple shoots were compared with those from non-etiolated shoots for frequency of shoot organogenesis and for efficiency of β-glucuronidase (GUS) expression after cocultivation of explants with Agrobacterium tumefaciens strain EHA105 (p35SGUSint). First (youngest) internodal explants from etiolated shoots produced 2-, 8- and 73-fold numbers of shoots compared to second, third, and fourth internodal explants, respectively. Moreover, these explants produced sevenfold the number of shoots as similar explants from non-etiolated shoots. All first internodes from etiolated shoots exhibited GUS-expressing zones and yielded fourfold as many GUS-expressing zones as commonly used leaf explants from non-etiolated shoots, which exhibited GUS-expressing zones in only 63% of the explants. An average of 9.8 GUS expressing calli per explant were observed on first internodes from etiolated shoots 2 weeks after cocultivation with A. tumefaciens. Received: 17 February 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998     

Abscisic acid controls dormancy development and bulb formation in lily plantlets regenerated in vitro

Plantlets of lily regenerated in vitro from scale explants consist of scales and leaves from which the base of the petiole has swollen to a scale. Fluridone, an inhibitor of ABA-synthesis, applied during culture in vitro, inhibited the swelling of the petioles and promoted leaf formation. At high fluridone concentrations (10 or 33μ M ), swelling was completely blocked, and plantlets consisted of leaves only. Addition of ABA during the regeneration in vitro had the opposite effect and resulted in plantlets with scales only. When applied simultaneously with fluridone, ABA nullified the effect of fluridone. This demonstrates that bulb formation in lily is under the control of ABA. Lily plantlets regenerated in vitro on scale explants at 20 or 25°C were harvested after 11 weeks, and the leaves were removed from the bulblets. The bulblets were dormant and required a cold treatment to achieve rapid emergence after planting in soil. Fluridone added during the culture in vitro prevented the development of dormancy, and the bulblets did not require a cold treatment. The effect of fluridone was nullified by simultaneous addition of ABA. Bulblets harvested after 6 weeks of culture at 20°C had not yet developed dormancy. Bulblets regenerated at 15°C were only slightly dormant. In both types of bulblets, it is unlikely that the lack of dormancy was due to low ABA-levels since addition of ABA did not affect the dormancy status. These data indicate that the level of endogenous ABA and an unknown additional factor play major roles in the development of dormancy.     

Adventitious shoot regeneration from leaf explants and stem nodes of Lilium

A method for the regeneration of lily plantlets (Lilium spp.) through different morphogenic pathways is described. Plant regeneration was obtained from in vitro cultured leaves of four lily hybrids, cultured on Murashige and Skoog's basal medium supplemented with cytokinins (TDZ and BA) and auxins (NAA and IBA) at different concentrations. Direct shoot regeneration occurred with all tested media for the Asiatic lilies `Elite' and `Pollyanna' and also for the Oriental hybrid `Star Gazer'. Callus developed on TDZ-enriched medium from leaf segments of L. longiflorum cv. `Snow Queen' regenerated by direct organogenesis. This occurred on a medium with auxin/ cytokinin balance which was lower than other genotypes. There were fewer problems of sterilization with leaves from sprouted bulbs than in vitro scale culture. This suggests that the leaf-segments obtained in this way could be an alternative to the scales as a source of material for propagation. A protocol for micropropagation based on bulblets from in vitro shoot-tip-derived stem nodes was also used. The development of pseudo-bulbets is particularly advantageous since it allows for structures characterised by absent or low dormancy. Regenerated shoots have been rooted and successfully acclimatized to greenhouse conditions where they flowered after the second year giving plants with true-to-type shape and colour.     

A culture unit system for the study of responses of mycorrhizal and non-mycorrhizal seedling to treatments

The time-dependence of Mn accumulation was confirmed in potato foliage (Solanum tuberosum. L.cv. Norland) grown in solution culture. Older leaves grown at 0.61 mM Mn had substantially higher Mn concentrations than younger leaves and stem samples. Levels of Mn in older leaves increased steadily from 4000 µg g^–1 at one week to 8–10,000 µg g^–1 at 6 weeks, but were relatively constant in the emerging leaves. Even foliage grown at low Mn levels (0.01 mM Mn) had 4 fold gradients in Mn concentration from younger (40 µg g^–1) to older leaves (180 µg g^–1).At 0.61 mM Mn, concentrations of 3–4000 µg g^–1 in the youngest fully-developed leaves did not bring about any decline in yield, and levels of up to 5000 µg g^–1 occurred in individual potato leaves before Mn toxicity symptoms were observed. Potato foliage grown at the high Mn had similar leaf numbers, but showed an increased stem length and smaller leaves than foliage grown at 0.01 mM Mn. In particular, the leaf area of the middle and lower leaf fractions were affected by the high Mn level.The ability of rapidly growing plants to withstand high concentrations of Mn is discussed in relation to the pattern of dry matter and Mn accumulation shown by potato foliage.     

Identification of microspore-derived plants in anther culture of flax (Linum usitatissimum L.) using molecular markers

The microspore origin of anther-culture-derived plants of flax was determined using inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers. Polymorphic fragments between the two parents of the F₁ donor plants were identified and their segregation patterns in anther-culture-derived plants were used to elucidate the origin of those plants and to determine the degree of independence of plants regenerated from the same callus. Using one ISSR primer (UBC 889) and two RAPD primers (UBC 556 and 561), 12 out of 16 plants were unequivocally identified as being derived from microspores. Plants derived from the same callus had identical PCR patterns at five polymorphic loci and thus were likely derived from the same microspore. Therefore, it is proposed that the number of calli forming shoots be used to describe the anther culture efficiency in flax. Received: 3 February 1998 / Revision received: 8 June 1998 / Accepted: 8 July 1998     

The effect of triacontanol on micropropagation of balm, Melissa officinalis L.

 Triacontanol, a long-chain primary alcohol was found to be an effective growth regulator in the micropropagation of balm, Melissa officinalis. In both the multiplication and the rooting phase, concentrations of 2, 5, 10 and 20 μg triacontanol per liter were applied. After 4 weeks of culture, the fresh weight of shoots was measured in the multiplication phase and root formation, photosynthetic activity, chlorophyll content and the fresh and dry weights of shoots were analyzed in the root induction phase. In the multiplication phase, 5 μg/l triacontanol was found to be the optimal concentration, while in the rooting phase 2 μg/l was the most effective. Triacontanol increased the number and length of roots, and it enhanced shoot growth, fresh weight, and the chlorophyll content, but it had no effect on the dry weight and the photosynthetic activity of the plants. Results of our work demonstrate that triacontanol can be applied as an effective growth regulator in the tissue culture of balm. Received: 3 December 1997 / Revised: 24 February 1998 / Accepted: 26 February 1999