贯叶金丝桃组织培养的研究

分别以甘肃天水贯叶金丝桃的幼根、幼茎、幼叶为外植体．在1／2MS培养基上附加各类激素,进行贯叶金丝桃的组培实验。研究发现各外植体的增殖速率由高到低分别为幼茎、幼根、幼叶,且得到贯叶金丝桃组培各阶段的最佳培养基成分。诱导愈伤组织的培养基为1／2MS 1．3～1．6mg／L BA 0．2mg／L NAA;培养基1／2MS 1．3～1．6mg／L BA 0．15mg／L NAA有利于不定芽的形成;诱导不定根的培养基为l／2MS IBA0．5～O．8mg／L 蔗糖2．0％。向1／2MS培养基中添加不同的生长素(IAA,IBA,NAA,2．4-D)．在不同浓度梯度的培养基上进行诱导贯叶金丝桃的愈伤组织及不定根的试验,结果表明：生长素IAA,IBA既可诱导愈伤组织,又可以诱导不定根的产生。生长素NAA,2,4-D可诱导产生愈伤组织,但对不定根的诱导作用较差。     

Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of <Emphasis Type="Italic">Periploca sepium</Emphasis> Bunge

In order to study the capabilities of Periploca sepium adventitious root induction in different types of explants, we selected leaves, roots and stems with or without buds. The growth of adventitious roots and periplocin content in these roots were determined. In order to investigate the suitable media salt strength, we cultured the adventitious roots in different salt strength (0.25, 0.50, 1.0, 1.5, 2.0) of Murashige and Skoog (MS) media supplemented with 1 mg/l indole butyric acid (IBA) and 30 g/l sucrose. The results showed that both leaf and root explants were proven suitable for the adventitious root induction; however, the stems could hardly induce adventitious roots no matter whether the stems had buds or not. Further studies reported that adventitious root proliferation and periplocin production derived from root explants were higher than those derived from leaf explants. So the root explants were the optimum explants for adventitious root induction, growth and periplocin production. The salt strength experiment showed that with the increasing salt strength (1.0–2.0 MS), adventitious root growth decreased significantly, as well as periplocin content in comparison with lower (0.25–0.5 MS) salt strength media.     

GIS-facilitated in vitro propagation and ex situ conservation of Achillea occulta

A Geographical Information Systems (GIS)-facilitated approach for the in vitro propagation and ex situ conservation of the conservation priority species Achillea occulta is presented. To realize the species?? ecological requirements, the coordinates of the original habitat were linked with thematic layers derived from digital databases in a GIS environment. From wild plants, shoot tips were established in vitro in a basal medium with 4???M 6-benzyladenine (BA) and 0.5???M indole-3-butyric acid (IBA). A modified basal MS medium (modMS, double amount of Fe) proved to be the most effective for in vitro adventitious shoot production. The effect of BA in combination with ??-naphthaleneacetic acid (NAA) or IBA on shoot proliferation was also tested. The highest number of new microshoots/explant (3.5), with 0.93?cm shoot height was obtained when the modMS was supplemented with 5???M BA and 2.5???M IBA. To evaluate the root induction ability, microshoots produced were transferred to modMS media supplemented with 0?C20???M IBA and 0?C20???M NAA. Rooting proved to be very difficult and only by adding 20???M IBA, a 12.5% rooting percentage was achieved. Acclimatization succeeded only during early spring. Young plants transplanted at the Balkan Botanic Garden of Kroussia produced flowers and seeds in the first year. This GIS-approach provided useful guidelines for A. occulta??s (a) effective propagation (selection of greenhouse temperatures, temperatures during in vitro culture, suitable period for cuttings and acclimatization of plantlets), and (b) ex situ cultivation (selection of watering regime, temperatures, locations and exposures for growing sites).     

Establishment of culturing conditions and assessment of antioxidant activity and somaclonal variation in the adventitious root suspension cultures of <Emphasis Type="Italic">Oplopanax elatus</Emphasis> Nakai

Oplopanax elatus Nakai, a plant traditionally used in folk medicine, is currently in population decline due to uncontrolled harvesting. In the present study, we investigated the factors affecting O. elatus adventitious root production, including hormones (alone or in combination), explant type, basal salt type and strength, sucrose concentration, pH, and temperature. Results revealed that adventitious root formation was optimal with root explants grown on 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^?1 Indole-3-butyric acid (IBA) (pH 5.8) at 25 °C. Chlorogenic acid concentration was highest in roots propagated in 1/2 MS medium containing 0.5 mg L^?1 IBA; vanillin, another phenolic compound, was also detected in cultures. Liquid media containing 3% sucrose exhibited the highest radical scavenging activity and total phenolic compound contents. X-ray diffraction revealed significant differences in the elemental intensity between adventitious root and field-grown plantlet extracts. Analysis of simple sequence repeats confirmed that adventitious roots regenerated in vitro were genetically similar to their mother plant. Thus, we identified the optimal conditions for proliferation of O. elatus adventitious roots in liquid culture, from which, secondary metabolites, particularly bioactive compounds associated with the medicinal use of this plant, can be mass produced without further population deterioration.     

Lateral root development and saponin accumulation as affected by IBA or NAA in adventitious root cultures of <Emphasis Type="Italic">Panax ginseng</Emphasis> C.A. Meyer

Summary The effect of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) on lateral root formation was investigated in adventitious root culture of Panax ginseng. Lateral root formation was affected by IBA (24.6 μM) or NAA (9.8 μM). Lateral root primordia emerged from the explant root pericycle after about 7 d of culture when the roots were cultured on Schenk and Hildebrandt (SH) medium supplemented with 24.6 μM IBA or 9.8 μM NAA. However, no changes were observed in the explant root pericycle on auxin-free medium. The IBA treatment was more effective for lateral root induction and root growth compared to NAA. In morphological and histological aspects, the lateral roots formed under IBA treatment developed normally, while NAA-treated roots exhibited abnormal growth. The accumulation of total saponin was greater in roots treated with IBA than with NAA.     

Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.     

Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots ofEchinacea angustifolia

We investigated different concentrations of auxins (1AA, IBA, NAA), the strength of the MS medium, sucrose and ammonium/nitrate contents, initial medium pH, and inoculum size to determine their effects on biomass increase and the accumulation of total phenols and flavonoids in adventitious roots ofEchinacea angustifolia. These roots were cultured under darkness in shake flasks for 4 weeks. IBA proved the best auxin for inducing root proliferation. Root growth was inhibited when the initial pH was maintained below 5.0 or above 6.0. Nitrate, rather than ammonium, was more necessary for root growth and phenolics accumulations. Overall, biomass increased and total phenol and flavonoid contents were maximized under the following conditions: half-strength MS medium supplemented with 2 mg L^-1 IBA, 5% (w/v) sucrose, 5:25 (mM) ammonium/nitrate ratio, pH adjusted to 6.0 before autoclaving, and an inoculum size of 10 g L^-1 FW. These results indicate that the type ofin vitro environment strongly affects growth and the accumulation of phenolics from adventitious root cultures ofE. angustifolia. Such optimization is beneficial to large-scale production of biomass and secondary metabolites in that species.     

Effect of plant growth regulators on plant regeneration of Dioscorea remotiflora (Kunth) through nodal explants

Dioscorea remotiflora (Kunth) is an important wild plant that produces tuberous roots used as a source of food in the Western part of Mexico. Lack of planting material and inefficiency of traditional methods of propagation are the main constraints for implementing large-scale cultivation. In contrast, tissue culture techniques allow increasing multiplication and rapid production of plant material. In this regard, leaves or nodal segments were incubated on MS, B5 and WPM culture media with different PGRs in order to obtain an efficient micropropagation protocol. Leaves explants were unable to inducing shoots or callus. However, nodal segments produced axillary shoots and/or callus in all culture media. MS containing 2.33???M KIN was the most suitable to inducing shoots; an average of 6.6 shoots per segment for 100?% explants was obtained, which displayed also the greater number of nodes (5.0) and leaves (7.9) per segment. A decrease on shoot proliferation was observed combining BA or KIN with 2,4-D or NAA. However, small brownish callus were induced on 100?% of segments using 2.33???M KIN with 5.37???M 2,4-D or 9.30???M KIN plus 2.69???M NAA. In contrast, by adding 2.69???M NAA, 66.4?% of the nodal segments formed shoots and produced also yellowish friable callus on the base of the shoots. Shoots were easily rooted with 8.28???M IBA (96.9?%), displaying the greatest root and shoot biomass, but maximum number of tuberous roots, and root or tuberous root biomass was produced increasing IBA (20.7???M).     

In vitro morphogenetic studies on three apomictic species of Garcinia

Comparison of morphogenetic potential of three important Indian species of Garcinia??G. indica, G. cambogia and G. xanthochymus has been reported. Apomictic seeds of G. indica were found to be morphogenetically most potential followed by G. cambogia. The explants of G. xanthochymus were highly recalcitrant towards in vitro conditions and failed to induce adventitious buds on any of the media tested. High frequency direct shoot bud differentiation was induced in aseptic seed cultures of G. indica and G. cambogia on MS medium supplemented with cytokinins (BAP, kinetin or TDZ). Amongst the three cytokinins tested, TDZ (0.1?C0.5???M) was most effective for adventitious bud differentiation in both G. indica and G. cambogia, however, the proliferating buds failed to elongate. Substantial number of buds induced on BAP supplemented media elongated into shoots after subculture on elongation medium. Addition of NAA along with cytokinins in the induction medium enhanced callusing without improvement in bud induction response. The induced adventitious buds were elongated on MS basal medium containing 0.2% activated charcoal. Direct rooting was achieved in both G. indica and G. cambogia on auxin supplemented media with best response at 10???M IBA concentration in both the species. The in vitro raised plantlets showed 90% survival in the field when transferred after hardening and acclimatization.     

Studies on the in vitro regenerative competence of aerial roots of two horticultural important Cymbidium species

In the present study aerial roots were successfully used as explants source for in vitro propagation of Cymbidium aloifolium and Cymbidium iridioides. Aerial roots of ~5?C6?week old from axenic cultures were cultured on MS medium adjuncts with different additives. In C. aloifolium within 20?days of culture ~60% of explants responded positively on MS medium containing sucrose (3%, w/v) and Kn (3???M) and formed PLBs. While in C. iridioides ~50% root explants responded positively on medium enriched with sucrose (3%), AC (0.1%) and IAA (3???M) after 40?days of culture. The shoot buds/PLBs converted into plantlets when maintained on regeneration medium. Of the three basal media tested, MS medium supported optimum regeneration and culture proliferation in both the species. In C. aloifolium ~12 shoot buds developed on medium nourished with sucrose (3%) and BA (3???M) but in C. iridioides optimum regeneration was achieved when medium supplemented with sucrose (3%), CW (15%), CH (100?mg?L^?1) and ~20 shoot buds formed per subculture. The well rooted plantlets were acclimatized for 3?C4?week in 1/10th MS salt solution containing sucrose (1%), charcoal pieces, brick pieces and chopped mosses as support under normal laboratory conditions. The hardened plants were transferred to potting mix where 80 and 75% of transplants survived in C. aloifolium and C. iridioides respectively after 2?months of transfer.     

IBA及NAA对山杜英组培苗生根过程中POD、PPO活性的影响

本文研究山杜英组培苗根诱导分化过程中过氧化物酶、多酚氧化酶活性变化。结果表明,在使用IBA和NAA后,对生根有显著影响,过氧化物酶活性在0~4d迅速下降,然后逐步上升,在根开始伸长时,活性又开始下降;不使用任何生长调节剂的情况下不生根,过氧化物酶活性在0~16d上升,然后开始下降;多酚氧化酶活性在整个根诱导中都维持在较低水平。     

Root Development Enhanced by Using Indole-3-butyric Acid and Naphthalene Acetic Acid and Associated Biochemical Changes of In Vitro Azalea Microshoots

Based on the importance of producing in vitro adventitious roots, this study was carried out to investigate the effects of indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) at a concentration of 2 mg L^?1 on the formation of adventitious roots of azalea and their impact on biochemical changes and endogenous hormones. The rooting percentage, root number, and root length were increased in the microshoots of both studied cultivars (‘Mingchao’ and ‘Zihudie’) when the growth medium was supplemented with IBA. Additionally, peroxidase, indole acetic acid oxidase, hydrogen peroxide, and soluble protein contents were improved in both cultivars by auxin treatments especially during the first 7 days of the rooting period. However, application of IBA and NAA increased catalase and polyphenol oxidase in both cultivars during the first 14 and 28 days of culture. The increase in endogenous indole acetic acid (IAA) levels was accompanied by low activity of IAAO during most periods of root induction of microshoots in all treatments. Endogenous gibberellic acid levels were increased after 7 days of culture and then increased again after 28 days of culture. In contrast, the levels of endogenous zeatin riboside and isopentenyl adenosine were decreased with auxin treatments in the first period of the rooting process and then increased after 21 and 28 days of culture. The present study demonstrated that IBA at a concentration of 2 mg L^?1 has a strong effect on azalea rooting. Moreover, the efficiency of IBA and NAA effects on biochemical changes during adventitious root induction was investigated, which may provide new horizons of in vitro rooting production and provide valuable information for the micropropagation of Rhododendron plants.     

Additive effects of three auxins and copper on sorghum in vitro root induction

A healthy root system is vital for tissue culture plantlet survival and rapid adaptation from the in vitro microenvironment to glasshouse conditions. Optimization of the root induction medium is an effective way to promote root induction and elongation. Levels of three auxins (α-naphthaleneacetic acid [NAA], 3-indoleacetic acid [IAA], and 3-indolebutyric acid [IBA]) and copper sulfate (CuSO₄) have been investigated in a series of experiments with a sorghum inbred line, Tx430. Significant improvement in root proliferation and shoot growth were observed on Murashige and Skoog (MS) medium supplemented with 1 μmol/L CuSO₄, 1 mg/L NAA, 1 mg/L IAA, and 1 mg/L IBA. On average, one explant (the original in vitro-derived shoot) of Tx430 regenerated 56.7 roots, which was 20-fold higher on the optimal medium than on MS control medium. Another tested genotype SA281 showed similar response patterns as Tx430 across media. In addition, 100% of Tx430 and SA281 plantlets originating from the optimized root induction medium all survived after being transferred to potting soil in the glasshouse. The results demonstrate that a combination of auxins (NAA, IAA, and IBA) and CuSO₄ together at optimal concentrations provide additive effects on promoting root proliferation and explant growth of in vitro sorghum in root induction medium, and subsequently resulted in 100% survival rate of plantlets ex tissue culture. Compared with two published and frequently used root induction media, the optimized medium significantly enhanced root induction and plantlet growth.     

Adventitious rooting performance in micropropagated Cornus mas

Axillary buds sampled from a mature 27-year-old Cornus mas cv. Macrocarpa were grown in vitro on modified woody plant medium (WPM). Adventitious rooting performance of microshoots was assayed on half-strength WPM supplemented with 1-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) under various pH. NAA induced significantly higher rooting frequencies than IBA. The pH of 6.8 inhibited rooting, and differentiated roots were extremely thick and fragile. The highest rooting frequency was recorded on half-strength WPM supplemented with 5.37 µM NAA at the pH value adjusted to 6.2 (73 % of rooted shoots). In the presence of IBA, the formation of adventitious roots was observed only in the basal part of the microshoot dipped into rooting medium. In the case of NAA, however, adventitious roots arose also from the parts of microshoots that were not in contact with medium. The growth of aerial roots was always positively gravitropic. The nuclear microsatellite Cf-G17 gave a monomorphic fingerprinting pattern across the mother shrub and micropropagated plantlets. Acclimatized plants did not show any visually detectable morphological variation and the aerial adventitious root formation was no longer observed.     

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.     

Influence of galactoglucomannan oligosaccharides on root culture of <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis>

Galactoglucomannan oligosaccharides (GGMOs) activity in K. humboldtiana root culture has been determined. GGMOs inhibited adventitious root growth and lateral root induction in contrast to IAA, IBA, and NAA stimulating effect in these processes. Similarly, the combination of GGMOs with natural auxins (IAA, IBA) evoked an inhibition of adventitious root growth and lateral root induction that depended on the oligosaccharides concentration and the type of auxin. The growth stimulating effect of the synthetic auxin, NAA, in adventitious roots was negatively affected by GGMOs, but they were without influence on lateral root induction. The presence of oligosaccharides triggered lateral root position on adventitious roots and the anatomy of adventitious roots (diameter, proportion of primary cortex to the central cylinder, number and size of primary cortical cells, intercellular spaces, and the number of starch grains in cells of primary cortex) in dependence on their coactions with auxin.     

Effect of auxins and plant oligosaccharides on root formation and elongation growth of mung bean hypocotyls

The interaction of auxins – IAA, IBA or NAA – with galactoglucomannan oligosaccharides (GGMOs) on adventitious root formation and elongation growth of mung bean hypocotyl cuttings was studied. GGMOs induced adventitious roots in the absence of auxins; however, their effect was lower compared with IBA or NAA. On the other hand, in the presence of auxins, GGMOs inhibited adventitious root induction. Their effect depended on the concentration of oligosaccharides and the type of auxin used. The highest inhibition effect of GGMOs at a concentration of 10⁻⁸ M in the presence of IBA and NAA was observed. In the presence of IAA their inhibition was non-significant in regard to the concentration. The interaction of auxins with GGMOs resulted in the formation of adventitious roots on a shorter part of hypocotyls compared with the effect of auxins alone. However, roots were induced more extensively along the hypocotyls treated with GGMOs compared with the control. GGMOs inhibited the length of induced adventitious roots in the presence of IAA, while in combination with IBA or NAA they were ineffective. The elongation of hypocotyls induced by IAA or IBA was inhibited by GGMOs, too. However, in the presence of NAA or by endogenous growth they were without any significant effect on elongation growth. These findings suggest that GGMOs in certain concentrations might inhibit rooting and the elongation process dependant on auxin used.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Rhododendron wattii</Emphasis> Cowan—a critically endangered and endemic plant from India

Rhododendron wattii Cowan is a rare and endangered plant found in northeast India. In an effort to boost specimen numbers, experiments of in vitro seed germination, shoot induction on different media supplemented with the cytokinin isopentenyladenine (2iP), and root induction with auxins α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) in woody plant medium (WPM) were carried out. A maximum mean shoot number of 7.72 per explant were obtained from nodal explants cultured on WPM and 39.36 μM 2iP with a maximum mean shoot length of 2.30 cm per explant. Among the auxins investigated for root induction, IBA at 2.45 μM was found to produce the most and the longest roots, when compared to other treatments. However, WPM supplemented with 0.2% (w/v) activated charcoal also showed 100% root formation with shoots having broader leaves compared to auxin treatments. About 60% of in vitro rooted plantlets transferred from lab to greenhouse conditions survived. Sixty acclimatized plants were reintroduced in the vicinity of their natural habitat at Naga Heritage Village, Kisama, Nagaland, in May 2016 for ex situ conservation. Survival of the reintroduced plants was confirmed during the field visit conducted in November 2016.     

碳源、生长素及诱导子对木豆不定根生长和次生代谢产物合成的影响

以1/2MS为基本培养基,研究了蔗糖、IBA、茉莉酸甲酯（MJ）及水杨酸（SA）对木豆不定根生物量和次生代谢产物合成的影响。研究结果表明,蔗糖浓度对木豆不定根的生物量和次生代谢产物合成有显著影响,蔗糖浓度为30 g·L^-1时,木豆不定根的生物量和次生代谢产物的含量均达到最大值。低浓度的IBA有利于木豆不定根的生长和次生代谢产物的积累,高浓度的IBA表现出抑制作用。IBA浓度为0.1 mg·L^-1时,生物量、染料木素及芹菜素含量均为最大值,分别为对照组的1.1、1.1和2.8倍。在0~200 μmol·L^-1浓度范围内,MJ对不定根的生长几乎无影响（P>0.05）,但对次生代谢产物的合成有重要影响。MJ浓度为100 μmol·L^-1时,染料木素和芹菜素的含量均达到最大值,分别为对照组的1.9和2.1倍。SA抑制木豆不定根的生长和染料木素的合成,但对芹菜素的合成有一定促进作用,SA浓度为100 μmol·L^-1时,芹菜素的含量最高,为对照组的1.5倍。木豆不定根的悬浮培养是获得次生代谢产物的一条有效途径,为大规模生产染料木素和芹菜素提供了很好的思路。