裂叶悬钩子组织培养研究 Ⅰ.裂叶悬钩子叶外植体培养直接再生不定芽及植物激素对它的影响

本文首次报道裂叶悬钩子(Rubus laciniatus Wild)叶外植体培养在改良的NN~(69)培养基上附加2—4mg/1 6-BA和0.1mg/1 NAA或1—3mg/1 2,4-D和0.1mg/1 NAA,两者都可直接从完整叶片、叶片下切段或叶柄诱导出不定芽。诱导频率达20—48%。而不定芽绝大部分发生在叶轴处或叶柄基部。完整叶片的不定芽诱导率与叶片下切段无差别,但比叶柄基部诱导率要高。6-BA对叶轴处不定芽诱导率比2,4-D的要高。此外,不需继代培养,不定芽数可达10—20个,继代培养一个月左右,每个不定芽能形成丛生芽数可达40一60个。另外,本文还讨论了细胞分化过程中的极性现象。     

Effect of growth regulators and role of roots in sex expression in spinach

When 7-d-old plantlets of spinach (Spinacia oleracea L.) were immersed with their roots for 24 h in 25 mg/l gibberellic acid (GA₃), or 15 mg/l 6-benzylaminopurine (6-BAP), or 15 mg/l indole-3-acetic acid (IAA), or 10 mg/l abscisic acid (ABA) and subsequently grown on long (18-h) days, the ratio of plants with male and female flowers, which in the controls was almost 1:1 (48 and 52%, respectively), was greatly altered. The treatments with 6-BAP, IAA and ABA raised the percentage of female plants to 88, 76 and 71%, respectively; the GA₃ treatment increased the percent of male plants to 79%. When young, vegetative spinach plants (3 visible leaves) grown in 18-h days were cut a the root neck, and the shoots grown with their bases in nutrient solution, with adventitious roots either being allowed to develop or being systematically removed, 85% of the plants without roots became males, 85% of those with roots became females. But if the cut shoots were first, for 28 h, placed in a 15-mg/l 6-BAP solution and then grown in the absence of roots, the percent of female plants was restored to 84. These results fully agree with those obtained previously with hemp, namely, that plant growth regulators exert a regulating effect on the sex expression of dioecious plants when applied through the roots in early stages of development; that the root system plays an important role in determining the sex of these plants, that this role of the roots is associated with the synthesis of cytokinins in them. Dioecious short- and long-day plants do not differ in these respects.     

In vitro propagation of Coleus forskohlii Briq., a threatened medicinal plant

In vitro clonai multip1ication of Coleus forskahlii Briq a threatened plant, has been achieved on MS medium supplemented with Kn (2.0 mg/l) and IAA (1.0 mg/l) using nodal segments as explants, Shoots multiplied at a rate of 12 — fold every six weeks. Rooting was achieved upon transfer of shoots onto MS medium containing IAA (1.0 mg/l). The micropropagated plants were successfully established under field conditions. Forskolin content in tubers of plants obtained by micropropagation was found to be 0.1%, the same as that found in wild plants. This micropropagation procedure should be useful for conservation as well as production of this important plantAbbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) basal medium - NAA -naphthalene acetic acid     

裂叶悬钩子组织培养研究——Ⅰ.裂叶悬钩子叶外植体培养直接再生不定芽及植物激素对它的影响

本文首次报道裂叶悬钩子(Rubus laciniatus Wild)叶外植体培养在改良的NN⁶⁹培养基上附加2—4mg/1 6-BA和0.1mg/1 NAA或1—3mg/1 2,4-D和0.1mg/1 NAA,两者都可直接从完整叶片、叶片下切段或叶柄诱导出不定芽。诱导频率达20—48%。而不定芽绝大部分发生在叶轴处或叶柄基部。完整叶片的不定芽诱导率与叶片下切段无差别,但比叶柄基部诱导率要高。6-BA对叶轴处不定芽诱导率比2,4-D的要高。此外,不需继代培养,不定芽数可达10—20个,继代培养一个月左右,每个不定芽能形成丛生芽数可达40一60个。另外,本文还讨论了细胞分化过程中的极性现象。     

Variation and morphogenetic characteristics of different Stachys species during microclonal propagation

Morphogeneses of Stachys different species introduced in culturing in vitro have been compared. The frequency of altered forms have been demonstrated to be related to the plant genotype. All regenerants of S. sieboldii, which reproduces in vivo only vegetatively, are phenotypically normal, irrespective of the concentrations of plant growth regulators at which they have been obtained. Only changes in isozyme patterns have been observed in the regenerants grown in media containing at least 10 mg/l benzyl aminopurine (BAP); most of these changes are the absence of a particular component of the pattern. The cross-pollinating species Stachys ocymastrum, which typically reproduces by seeds, has yielded morphologically altered forms even in phytohormone-free media; its isozyme patterns often contained a new component. Analysis of the isoperoxidase patterns of regenerants of both Stachys species obtained with the use of high phytohormone concentrations has demonstrated qualitative and quantitative changes suggesting the appearance of somaclonal variants even in the course of plant regeneration directly from nodal segments, bypassing callus formation. Changes have also been found in Stachys plants regenerating from the callus tissue.     

Apical callus formation and plant regeneration controlled by plant growth regulators on axenic culture of the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta)

Axenic cultures of Gracilariopsis tenuifrons (Bird et Oliveira) Fredericq et Hommersand (Gracilariales, Rhodophyta) were established in ASP12‐NTA solid medium (0.4% agar and 1.0% sucrose) supplemented with plant growth regulators to evaluate the effects on apical callus formation and plant regeneration. Indole‐3‐acetic acid (IAA), 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 6‐benzylaminopurine (BA) were added individually or in combinations (IAA : BA) over a range of concentrations from 0.5 to 5 mg L^?1. Growth of apical and intercalary segments was stimulated by high concentrations of 2,4‐D (5 mg L^?1) and a high IAA to BA ratio (IAA : BA = 5:1 mg L^?1) respectively. Apical calluses were originated from divisions of apical and cortical cells located at apical regions of thallus segments and lateral branches. Low concentration of IAA (0.5 mg L^?1) or a high IAA to BA ratio (IAA : BA = 5:1 mg L^?1) were the optimal treatments for inducing apical callus formation in apical segments, while high concentration of IAA (5 mg L^?1) stimulated the highest callus induction rate in intercalary segments. Conversely, equal parts IAA and BA (IAA : BA = 1:1 mg L^?1) and low concentration of 2,4‐D (0.5 mg L^?1) stimulated growth of apical calluses from apical and intercalary segments, respectively. Two processes of regeneration were observed: direct regeneration (upright axis originated from cells of proximal region of intercalary segments) and indirect regeneration (adventitious plantlet originated from cells of apical calluses). Direct regeneration was promoted significantly by treatment with a low IAA to BA ratio (IAA : BA= 1:5 mg L^?1), and treatments with IAA (0.5 mgL^?1) or 2,4‐D (0.5 or 5 mg L^?1) significantly stimulated the elongation of upright axis. Plant growth regulators are essential to inducing indirect regeneration, and a high concentration of IAA (5 mg L^?1) and BA (5 mg L^?1) were the optimal treatments for inducing the regeneration of plantlets from apical calluses in apical and intercalary segments, respectively. Regenerating plantlets grew into plants morphologically similar to those formed from germinating spores, and became fertile after 6 weeks. The results suggest that auxins and cytokinins are involved in developmental regulatory processes in G. tenuifrons. The regeneration process from calluses in species of Gracilariales was observed for the first time in the present study. The culture system described for G. tenuifrons could be useful for micropropagation and for biotechnological applications in agarophytic algae.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.     

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

Micropropagation of meadowfoam (Limnanthes spp.)

Summary A rapid micropropagation system was developed for meadowfoam (Limnanthes spp. Brown) using four genotypes of three species. Murashige and Skoog (MS) medium supplemented with N⁶ benzyladenine (BA) and indole-3-acetic acid (IAA) at 0, 0.1, 0.5, 1.0 and 2.0 mg/l was tested for multiplication, shoot elongation and rooting. Expiants were taken from pot-grown plants. The most useful level for shoot growth and multiplication of both floral induced and non-induced plants was 0.5 mg/l BA. IAA failed to affect shoot growth or multiplication. Expiants from non-induced plants multiplied at moderate to high rates on 0.5 mg/l BA, while those from induced plants multiplied slowly and tended to elongate and flower. Non-induced plants on 2 mg/l BA produced large numbers of tiny shoots; induced plants did not respond. Shoots of all genotypes rooted on MS medium without hormones and all plants grew normally after transplanting to soil. This system provides a new tool for the development of meadowfoam as a crop plant.Abbreviations (BA) N ⁶ -benzyladenine - (IAA) indole-3-acetic acid - (MS) Murashige and Skoog medium, 1962     

Bulgarian golden root <Emphasis Type="Italic">in vitro</Emphasis> cultures for micropropagation and reintroduction

Rhodiola rosea is an endangered medicinal plant used for cancer, cardiovascular, and nervous system diseases therapy. Due to its limited distribution and sustainability alternative methods for production of its valuable substances are under investigation. Using in vitro techniques apical and rhizome buds, leaf nodes, stem and radix segments from wild plants and in vitro seedlings were plated on 24 modified Murashige and Skoog (1962) media. Decontamination of plant material was successful only in 21% of the schemes. The best shoot induction was obtained from seedling explants on media containing 2 mg/l zeatin or N⁶-benzylaminopurine, each. Their reduction stimulated shoot formation in the next passages (multiplication rate up to 5). Efficient rooting was induced on half-strength MS with 2 mg/l Indole-3-butyric acid and stimulated by adding 0.2 mg/l Indolyl-3-acetic acid. Regenerants rooted in perlite, peat, and soil (1:1:2), adapted in greenhouse, and transplanted in the mountains survived (70%) and developed like the wild plants. Salidroside content of these plants after one or two years was high (0.64 and 0.61% in rhizomes and 0.62 and 0.53% in roots, respectively). This is the first established efficient scheme for micropropagation of Bulgarian R. rosea allowing habitats restoration, germplasm conservation, and potential application of biotechnology for production of valuable substances.     

A rapid procedure for plant regeneration from protoplasts isolated from suspension cultures and leaf mesophyll cells of wild Solanum species and Lycopersicon pennellii

A rapid procedure for protoplast isolation, culture and plant regeneration has been developed for two Solanum species (S. lycoperisicoides and S. verrucosum) and Lycopersicon pennellii. Freshly isolated protoplasts were initially cultured in liquid Solanum Culture Medium (SCM), containing 2,4-dichlorophenoxy acetic acid (2,4-D). Subsequent dilution with fresh culture medium without auxins appeared to be essential to obtain rapid regeneration medium later on. The resulting micro calli were first grown in a culture medium containing 0.5 mg/l 6-BAP and 0.05 mg/l NAA and 0.2 M mannitol and 7.3 mM sucrose to induce greening, at a lower osmolarity (300 mOsm · kg⁻¹). Then, the green micro calli were transferred to shoot induction medium, containing 2 mg/l zeatin, 0.1 mg/l IAA and 2% sucrose (150 mOsm · kg⁻¹). In this way plants could be regenerated from leaf mesophyll protoplasts and suspension cell-derived protoplasts of L. pennellii and S. lycopersicoides within 2 months. Shoot regeneration from leaf mesophyll protoplasts of the two lines of S. verrucosum could be obtained 3 months after protoplast isolation.     

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans [Glycine max (L.) Merr.]

A successful, efficient system for multiple soybean shoot induction of soybean [Glycine max (L.) Merr.] is reported. Multiple shoots were induced from cotyledonary nodes and hypocotyl segments cultured on media supplemented with 2 mg/l thidiazuron (TDZ) or 1.15 mg/l benzyladenine (BA). It was found that TDZ induced adventitious shoots more efficiently than BA and that hypocotyl segments promoted more adventitious shoots than cotyledonary nodes. The optimal TDZ concentrations for shoot organogenesis from hypocotyl segments were between 1 and 2 mg/l. Basal media also influenced the efficiency of shoot organogenesis. The frequency of adventitious shoot formation tended to increase when the salt concentration in the basal media supplemented with 2 mg/l TDZ was reduced. Two media (1/2B5 and 1/2L2) stimulated shoot organogenesis efficiently from hypocotyl segments. This method can thus be advantageously applied in the production of transgenic soybean plants. Received: 3 July 1996 / Accepted: 9 May 1997     

Plant regeneration from protoplasts of a wild lettuce species (Lactuca saligna L.)

Protoplasts isolated from young, unexpanded leaves of the wild lettuce species, Lactuca saligna, divided to give colonies, when plated at low density in KP8 medium solidified with 1% (w/v) agarose. Sustained colony development was dependent upon the incorporation of the bead culture approach. Plant regeneration, via organogenesis, followed the transfer of colonies firstly to K8 medium and then to M/S medium supplemented with IAA and 6-BAP, both solidified with 1% (w/v) agarose. Excised shoots were rooted on agar-solidified M/S medium lacking phytohormones. The potential of this species as a source of race-non-specific mildew resistance for transfer into lettuce (L. sativa) via somatic hybridisation is discussed.Abbreviations 6-BAP 6-benzylamine purine - IAA indoleacetic acid - M/S Murashige and Skoog (1962) - fwt fresh weight     

睡菜离体快繁技术的研究

以睡菜的幼嫩茎段为外植体,接种到附加不同浓度激素配比(6-BA/NAA)的MS培养基,诱导睡菜愈伤组织、芽及根的生长。研究发现,外植体在1.0mg/L 6-BA+0.1mg/L NAA+MS的培养基上培养10d,可观察到浅绿色的愈伤组织。愈伤组织转接到4.0mg/L 6-BA+0.3mg/L NAA+MS培养基上2周左右可生成芽。对带芽的愈伤组织再进行诱导生根进而形成完整再生植株,最适根诱导培养基为0.3mg/L 6-BA+1.0mg/L NAA+MS培养基。该实验采用植物离体快繁技术成功建立了睡菜再生体系,为睡菜种苗规模化奠定了技术基础。     

Role of Exogenous Hormones in Inducing Cells in different Positions of Perianth Explants to Regenerate Flower Buds in Hyacinthus orientalis

Role of the exogenous hormone in inducing different position cells of perianth explants of hyacinth to regenerate flower buds was studied. Experiments showed that (1) Exogenous hormones are necessary for inducing cells of the perianth explant to regenerate the flower buds; (2) Only cytokinine alone could induce the regeneration of the flower buds, the auxin was not necessary; (3) Exogenous hormones in different concentrations could induce cells in the different parts of the perianth explants to differentiate the flower buds: 6-BAP or zeatin 2 mg/L alone could induce cells located at the lower part of the perianth to differentiate flower buds. Combination of 6-BAP or zeatin 2 mg/L and 2, 4-D 0.1 mg/L was advantageous to cells located middle part of the perianth to regenerate the flower buds. Combination of 6-BAP or zeatin 2 mg/L and 2, 4-D 1.0 mg/L could promote cells located at the upper part of the perianth to differentiate flower buds.     

Micropropagation of 21 species of Mexican cacti by axillary proliferation

Summary We have developed micropropagation systems for 21 species of Mexican cacti using explants from seedlings germinatedin vitro or shoot segments of juvenile 2–3-yr-old greenhouse plants. The species propagated belong to the generaAstrophytum, Cephalocereus, Coryphantha, Echinocactus, Echinocereus, Echinofossulocactus, Ferocactus, Mammillaria, Nyctocereus, andStenocactus. Multiple shoot formation from areoles was achieved in Murashige and Skoog (MS) medium supplemented with either 1 or 2 mg N⁶-benzyladenine (BA) per 1 (4.44 or 8.87 μM) or BA at 1 or 2 mg/l plus naphthaleneacetic acid at 0.1 or 1 mg/l (0.54 or 5.37 μM). The requirements of growth regulators for optimal shoot proliferation, the velocity of the response, and the number of buds produced by explant were different among the genera and species studied. Rooting of the shoots generatedin vitro was achieved in MS medium supplemented with indoleacetic acid at 0.5–1 mg/l (2.85–5.71 μM) or indolebutyric acid at 0.5–1 mg/l (2.46–4.90 μM). Finally, 70–95% of the rooted plants transferred to potting medium survived.     

Plant regeneration from callus-derived protoplasts of Pelargonium x domesticum

A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. Melissa) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×10⁵ protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×10⁵ protoplasts/ml in a mixture (11 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm³ developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - CW Calcofluor White - FDA fluorescein diacetate     

Micropropagation of Zeyheria montana Mart. (Bignoniaceae), an endangered endemic medicinal species from the Brazilian cerrado biome

Zeyheria montana Mart. has become endangered, primarily because of deforestation of its habitat, its use as a medicinal plant extract, and the strong endemism of the species. In this study, an efficient protocol was established for the micropropagation and conservation of Z. montana germplasm using isolated mature zygotic embryos as explants. Embryos germinated in vitro 4 d after isolation and inoculation on modified Murashige and Skoog (MS) medium containing 2.0 mg/l gibberellic acid (GA₃). The addition of GA₃ also improved the germination index and accelerated the process of germination. Nodal segments from seedlings were placed on modified ¼-strength MS medium containing 0.1 mg/l 6-benzyladenine and 0.5 mg/l GA₃. Nodal segments produced 7.3 shoots per explant within 60 d. Following transfer of shoots to a medium containing 1.5 mg/l indole-3-butyric acid, roots formed. All plantlets obtained were successfully acclimatized under greenhouse conditions, and approximately 68.5 acclimatized plants could be obtained per seed each year. This protocol provides a method to preserve this rare and endangered medicinal plant.     

Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high-frequency axillary shoot proliferation

A protocol is described for rapid and large-scale propagation of the woody aromatic and medicinal shrub Vitex negundo by in vitro culture of nodal segments from mature plants. Of the three different cytokinins – N⁶-benzyladenine (BA), kinetin, and thidiazuron – evaluated as supplements to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.0 mg/l was most effective in inducing bud break. Although callus-free multiple-shoot formation was a function of cytokinin activity alone, faster bud break coupled with an enhanced frequency of shoot development (92%) and internode elongation were dependent on the synergistic influence of gibberellic acid (GA₃) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l). The frequency of shoot proliferation was markedly influenced by the explanting season. By repeated subculturing of nodal segments harvested from the in vitro-formed axenic shoots on MS containing 1.0 mg/l BA and 0.4 mg/l GA₃, prolific shoot cultures free from proximal callusing and showing a high-frequency multiplication rate were established. The percentage shoot multiplication (98–100%) as well as the number of shoots per node (six to eight) were highest during the first three culture passages, after which there was a gradual decline in shoot development. Rooting was best induced (94%) in shoots excised from proliferated shoot cultures on half-strength MS medium augmented with an optimal combination of indole-3-acetic acid and indole-3-butyric acid each at 1.0 mg/l. Vermi-compost was the most suitable planting substrate for hardening inside a plant growth chamber and its use ensured high-frequency survival (93%) of regenerated plants prior to outdoor transfer. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics as well as vegetative and floral morphology. Received: 10 January 1998 / Revision received: 17 June 1998 / Accepted: 8 July 1998