Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Callus Culture and Plant Regeneration from Seedling Explants of Pergularia daemia (Forsk) Chiov

Callus cultures were established from seedling explants of Pergularia daemia (Forsk) Chiov on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins. Optimal callus developed from leaf explants on MS medium supplemented with 2,4-D (2 mg l^?1) + 2iP (0.1 mg l^?1), was used for morphogenesis. Adventitious shoots were regenerated (70%) from the calli on MS medium supplemented with NAA (0.1 mg l^?1)+ BAP (2 mg l^?1). Individual shoots were rooted on half strength MS medium supplemented with 0.1 mg l^?1 IBA. Plantlets with well developed roots were successfully transferred to soil and 50% of the transferred plants survived.     

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10⁴ protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.     

影响沙田柚叶片离体培养的因素研究

研究了离体培养条件下影响沙田柚叶片愈伤组织诱导和分化的一些因素。结果表明 ,2 ,4 D可以诱导愈伤组织的形成 ,高浓度的蔗糖 (6% )显著提高叶片愈伤诱导率与愈伤组织重量 ,且在 2 ,4 D和蔗糖之间存在着相互作用。外源GA3处理抑制愈伤组织的诱导和生长 ,而CCC与ABA处理显著提高叶片的愈伤诱导率和愈伤组织生长量。愈伤组织转移到附加 3 .0mg/LBA的MS分化培养基上可以分化出芽 ,0 .2 5mg/LGA3的加入可以进一步提高愈伤组织的分化率和每块愈伤组织的再生芽数。     

裂叶悬钩子器官发生的细胞组织学观察

裂叶悬钩子(Rubus laciniatus W.cv.Thornless Evergreen)是蔷薇科悬钩子属一种重要的小果类植物。我们在以离体叶片诱导不定芽成功的基础上,对其叶片不定芽形态发生过程作了进一步观察。供接种的叶片从基部去掉叶柄,按前文的操作顺序进行灭菌,接种和培养。从培养第3天起,从叶片基部与叶柄接合处切取材料固定,以后每隔2天取样一次,一直取到20     

In vitro studies on Pea (Pisum sativum L.): I. Callus formation,regeneration and rooting

Abstract

Callus production, shoot formation via organogenesis and rooting of the regenerated shoots are reported in an Egyptian variety of Pisum sativum L. Calli were initiated from hypocotyl, leaf, root and mature embryo explants when cultured on MS medium containing B5 vitamins and supplemented with 2 mg/l 2,4-D+1 mg/l kin. Among the different types of explants, hypocotyl showed best potential for callus proliferation. Hypocotyl, leaf and immature cotyledon explants were used for shoot organogenesis. The best results of shoot formation were achieved when hypocotyl explants were cultured on MS-medium supplemented with 2 mg/l BA+1 mg/l NAA. However, immature cotyledon explants showed the highest frequency of shoot formation with 1 mg/l BA. Data of in vitro rooting showed that maximum root frequency occurred on culture medium containing half strength of MS salts, 40 g/l sucrose and 2 mg/l NAA.     

Somatic embryogenesis,organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA₃) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l^?1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l^?1 2,4-D and 1.6 mg l^?1 zeatin, the most efficient growth regulator combination.     

Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones.)

A reproducible and efficient callus-mediated shoot regeneration system was developed for the large-scale production of Valeriana jatamansi Jones., a highly medicinal plant species of global pharmaceutical importance. Effect of Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) on callus induction and production of valepotriates accumulation was studied by using different explants. In V. jatamansi, the degree of callus induction varied significantly depending on explants type and the growth regulators used. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. The callus induction frequency was found to be optimum in rhizome explants on media supplemented with 0.5 mg/l 2,4-D. The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. MS medium fortified with 0.75 mg/l thidiazuron in combination with 0.5 mg/l NAA showed the highest regeneration frequency (88.6 %) and produced the maximum number of shoot buds (15.20 ± 0.20) capable of growing into single plants. Vigorous callus obtained from MS medium supplemented with different concentrations of 2,4-D, NAA and IBA were used for industrially important valepotriates [acevaltrate (ACE), valtrate (VAL) and didrovaltrate (DID)] analysis. High performance liquid chromatography analysis of callus revealed that medium with 2,4-D (1 mg/l) was found responsible for increasing ACE and DID yield, whereas VAL production was higher in case of medium supplemented with NAA (1 mg/l). However, the accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. IBA was not beneficial for the valepotriate synthesis, as it helped to accumulate significantly lower concentration of ACE, VAL and DID. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 %. As Indian valerian is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present system can be utilized to obtain mass multiplication of the species as well as for the stable biomass and continuous valepotriate production for the pharmaceutical industries.     

Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.     

Callus initiation and plant regeneration in ragi (Eleusine coracana Gaertn.)

Callus was successfully initiated on root, mesocotyl and leaf base segments of 3- to 4-day-old seedlings of ragi (Eleusine coracana Gaertn.). 2,4-D along with casein hydrolysate for Murashige and Skoog's basal medium was found to be most effective for callus initiation and maintenance. Mesocotyl and leaf base tissue derived calli gave shoot buds in medium in which the 2,4-D concentration was lowered.     

Effect of culture medium and light conditions on the morphological characteristics and carbohydrate contents of Medicago strasseri calli

Using 6 culture media (12, 12D, 12G, 11, A and B) made up of MS medium (Murashige-Skoog, 1962) supplemented or not with glycerine, with different cytokinins, and/or 2,4-D, the morphological characteristics and contents in total carbohydrates, reducing sugars, sucrose and starch were studied in calli induced from explants (cotyledon, petiole, hypocotyl and leaf) obtained from Medicago strasseri seedlings. Callus formation was induced under photoperiod (16h light/8h darkness) conditions or in the absence of light. Considerable variability in the calli was observed, depending on the explants and media used. Under photoperiod conditions, medium A with KIN (1 mg/l) and 2,4-D (3 mg/l) induced many calli with the highest contents in total carbohydrates (886.1–889.3 mg/g DW), sucrose (132.1–188.2 mg/g DW) and starch (125.2–247.6 mg/g DW) and the lowest contents in reducing sugars (118.4–173.3 mg/g DW). In media 11, A and B, under conditions of darkness, calli degenerated at the start of culture. Calli developed in darkness generally had dry weights and total carbohydrate and starch contents lower than those cultured under photoperiod conditions. However, sucrose contents were greater in calli formed in darkness. At these cultures times, differentiation, in the form of organogenesis, was only seen using medium B with cotyledons, petioles and leaves as explants. It was also observed when petioles were cultured in medium A but with a less pronounced organogenic response.     

In vitro micropropagation of Ruscus aculeatus

We have developed three protocols for the rapid micropropagation of Ruscus aculeatus. The primary explants utilised were immature embryos, aerial buds excised from rhizomes and shoot buds regenerated from organogenic calli. In order to increase the plant regeneration from the primary explants, we used organogenic calli from cladode, stem and rhizome segments. We tested more than 20 culture media for callus induction and shoot regeneration and the best results were obtained when rhizome segments were cultured on Murashige and Skoog medium supplemented with 0.5 mg dm⁻³ 2,4-dichlorophenoxyacetic acid and 1 mg dm⁻³ kinetin.     

山茱英的组织培养与植株再生

目的：建立山茱萸的组织培养及植株再生体系。方法：分别以山茱萸的叶片、花柄和花托为材料,进行山茱萸不同外植体的离体培养研究,筛选最佳培养基组成。结果：适宜山茱萸叶片愈伤组织诱导的培养基组合为1／2MS,附加BA2．0mg/L、IBA0,5—1．0mg／L;适宜山茱萸花柄、花托愈伤组织诱导的培养基组合为1／2MS,附加BA1．0mg／L、2,4-D0．5mg／L;在1／2MS附加BA2．0mg／L、IBA0．05mg／L的培养基上,可诱导不定芽的产生;1／2MS附加IBA2．0mg／L的培养基有利于山茱萸试管苗生根。讨论：山茱萸的花托是进行组织培养的最适外植体,白色或翠绿色、结构致密的愈伤组织较易分化产生不定芽。     

In vitro regeneration from bulb explants of Indian squill,Urginea indica Kunth

Callus cultures were established from bulb explants of diploid Urginea indica Kunth (Indian squill) on a modified basal medium of Murashige and Skoog (1962) supplemented with either 2 mg/l^-1 2,4-D+15% (v/v) CM or 4 mg/l^-1 2,4-D+2 mg/l^-1 NAA+2 mg/l^-1 KN+1 g/l^-1 YE. Shoot primordia developed after 2–3 subcultures in that medium. Increased growth of shoot primordia was obtained in media containing less auxins and vitamins. Rooted bulbous plantlets obtained were maintained in MS medium with 0.5% sucrose.Adventitious shoots were induced from adaxial epidermal cells of outer scales of regenerated bulbs used as secondary expiants in presence of 1 mg/l^-1 of 2,4-D with slightly higher concentration of the three vitamins of MS medium. From each scale leaf, approximately 400 bulblets were produced in 18 weeks in liquid culture. 90% of the plants transferred to potted soil have survived.     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

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。     

High frequency plant regeneration from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture

An in vitro protocol for efficient plant regeneration has been developed from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture. Embryos with (endosperm-supported culture, ES) or without endosperm (non-endosperm-supported culture, NES) were excised from mature seeds and cultured on MS medium supplemented with various concentrations of 2,4-D (1–5 mg l⁻¹) for callus induction. The percentage of callus induction from ES explants was significantly (P < 0.05) lower than that from NES. The highest frequency (97.6%) of callus induction was obtained from NES explants on MS medium containing 3 mg l⁻¹ 2,4-D. When the primary calli were maintained at a reduced concentration of 2,4-D (0.5 mg l⁻¹) for 3 weeks, embryogenic calli were formed. The embryogenic calli were then transferred to MS medium supplemented with different concentrations of BA (1–5 mg l⁻¹) and 500 mg l⁻¹ casein hydrolysate (CH) for shoot regeneration. However, the capacity of plant regeneration from ES explant-derived calli was significantly (P < 0.05) higher than that from NES. The best response (81.3%) was observed from ES explant-derived calli on MS medium containing 2 mg l⁻¹ BA. Regenerated plantlets with well-developed root systems were transferred to pots where they grew well, attained maturity and produced fertile seeds. This method could be employed for genetic manipulation studies.     

Direct and indirect regeneration of plants from internodal and leaf expiants of<Emphasis Type="Italic">Hypencum bupleuroides</Emphasis> gris

Species of the genusHypencum are of considerable interest worldwide because of their medicinal properties.In- vitro culture is a useful tool for both multiplication of the genus and studying its economically important secondary metabolites. Here, we present an effectivein- vitro propagation method forH. bupleuroides. Leaf and internodal expiants excised from 9-week-old,in vitro-germinated seedlings were cultured on a Murashige and Skoog (MS) medium supplemented with benzyladenine (BA; 1.0 or 0.1 mg L^-1) and 2,4-dichlorophenoxyacetic acid (2,4-D; 1.0 or 0.1 mg L¹). Depending on the BA and 2,4-D combination used, these cultures produced adventitious shoot buds directly on the surfaces of both types of explants as well as excessive calli. Numerous shoots were obtained when the calli from both expiant types were cultured on an MS medium supplemented with 2 mg L^-1 BA. Internodal expiants were more responsive than leaf tissues to direct and indirect plant regeneration. After shoots that regenerated from either the calli or the expiant surface were excised, rooting was best on an MS medium lacking any growth hormones. These rooted plants were then acclimatized under greenhouse conditions, and 90% of regenerants had survived. Ours is the first report ofin- vitro plant regeneration fromH. bupleuroides.     

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。