Plantlet formation from embryonic tissue of chestnut grown in vitro

Development of axillary shoots was induced when isolated embryonic axes of chestnut (Castanea sativa Mill.) were cultured on a defined medium containing 6-benzyl-aminopurine (BAP). The optimal concentrations of BAP were determined for development of axillary shoots from both embryonic axes and subcultured shoots. After shoot multiplication a great number of shoots have been maintained sequentially without significant change in the proliferative rate for one year. Limited rooting has been obtained with excised shoots. Indole-3-butyric acid (IBA) at 2 mg/l was used to induce root primordia. After 8 days of treatment the plantlets were transferred to an auxin-free medium for root development.     

Enhanced Formation of Roots and Subsequent Promotion of Growth of Shoots on Cryopreserved Nodal Segments of Asparagus officinalis L

This study was designed to investigate the effects of cryopreservation on the survival, organogenesis, and growth of plants regenerated from nodal segments of asparagus (Asparagus officinalis L.) that had been cut from cultures in vitro. The addition of dimethylsulfoxide (Me2SO) to the freezing solution at 8-16% (v/v) with or without a sugar (glucose, sorbitol, or sucrose) was effective for successful cryopreservation by a slow prefreezing method. Frequencies of root formation (average, 59.3%) from cryopreserved and surviving nodal segments were significantly higher (P < 0.005) than those (average, 13.9%) from nodal segments that had only been treated with freezing solution supplemented with 12% (v/v) Me2SO and various sugars without freeze-thawing. The increased frequency of root formation from cryopreserved nodal segments appears to have been induced by the freeze-thaw step of the cryopreservation procedure. Numbers and lengths of shoots derived from cryopreserved nodal segments were initially lower but were subsequently higher, after 60-90 days of culture, than those of shoots derived from nodal segments without freeze-thawing. The promotion of growth of shoots from cryopreserved nodal segments seemed to have been due to the increased percentage of root formation. Histological observations revealed that only dome-shaped meristematic tissue and a few cells of cladophyll primordia survived in cryopreserved nodal segments that had been cultured for 5 days after thawing. Many mitochondria and well developed rER were observed in these cells. Disorganization and/or physiological changes might have occurred in the surviving tissues and/or cells of the cryopreserved nodal segments that were responsible for the subsequent increased formation of roots. Copyright 1998 Academic Press.     

Shikonin derivative formation on the stem of cultured shoots in Lithospermum erythrorhizon

Lithospermum erythrorhizon , which are capable of producing red pigments, have been established. The red pigments were formed on the stems of L. erythrorhizon shoots cultured both on solid and in liquid media without phytohormones at 25 °C in the dark. Thin-layer chromatography, high-performance liquid chromatography and ¹ H nuclear magnetic resonance analyses revealed that the red pigments which accumulated on the cultured shoots were shikonin derivatives. The effects of various basal media and phytohormones (indole-3-acetic acid, indole-3-butyric acid and kinetin) on the growth and the formation of shikonin derivatives were investigated. When the shoots were cultured on Murashige and Skoog solid medium, the addition of kinetin remarkably enhanced shikonin derivative accumulation in the shoots. However, these effects of kinetin were not observed in the liquid culture when cultured in Gamborg B5 medium. The maximum content of shikonin derivatives (2.3% as dry weight, ca. 1.5 mg/100 ml flask) was observed in the shoots cultured in phytohormone-free B5 liquid medium for 5 weeks. Received: 1 February 2000 / Revision received: 23 March 2000 / Accepted: 28 March 2000     

Protoplasts from cotyledon and hypocotyl of sunflower (Helianthus annuus L.): shoot regeneration and seed production

Summary Cotyledon and hypocotyl protoplasts of Helianthus annuus inbred line 47 302 bcd were embedded in alginate and plated on L4 medium (Lenée and Chupeau 1986). After one month, the calli were transferred on MSSH regeneration medium (Murashige and Skoog 1962; Schenk and Hildebrandt 1972) where they regenerated shoots (overall efficiency 10^–2%). The shoots were elongated on B5 (Gamborg et al. 1968) medium first without hormones, then supplemented with GA3 and BAP (both 0.05 mg/l). In order to overcome the difficulty to induce rooting by classical methods, the elongated shoots were grafted on a sunflower rootstock. The grafted shoots produced flowers and seeds. Different factors have been shown to have an important influence on the capacity to regenerate shoots: the genotype, the physical culture conditions at the callus regeneration step (e.g. protoplasts embedded in alginate), and the media composition.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - IBA indole-3-butanoic acid - IAA indole acetic acid - MES 2-N-morpholinoethane sulfonic acid - NAA 1-naphthalene acetic acid - 2,4D 2,4 dichlorophenoxyacetic acid     

Towards automation: Radiata pine shoot hedges in vitro

A novel system for in vitro shoot production has been developed whereby shoot hedges are maintained in one vessel. Monthly crops of shoots are produced for rooting. Radiata pine shoot hedges were maintained on Lepoivre (LP) nutrient agar medium for 18 months using a weekly liquid-nutrient replenishment system. In a separate experiment liquid-LP-nutrient replenishment of shoots twice weekly without transfers (D) resulted in better shoot growth and health than monthly transfers to fresh agar medium (B), monthly transfers to fresh agar medium plus aeration twice weekly (C), or no transfers and no liquid nutrient addition (A). Liquid nutrient replenishment twice weekly was better than 2 weekly or 4 weekly replenishment. The percentage of normal waxy (abundant tubular epicuticular wax) shoots harvested monthly increased significantly over the culture period from 41% at the first harvest to 93% at the eight harvest, and remained high at 97% from the ninth to twelfth harvest. The percentage of wet (no tubular epicuticular wax, small amounts of globular epicuticular wax) shoots harvested showed a corresponding decline—from 59%, to 7% at the eighth harvest. Shoots were harvested at a rate of 672/h (1.19 cents/shoot at a labour cost of NZ$8.00/h) and approximately 1100 shoots were produced per square metre of agar surface per month. Initial problems of contamination and crowding were overcome. These results will greatly facilitate progress towards automation of shoot production and reduction of costs of micropropagated trees. An automated system used in combination with other cost-saving techniques or robotics could potentially result in a substantial reduction in costs. This is the first report of a method of culturing shoots as hedges for a period of up to 18 months without manual subculturing.     

Mixed cropping with maize combined with moderate UV-B radiations lead to enhanced flavonoid production and root growth in faba bean

Abstract

Flavonoids have recently been proposed to function as developmental regulators and/or signaling molecules under biotic or abiotic stress. The aim of this study was to determine the composition and concentration of fiavonoid aglycones (kaempferol, luteolin, and quercetin) in faba bean shoots and roots, as affected by interspecific root interactions with maize and moderate UV-B radiation. Independent of the UV-B treatment, interspecific root interactions with maize enhanced the concentration of both quercetin and luteolin in the faba bean shoots and roots by 50 and 97.8%, respectively, and improved the root length by 14.6%. In addition, moderate UV-B radiation facilitated a systematic increase of both aglycones in both shoots and roots without affecting plant growth. To our knowledge, this report is the first work documenting the response of faba bean flavonoids to interspecific root interactions with maize and moderate UV-B radiation, and it provides a new perspective for understanding interspecific interactions.     

Oxalic acid enhances Cr tolerance in the accumulating plant Leersia hexandra Swartz

This study examined the relationship between oxalic acid and Cr tolerance in an accumulating plant Leersia hexandra Swartz. The plants grown in hydroponics were exposed to Cr at 0, 5, 30, and 60 mg/L (without oxalate), and 0, 40, and 80 mg/L concentrations of Cr (with 70 mg/L oxalate or without oxalate). The results showed that more than 50% of Cr in shoots was found in HCl-extracted fraction (chromium oxalate) when the plants were exposed to Cr. Cr supply significantly increased oxalate concentration in shoots of L. hexandra (p < 0.05), but did not increase oxalate concentration in roots. Under 80 mg/L Cr stress, electrolyte leakages from roots and shoots with oxalate treatment were both significantly lower than those without oxalate treatment (p < 0.05), indicating exogenous oxalate supply alleviated Cr-induced membrane damage. Oxalate added to growth solution ameliorated reduction of biomass and inhibition of root growth induced by Cr, which demonstrated that application of oxalate helped L. hexandra tolerate Cr stress. However, oxalate supply did not affect the Cr concentrations both in roots and shoots of L. hexandra. These results suggest that oxalic acid may act as an important chelator and takes part in detoxifying chromium in internal process of L. hexandra.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.)

 Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic shoots. Transformed shoots are invigorated and multiplied on a non-selective medium prior to grafting, thus assuring that plants can be recovered from transgenic shoots. We have constructed a binary vector, pBin34SGUS, with an intron-containing β-glucuronidase gene (uidA) under the control of the Figwort mosaic virus 34S promoter. The 34S promoter efficiently drives uidA gene expression both in transient assays and in transgenic Rio Red leaf tissue, although at levels five- to sevenfold lower than the Cauliflower mosaic virus 35S promoter. An untranslatable coat protein gene (uncp) of the Citrus tristeza virus strain SY568 and the Galanthus nivalis agglutinin gene (gna) were inserted into pBin34SGUS and transgenic plants have been obtained. Stable integration of the uncp and gna genes was confirmed by Southern hybridization and gna gene expression was confirmed by Western blot analysis. Received: 2 February 2000 / Revision received: 21 June 2000 / Accepted: 29 June 2000     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。     

Stimulation of shoot regeneration from jute cotyledons cultured with non-ionic surfactants and relationship to physico-chemical properties

Summary The effects have been studied of the non-ionic surfactants, Plutonic F-68, Tween 20 or Triton X-100, on shoot regeneration from cultured jute (Corchorus capsularis L.) cotyledons with attached petioles. This group of non-ionic surfactants was selected in order to determine a possible relationship between the physico-chemical properties of individual compounds and their observable effects on plant morphogenesis in cultured jute cotyledons. Supplementation of culture medium with 0.001–0.5% (w/v) Pluronic F-68 increased the mean percentage of cotyledons producing shoots and the mean number of shoots/cotyledon, with maximal responses at 0.5% (w/v). By contrast, Tween 20 produced maximal effects at 0.001% (v/v), with inhibition of shoot formation at 0.5% (v/v). In both cases, phenotypically normal plants were recovered which could be grown to maturity. Culture of cotyledons with 0.001% (v/v) Triton X-100 similarly increased both the percentage of cotyledons producing shoots and the number of shoots/cotyledon. However, these shoots did not develop into mature plants. Additionally, shoots did not regenerate from cotyledons cultured with Triton at 0.01–0.5% (v/v). These results demonstrate mat there is an apparent relationship between the hydrophilic-hydrophobic (HLB) balance of surfactants which determine their cell permeabilising properties and consequently, their effects on morphogenesis.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962)     

The architecture of Zeylanidium olivaceum (Podostemaceae) inferred from the structure of the primary shoots

Zeylanidium olivaceum (Podostemaceae-Podostemoideae) is the only crustose-rooted species of the genus that still develops prominent primary shoots from the seedling in addition to the secondary (root-borne) shoots forming the clonal plant body. The primary shoots are articulated into an up to 8.5 cm long and 4 mm thick stalk (hypocotyl) and a copiously foliated paint-brush-like shoot which is sympodially branched in the form of a helicoid cyme. The helicoid branching pattern indicates a transversal prophyll position, typical of the dicotyledons, but replaced in most other Podostemoideae by a median prophyll position. The short stems within the leafy head do not separate, but are fused to a dense aggregate (coenosome). Branches are mainly vegetative with a rosette of about 20 elongate subulate leaves. The primary shoots branch in the vegetative stage and thus differ from other Podostemoideae where ramification is confined to the floriferous shoots. The leaves adhere together at the base, forming an apical furrow-like hollow surrounding the shoot tip. The tiny shoot apex is one-layered, radially symmetrical, and develops leaf primordia in a decussate pattern. The erect primary shoots thus differ from the distichously foliated plagiotropic secondary shoots by the decussate phyllotaxis, and by the presence of more than 20 leaves on a shoot as compared to the about six leaves on the vegetative and floriferous secondary shoots. The features observed in the primary shoots are interpreted as primitive as compared to those of the secondary shoots. Z. olivaceum is thus characterised by heterobathmy, i.e., the occurrence of plesiomorphic (primary shoots) and apomorphic features (secondary shoots). The primary shoots exhibit primitive features that apparently have been lost in secondary and primary shoots of most other members of subfamily Podostemoideae.     

Optimized Shoot Regeneration System for the Commercial Korean Radish ‘Jin Ju Dae Pyong’

We have investigated the usefulness of hypocotyl (cultured on N1B2 medium) and cotyledon explants (on CR medium: Murashige and Skoog (MS) medium, 3% sucrose, 20 μm benzylaminopurine, pH 5.8) for the regeneration of shoots of the Korean radish ‘Jin Ju Dae Pyong’. The importance of ethylene (indirectly), polyamines and gelling agent were studied in both media. Although the addition of ethylene-inhibitors and silver nitrate to the culture media were beneficial towards shoot regeneration and agar-based treatments (0.8% w/v) being superior in shoot production compared to agarose treatments (0.4% w/v), both explants responded differently in culture. Hypocotyls cultured in the presence of silver nitrate or aminoethoxyvinylglycine (AVG) regenerated significantly (p < 0.05) more shoots compared to N1B2 medium alone; supplementation of 20 μM AVG to N1B2 medium gave optimal shoot production (40% of explants regenerating shoots). The addition of 10 μM AVG to CR medium produced maximum shoot regeneration from cotyledon explants (60% producing shoots). Plants derived from 3-month-old cultures produced greater seed weights, larger leaves and greater genetic variability (50–80% of cells having 20–40 chromosomes) compared to seed-derived (85–90% diploid) and plants from 1-month-old cultures (78–88% diploid). Our results show, that if prolonged culture of explants is avoided, a large number of phenotypically-normal plants can be produced, which in turn, could be utilized in the genetic improvement of radish.     

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     

In vitro plant regeneration from cotyledon explants of Swainsona salsula Taubert

An effective protocol has been developed for plant regeneration from cotyledon explants of Swainsona salsula Taubert (Saline swainsona), a medicinal and agronomic shrub. Adventitious shoots were obtained from 83.2% of cotyledon explants from 3-day seedlings cultured on Murashige and Skoog (MS) medium containing 2.0 mg l⁻¹ thidiazuron (TDZ), with an average of 9.3 shoots per explant. Individual elongated shoots were rooted on half strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid (IBA), with 59.3% success. Regenerated plants with well developed shoots and roots were successfully transferred to soil, without detectable variants. Histological observation revealed that shoots developed from cotyledon explants via organogenesis, with little callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of indole-butyric acid and electro-pulse on in vitro rooting and development of olive (<Emphasis Type="Italic">Olea europea</Emphasis> L.) microshoots

The effects of indole-butyric acid (IBA) and electro-pulses on rooting and shoot growth were studied in vitro, using olive shoot cultures. Tested shoots were obtained from seedlings belonging to three Spanish cultivars, ‘Arbequina’, ‘Manzanilla de Sevilla’ and ‘Gordal Sevillana’, which have easy-, medium- and difficult-to-root rooting abilities, respectively. The standard two-step rooting method (SRM), consisting of root induction in olive rooting medium supplemented with 0, 0.1 or 1 mg/l IBA followed by root elongation in the same rooting medium without IBA, was compared with a novel one-step method consisting of shoot electro-pulses of 250, 1,250 or 2,500 V in a solution of IBA (0, 0.1 or 1 mg/l) and direct transferral to root elongation medium. The rooting percentage of the seedling-derived shoots obtained with the SRM was 76% for ‘Arbequina’ and ‘Gordal Sevillana’ cultivars and 100% for ‘Manzanilla de Sevilla’ cultivar, whereas with the electro-pulse method, the rooting percentages were 68, 64 and 88%, respectively. IBA dipping without pulse produced 0% rooting in ‘Arbequina’ seedling-derived shoots. The electroporation in IBA not only had an effect on shoot rooting but also on shoot growth and development, with longer shoots and higher axillary shoot sprouting and growth after some of the treatments. These effects were cultivar-dependent. The electro-pulse per se could explain some of these effects on shoot development. I.M.G. Padilla and I. Vidoy contributed equally to this article.     

Totipotency of tomato protoplasts

Summary An efficient and reliable protocol for tomato protoplast isolation, culture, and plant regeneration has been developed. Fourteen diverse cultivars were tested. Fertile plants were regenerated from all 14 cultivars without any modification in the protocol. Plating efficiency (percentage of the protoplasts that formed minicalli) of up to 50% was achieved. Those mini-calli rapidly regenerated shoots at high frequencies. Regenerated shoots can be easily rooted on a basal medium with the appropriate auxin, and have been set to soil within two months after the isolation of the protoplasts.     

改良菜心离体培养植株再生体系的研究(简报)

菜心(Brassica campestris L.subsp.chinensis Makino var.parachinensis Tsen et Lee)为十字花科芸薹属芸薹种中国白菜亚种中的一个变种,又名“菜薹”。它是我国南方特产蔬菜之一,在蔬菜的周年供应上有重要地位。目前迫切需要培育抗病虫、抗逆和具有其他优良农艺性状的新品种,以提高菜心的     

Clonal propagation of Stevia rebaudiana Bertoni by stem-tip culture

Clonal propagation of Stevia rebaudiana has been established by culturing stem-tips with a few leaf primordia on an agar medium supplemented with a high concentration (10 mg/l) of kinetin. Anatomical examination has suggested that these multiple shoots originate from a number of adventitious buds formed on the margin of the leaf. Innumerable shoots can be obtained by repeating the cycle of multiple-shoot formation from a single stem-tip of Stevia. These shoots produce roots when transferred to a medium containing NAA (0.1 mg/l) without kinetin. The regenerated plantlets can be transplanted to soil.