An in vitro approach to investigate medicinal chemical synthesis by three herbal plants

Ever since regulatory changes introduced herbals into mainstream supermarkets and pharmacies, there has been an explosion of demand for herbal plants and extracts which can be used to improve human health and well being. Science still lacks a basic mechanistic understanding of how environmental triggers regulate phytochemical accumulation, but this gap can be bridged using in vitro models to examine herbal species responses. For St. John's wort (Hypericum perforatum), uniform in vitro shoot cultures were set up as a parallel to a previously established sand culture system for investigation of physical and chemical environmental factors that control hypericin accumulation. Cytokinin supplementation of shoot culture medium resulted in a proliferation of abundant leaf glands with enhanced levels of hypericin, as compared to controls. Cell cultures of echinacea (Echinacea angustifolia) were established, and hydrophilic pharmacological components (caffeic acid derivatives) were detected. A protocol of rigorous explant pretreatment, and use of newly emerging vegetative shoots permitted establishment of axenic kava (Piper methysticum) callus, which was used to regenerate roots (organogenesis). Kavapyrone synthesis was achieved in both undifferentiated cell cultures and in cultured roots, although at lower levels than found in in vivo root systems. The predominance of kavain and methysticin in both forms of the in vitro cultures was parallel to the relative proportions from kava roots in vivo. The cell and organ cultures of all three herbal medicinals provide advantageous, easily-manipulated models to decipher environmental controls of phytochemical biosynthesis.     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Role of origin and endophyte infection in browning of bud-derived tissue cultures of Scots pine (Pinus sylvestris L.)

Callus tissues originating from buds of mature Scots pine (Pinus sylvestris L.) trees exhibit the typical problem of browning, which leads to degeneration and death of the tissues. The effects of medium, origin (tree and location) and endophyte infection were studied on the browning and growth of bud-derived tissue cultures. The calli growing on medium with higher kinetin content and source of organic nitrogen, and originating from the southern location grew better and exhibited less browning. Endophytic microbial cells were detected in the brown callus tissues by transmission electron microscopy. The natural endophyte infection frequency of Scots pine buds was studied and found dependent on the tree, but not on the location. A well-growing, green callus line was artificially infected by an endophytic strain of Methylobacterium extorquens, and browning was not observed on solid media compared to the uninfected control clones of the same callus. However, suspension cultures started from the infected callus died faster than cultures started from the uninfected callus. The endophyte species composition and plant genotype together with tissue culture conditions are the key factors for gaining plant tissue cultures with high regeneration capacity.     

Production of hairy roots in Aconitum heterophyllum wall. using Agrobacterium rhizogenes

Summary A method for the production of hairy roots of Aconitum heterophyllum wall. is reported for the first time. Embryogenic callus cultures were successfully transformed using Agrobacterium rhizogenes strains viz. LBA 9402, LBA 9360, and A4 for the induction of hairy roots. The transgenic nature of hairy roots was confirmed by mannopine assay using paper electrophoresis. Best growth of transformed roots was obtained on 1/4 MS (Murashige and Skoog, 1962) medium with 3% sucrose. Total alkaloid (aconites) content of transformed roots was 2.96%, which was 3.75 times higher compared to 0.79% in the nontransformed (control) roots. Thin layer chromatography (TLC) analysis of the components of aconites in the transformed roots revealed the presence of heteratisine, atisine, and hetidine.     

In vitro selection and characterization of Ni-tolerant callus lines of Setaria italica L

Nickel tolerant callus lines of Setaria italica L. were developed from callus cultures grown on MS medium supplemented with 0.5 mg·dm⁻³ kinetin+2.0 mg·dm⁻³ 2,4-D+2.0 mg·dm⁻³ Ni⁺². Standard growth parameters such as callus fresh and dry weight, growth tolerance index were used as indicators of nickel toxicity. Measurements as early as 2 weeks after the beginning of the treatments did not yield consistent results. However, growth tolerance index at 4, and 8 weeks after the beginning of treatments yielded significant differences among the non-tolerant and tolerant calli. The tolerant calli has enhanced growth at 2.0 mg·dm⁻³ Ni⁺² while non-tolerant calli showed a reverse trend in growth in the presence of 2.0–2.5 mg·dm⁻³ of nickel. The tolerant calli differentiated into mass of embryogenic calli within 4 weeks of culture which could be maintained for prolonged period without loss of regenerative capacity.     

八角莲组织培养的器官发生途径研究

为探究小檗科植物八角莲组织培养的器官发生方式,该研究以八角莲离体叶片、叶柄在MS培养基上诱导产生的愈伤组织、不定芽、不定根为对象,用连续石蜡切片技术分析八角莲组织培养的器官发生途径。结果表明:八角莲愈伤组织形成的解剖学特征是靠近表皮的薄壁细胞经激素刺激恢复分裂能力,继续培养形成拟分生组织。拟分生组织可形成许多分化中心。通过对八角莲组织培养产生的不定芽细胞组织学观察发现芽原基起源于愈伤组织外侧的几层薄壁细胞,芽原基背离愈伤组织中央生长形成不定芽,故八角莲脱分化形成的芽起源方式为外起源。而八角莲的根原基起源于组织深处髓部薄壁细胞和部分维管形成层细胞,进而形成类似球形或楔形并朝韧皮部突起的根原基轮廓,根原基继续发育会突破表皮生成不定根,起源方式为内起源。八角莲离体再生途径为器官发生型,在组培苗生长过程中先诱导形成不定芽,再诱导形成不定根,在愈伤组织上形成维管组织将不定芽和不定根连接成完整植株。     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.     

Tissue cultures of Artemisia pallens: Organogenesis,terpenoid production

Germinated seedlings of Artemisia pallens gave three types of cultures on MS medium supplemented with different plant growth hormones. Medium containing BA+2,4-D stimulated unorganized callus; BA+IAA medium, semi-organized tissues interspersed with shoot buds; and BA+NAA+IAA medium, multiple shoot cultures. The in vitro shoots developed roots in medium devoid of growth hormones. TLC and GLC analysis of the tissue extracts showed that linalool was present in the cultured tissues, with maximum concentration in the unorganized tissue. Although the TLC profiles of the three culture extracts were similar, the extracts did not contain the major polar compounds of the plant. The plant extracts contained more polar compounds and gave the characteristic fragrance of davana.Abbreviations MS Murashige & Skoog's basal medium - BA benzyladenine - Kn kinetin - NAA naphthaleneacetic acid - IAA indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje

Agrobacterium rhizogenes transformed and control roots of the tetraploid potato cv. Bintje were compared. Transformed roots were obtained after infection by A. rhizogenes 15834 or 1855. Both in leaf and stem segments, more roots were formed at the basal side of the segments, indicative for a polarity in root formation. As compared to control roots the transformed roots are characterized by smaller and more densely stained cells, a zone of cell division, and smaller statoliths. These characteristics are correlated with vigorous growth, high branching incidence and diminished geotropism. The plant regeneration procedure according to Ooms et al. [1] was modified. The transformed roots required less 2,4-D than control roots for the induction of shoot-competent calli. The callus and shoot induction phases were reduced from 8 and 6 weeks to 3 and 3 weeks, respectively. Upon induction, 25%, 58% and 61% of the root clones originating from tuber, stem and leaf, respectively, produced shoots, whereas all of the control roots produced shoots. Shoot outgrowth occurred on liquid MS medium in the absence of hormones.Abbreviations Ri-root Agrobacterium rhizogenes transformed root - BAP benzylaminopurine - IAA indoleacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid - 2,4-D 2,4 dichlorophenoxyacetic acid     

In vitro propagation of two species of commelinaceae from bud cultures and repeated subcultures on a growth regulator-free medium

Summary In our wide-ranging research on in vitro propagation of some monocotyledonous plants, two Commelinaceae species were studied: Tradescantia fluminensis var. foliis variegatis and Tradescantia pallida. Initial cultures were established successfully using nodal and apical meristems that produced single shoots, many roots, and no callus, by utilizing growth regulator-free MS medium. Addition of growth regulators did not increase the activity of explants that produced single or multiple shoots, atypical roots, and no callus. Consecutive cultures were possible using the apical and nodal meristems of the previous generation. The behavior of the different generations in culture did not change and was similar to the initial cultures. Their growth capacity was maintained over many months, also on a growth regulator-free medium. In both species, the chromosome number in the root tips of the mother plant and all morphologically stable in vitro plantlets confirmed a constant ploidy level, in T. fluminensis 2n=72, and in T. pallida 2n=24.     

Plant regeneration from Bulgarian rose callus

Plant regeneration capacity of Bulgarian rose callus tissue was examined. Adventitious bud formation could be successfully attained, depending on the kinds of mineral salts used in the medium, auxin and cytokinin used. When callus tissues were cultured on the medium without ammonium nitrate and contained indoleacetic acid and benzyladenine, buds were formed in the callus. The number of buds were significantly increased by the simultaneous addition of calcium ionophore. When the cultures were transferred to the medium without cytokinin, roots were formed in the basal part of the buds.Abbreviations BA benzyladenine - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid     

In vitro plant regeneration in Holarrhena antidysenterica wall., through high-frequency axillary shoot prolieferation

Summary An efficient, rapid and large-scale propagation of the woody, aromatic and medicinal shrub, Holarrhena antidysenterica, through in vitro culture of nodal segments with axillary buds, is described. N⁶-benzyladenine used at 15 μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation at the rate of 43 microshoots per nodal explant with axillary buds, after 30 d of eulture. By repeated subculturing of nodal explants with axillary buds, a high-frequency multiplication rate was established. Efficient rooting was achieved with 35 μM indole-3-butyric acid which was the most effective in inducing roots, as 80% of the microshoots produced roots. Plantlets went through a bardening phase in a controlled plant growth chamber, prior to ex vitro transfer Micropropagated plants established in garden soil were uniform and identical to donor plants with respect to growth characteristics and vegetative morphology.     

细枝木麻黄离体培养过程中愈伤组织和再生芽的细胞组织学观察

为探讨细枝木麻黄(Casuarina cunninghamianaMiq.)愈伤组织分化过程的细胞组织学,对离体培养条件下的愈伤组织进行扫描电子显微镜和石蜡切片观察,分析愈伤组织的细胞分裂、分化以及芽再生的发生过程。结果表明,新鲜外植体培养于愈伤组织诱导培养基上,伤口处的薄壁细胞开始脱分化,培养1周后形成明显的愈伤组织;继续培养2周后,胚性愈伤组织形成,且表层细胞启动分化形成芽原基;培养4周,可肉眼观察到胚性芽原基,数量增多并逐渐分化形成不定芽;培养至第6周,生成不定芽,并大量增殖和分化。因此,细枝木麻黄是通过愈伤组织分化形成胚状体的途径进行植株再生的,为建立细枝木麻黄组织培养高效再生体系提供了理论依据。     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

General features of chromosome substitutions in Triticum aestivum x T. timopheevii hybrids

Summary Tissue culture of the Zea mays inbred line A188 resulted in the regeneration of plants having a high level of phenotypic variation compared to seed-grown control plants. To determine how such variation was induced and whether this could be related to specific in vitro culture methods, callus cultures were established and maintained on different, commonly used culture media. Plants were regenerated and the genomic DNA of callus cultures and regenerants analysed for RFLP differences. The results show that regardless of the gene probe used, callus formation resulted in significant deviations from the DNA pattern normally found in seed-grown control plants. Alterations in gene copy number also occurred. As differentiation and organogenesis began, the level of DNA variation fell, and most of the regenerated plants showed a genetic similarity to the controls; those with RFLP differences were the somaclonal variants.     

The developmental morphology ofIndotristicha ramosissima (Podostemaceae,Tristichoideae)

The developmental morphology ofIndotristicha ramosissima, a submerged rheophyte from South India, is described. Besides creeping organs (called roots) there are branched shoots with two kinds of short-lived photosynthetic appendages: scales and compound structures (called ramuli). These ramuli may be interpreted as leaf-stem intermediates because they combine typical leaf characters (extra-axillary position, determinate growth, subtending an axillary bud) and typical stem characters (nearly radial symmetry, acropetal development with apical meristem, arrangement of the scaly subunits helical or irregular). Floral shoots arise from axillary exogenous buds along the vegetative shoots, occasionally also from endogenous buds along the roots and vegetative shoots. The uppermost scales and ramuli of each floral shoot form a cup-like structure around the base of the terminal flower.Indotristicha is thought to be primitive within theTristichoideae (Podostemaceae). Some morphogenetic switches are postulated in order to deriveIndotristicha from a putative ancestor that still showed the classical root-shoot model typical of most angiosperms.     

In vitro neoplastic transformation of plant callus tissue by γ-radiation

Tumors have been induced by γ-radiation in callus tissue derived from a monocotyledonous flowering plant, Haworthia mirabilis Haw. The transformed tissue exhibited compact texture, excessive cell proliferation and loss of capacity for organogenesis. Tumors were characterized by their ability to undergo continuous autonomous growth on minimal media in the subsequent 4 generations of subculture. In contrast, the nonirradiated control tissue grew with friable texture, required inositol or growth hormones and showed prolific differentiation of vegetative buds.     

Gibberellic acid causes earlier flowering and synchronizes fruit ripening of coffee

The effect of 100 mgl^–1 gibberellic acid (GA₃) on flowering and fruit ripening synchrony, fruit set, fruit fresh weight, and vegetative growth were studied for different size classes of coffee (Coffea arabica L. cv. Guatemalan) flower buds. Flower buds that were > 4 mm, but not developed to the candle stage at the time of GA₃ treatment, reached anthesis 20 days earlier than the controls, and their development was independent of precipitation, unlike the controls. Fruit from buds that were treated with GA₃ at the candle stage showed earlier and more synchronous ripening than the control, although no differences in flowering were found during anthesis. Buds that were smaller than 4 mm at the time of treatment did not respond to GA₃ applications. Treatment with GA₃ did not affect fruit set, fresh weight of fruits, or vegetative shoot growth.     

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine