Comparative histochemical localization of secondary metabolites in seed-raised and in vitro propagated plants of Excoecaria agallocha Linn. (Euphorbiaceae), the milky mangrove tree of historical significance

Abstract

Mangroves synthesize novel secondary chemicals that are poorly understood. Among the euphorbiaceous mangrove species, Excoecaria agallocha Linn. produces novel terpenoids and alkaloids of medicinal importance. We conducted a comparative tissue level histochemical study of E. agallocha L. to determine whether in vitro propagation alters the content of phytochemicals within the plant parts. Transverse sections of the root, stem and leaves of seed-raised saplings and in vitro propagated plants stained with 10% vanillin-perchloric acid revealed accumulation of terpenoids in the cork cambium. Alkaloids were localized using Dragendorf's reagent in the cortex of the root sections as brown layers. Methylene blue staining revealed that seed-raised plants possessed more lignified cells, distinct latex ducts and ellipsoidal guard cells compared to the plants propagated in vitro, which revealed abnormal, circular guard cells. The phytochemical content of E. agallocha propagated by the in vitro method was comparable to the seed-raised plants. Phytochemical studies of the species of E. agallocha propagated in vitro would confirm whether the species could be used for its medicinal compounds.     

An efficient system for the production of clonal plantlets of the medicinally important aromatic plant: Salvia africana-lutea L.

An in vitro cultivation protocol was developed for S. africana-lutea a species threatened by over collection due to its importance as an aromatic medicinal plant in the Western Cape of South Africa. Adventitious shoot induction was most successful using hypocotyls as explants for propagation on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 4.4 μM BA only; 2.7 μM NAA and 4.4 μM BA; or 2.9 μM IAA and 9.3 μM kinetin respectively. For continuous subculture, IAA and BA (μM) at a ratio of 2.9:4.4 or 2.9:8.9 had the best regeneration potential producing approximately three plantlets per nodal explant. Plantlets had 4–5 nodes that could be utilized for the following subculture phase to induce axillary shoots. The tissue culture of S. africana-lutea not only favoured rapid multiplication but was also characterized by seasonal in vitro flowering that was in synchrony with that of plants growing in the wild. This propagation regime has the capacity for producing 2000–3000 plants from one shoot after 3 four-week long subculture cycles, making it highly attractive for implementation as an in vitro conservation strategy. The micropropagated plants were easily acclimatized (88%) within a month after rooting in vitro and planted ex vitro in a sand:soil:peat moss:vermiculite (1:1:1:1; v/v) mixture.     

Ring width and vessel features of the mangrove Excoecaria agallocha L. depend on salinity in the Sundarbans,Bangladesh

The Bangladesh Sundarbans is the largest continuous mangrove in the world that providing crucial environmental services, particularly related to coastal protection and livelihoods of millions of people. However, anthropogenic disturbances, diseases infestation and environmental changes including sea level rise (SLR) and fresh-water flux into the delta are threatening the Sundarbans and other mangrove ecosystems worldwide. Protection of mangrove ecosystems requires knowledge on factors that mainly drive growth and vitality of tree species to evaluate which consequences can be expected from, mainly hydrology-related, environmental changes. In this study, we assessed the nature and periodicity of tree rings in Excoecaria agallocha, a wide spread mangrove species in the Bangladesh Sundarbans. We also analysed the influence of climatic factors, such as precipitation, temperature and vapor pressure deficit (VPD), and river discharge, as a proxy of salinity on ring width (RW) and vessel features, such as mean vessel area (MVA) and mean vessel density (MVD). E. agallocha forms distinct tree-ring boundary that characterized by a narrow (2–4 cells wide) band of radially flattened fibres. The RW as well as the MVA and MVD are crossdatable. The RW is mainly driven by salinity which is influenced by freshwater inputs through precipitation during monsoon along with river discharge January to April. The MVA and MVD responded to similar seasons and months as RW, but mostly with opposite signs in MVD. The results suggest that fresh water inputs through precipitation and river discharge positively influence the radial growth of E. agallocha in the Sundarbans. The RW and vessel features can be used as proxies to explore the growth dynamics of this species, especially in relation to global environmental changes.     

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White’s, Gamborg’s B₅ or woody plant medium (WPM) supplemented with 2.5 µM benzyl adenine (BA) and 0.1 µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5 µM) and NAA (0.1 µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83 cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13 cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2 mM indole-3-butyric acid (IBA) for 5 min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.     

Mother plant age and seasonal influence on in vitro propagation of <Emphasis Type="Italic">Quercus euboica</Emphasis> Pap., an endemic,rare and endangered oak species of Greece

A micropropagation method for Quercus euboica Pap. was developed. Nodal explants from seedlings gave higher multiplication rates than explants from adult plants. Cultures initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate. Woody Plant Medium (WPM) salts, with 100 mg l⁻¹ myoinositol, 1 mg l⁻¹ thiamine, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ nicotinic acid and 3% sucrose was used as basal medium and several cytokinins at various concentrations were evaluated for their effect on shoot multiplication. The highest shoot multiplication rate was obtained with 4.44 μΜ BA. IBA at 9.84 μΜ in the culture medium during the first week of culture, and if followed by culture in hormone-free medium, gave the best rooting results. Darkness at the beginning of the rooting period did not improve rooting. The use of plastic wrap as a cover material of the culture vessels enhanced rooting percentage and root number. Plantlets acclimatized ex vitro in soil from the natural environment of the species survived at a higher percentage (up to 93%) and had more vigorous growth than plantlets grown in a compost–perlite (2:1 v/v) medium (up to 36%).     

In vitro propagation from axenic explants of <Emphasis Type="Italic">Lilium oxypetalum</Emphasis> (D. Don) Baker,an endemic bulbous plant of high altitude Himalaya

In vitro propagation protocol for Lilium oxypetalum, a high altitude Himalayan endemic lily, has been developed. Effect of explant types (i.e., callus and in vitro bulblet scales) and sucrose concentration [0–6.0% (w/v)] on in vitro bulblet regeneration of L. oxypetalum was tested in previously optimized Murashige and Skoog basal medium supplemented with 2.0 μM 6-benzyladenine and 0.1 μM α-naphthaleneacetic acid. Callus explants produced significantly (P < 0.01) higher number of bulblets per explant than bulblet scale explants. Of the different concentrations of sucrose tested, 4.5% (w/v) sucrose showed significantly (P < 0.01) higher percentage regeneration (i.e., 70.8 ± 4.2 and 79.2 ± 4.2% regeneration on callus and bulblet scale explants, respectively), and produced higher number of bulblets per explant (i.e., 9.0 ± 0.4 and 5.4 ± 0.5 bulblets on callus and bulblet scale explants, respectively). Regenerated bulblets developed 2–3 leaves when subcultured for 4 weeks and were subsequently transferred ex vitro with a survival rate of 66.7% after 6 weeks. Leaves of the survived plantlets became dry after growing ex vitro for 10 weeks, amongst which 86.4% re-sprouted after remaining dormant for 5–6 weeks and produced 1.5 bulblets per explant. Findings of the present study hold promise for efficiently multiplying the target species in view of its potential economic and conservation significance.     

An improved protocol for in vitro multiplication of Bacopa monnieri (L.)

A cost-effective and efficient protocol has been described in the present work for large scale and rapid in vitro propagation of a valuable medicinal herb Bacopa monnieri (L.) by shoot bud proliferation on internodal segments isolated from field grown mature plants. This shooting was achieved on low concentration of cytokinin with BAP 1.0 mg/l and Kn 0.5 mg/l within 3 weeks of inoculation contrary to earlier reports where auxin is also used in combination with cytokinin. Normally internodes gives rise to a clump of shoots (18 shoots). On an average within a period of three subcultures the internode explant generated 324 shoots thereby improving the economics of the cost of the plants produced and time required. For rooting this is a first report of its growth in a liquid culture. The regenerated plants resemble the mother plants in the general habitat without any morphological variation. This paper reports a very simple, reproducible procedure for in vitro propagation of Bacopa monnieri (L.) that can be used to form a foundation stock of elite plant material for large-scale cultivation.     

Rapid in vitro propagation,conservation and analysis of genetic stability of Viola pilosa

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal + 0.5 mg/l BA + 0.5 mg/l TDZ + 0.5 mg/l GA₃ gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10 °C than at 4 °C after conservation of in vitro multiplying shoots. In cryopreservation–vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4 °C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10 °C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.     

Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species

The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined.     

Plant in vitro culture for the production of antioxidants--a review

Plant in vitro cultures are able to produce and accumulate many medicinally valuable secondary metabolites. Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting detrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used for increased biosynthesis and the accumulation of antioxidant compounds in plant cells. In the present review some of the most active antioxidants derived from plant tissue cultures are described; they have been divided into the main chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes are presented. The strategies used for improving the antioxidants in vitro production efficiency are also highlighted, including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up.     

In vitro propagation of Stackhousia tryonii Bailey (Stackhousiaceae): a rare and serpentine-endemic species of central Queensland, Australia

Stackhousia tryonii Bailey, a rare species whichhyperaccumulates nickel and with a potential to be exploited inphytoremediation/phytomining, is difficult to propagate via seeds. This studyinvestigated the development of a micropropagation protocol for the productionof large stocks of S. tryonii. Disinfested shoot tips andnodal buds were precultured on Gamborg's (B₅) basal medium toobtain aseptic shoots for the optimisation of the protocol. 6-Benzyl aminopurine(BAP) at 1.0 mg l^–1 produced the highest number ofshoots per explant in B₅ medium. Comparison betweenB₅ and MS media showed similar responses, but with marked influenceof BAP concentration on shoot numbers. Transfer of shoots from MS(multiplication) medium to MS medium supplemented with indole-3-acetic acid(IAA) and indole-3-butyric acid (IBA), individually or in combination, indicatedthat a combination of IAA and IBA (0.75 mg l^–1each) is required to produce roots on young shoots (75%) compared to IBA(15–45%) or IAA (0–10%) alone. This study demonstrated that by usingthis protocol, a high multiplication rate (up to 18 shoots per explant) could be produced within 4 weeks, andthey can be readily hardened (98% survival) in a glasshouse by transplantingthem into a potting mixture of sand and perlite (4:1).     

In vitro propagation of Litsea cubeba (Lours.) Pers., a multipurpose tree

 A rapid clonal propagation system has been developed for Litsea cubeba. Following investigation of a range of cytokinins and a variety of explant sources (shoot tip, node, leaf and petiole) it was established that 6-benzyladenine with shoot tip explants gave optimal multiple-shoot induction. In vitro rooting on growth-regulator-free medium was possible and over 100 plantlets were successfully weaned to the glasshouse. Received: 11 July 1996 / Revision received: 24 December 1996 / Accepted: 22 March 1999     

Requirements for in vitro propagation of seven nitrogen-fixing Alnus species

Studies on the in vitro propagation of Alnus crispa, A. glutinosa, A. incana, A. japonica, A. rubra, A. sinuata and A. viridis indicated interspecific as well as intraspecific variations in their requirements for in vitro culture. The WPM and Blaydes media supported, respectively, growth of A. glutinosa and A. crispa but not that of both species, while the MS medium induced equal or significantly better growth than WPM and Blaydes media for both species. The optimum type and concentration of sugar to be used in the multiplication medium varied with species. Only A. glutinosa showed good growth on sucrose while glucose was optimum for all other species but at different concentrations. All species rooted in 3 weeks on half-strength MS medium including 1 M IBA. All clones of A. glutinosa and A. rubra rooted 100%, whereas easy-to-root and difficult-to-root clones were observed in the other species. In the rooting medium, glucose promoted rooting of the difficult-to-root clones better than sucrose. Survival following transfer to an artificial substrate was 100% for all species. Nodulation tests using pure cultures of two Frankia strains showed 100% nodulation on all Alnus clones.     

Effect of photoperiod and light intensity on in vitro propagation of Alocasia amazonica

Plantlets of Alocasia amazonica regenerated under a photon flux density (PFD) of 15 or 30 μmol m⁻² s⁻¹ showed better growth and development than those grown under higher PFDs. While chlorophyll a and chlorophyll b decreased, the number of stomata increased with increasing PFD. Photoperiods also affected plantlet growth and stomatal development. Highest growth was observed for the short photoperiod (8/16 h) and for equinoctial (12/12 h) light and dark periods. Very few stomata developed in the leaves of plantlets grown under a short photoperiod (8/16 h) and the number of stomata increased with increasing light period. In conclusion, both light intensity and photoperiod independently affect growth of A. amazonica and development of stomata, depending on the intensity and duration of light treatment.     

黄花蒿组培快繁与种质离体保存的研究

以带侧芽的黄花蒿(Artemisia annua L.)茎段为外植体,以MS为基本培养基,进行组织培养和种质保存研究.结果表明,培养基MS 6-BA 1.0 mg L-1 IBA 0.1 mg L-1、MS 6-BA 0.5 mg L-1 IBA 0.1 mg L-1和MS NAA0.1 nag L-1 IBA 0.5 rng L-1可分别用于黄花蒿的芽诱导、增殖和生根培养,培养20 d的增殖倍数为5.5倍,生根率98.3%.培养基MS CCC 1.0 mg L-1、MS CCC 2.0 nag L-1、MS PP3334.0 mg L-1可用作离体保存,连续保存200 d的存活率分别达72.3%、77.0%、69.2%.活力检测表明,黄花蒿种质经保存后的增殖、生根能力没有下降.因此,可通过诱导腋芽增殖建立黄花蒿快繁体系,及在培养基中添加CCC或PP333拼能使材料长期保存.     

Rapid in vitro multiplication of Drosera indica L.: a vulnerable, medicinally important insectivorous plant

An efficient protocol is described for rapid in vitro multiplication of the vulnerable medicinal herb Drosera indica L. by enhanced axillary bud proliferation from shoot tips as explants. In order to standardize in vitro multiplication of D. indica, the effects of different strengths of Murashige and Skoog (MS) medium (1/4, 1/3, 1/2 and full strength), different percentages of sucrose (1, 2 and 3%), various pH (3.7, 4.7, 5.7 and 6.7) and MS basal medium fortified with different concentrations of zeatin (Z), kinetin (KN) (0.1, 0.5, 1.0 and 2.0 mg/l) and 6-benzylaminopurine (BA) (0.01, 0.05 and 0.1 mg/l) were tried. Multiple shoot production was independent of different strengths of MS, various percentages of sucrose and also when pH was altered. Although the number of multiple shoots developed on MS medium supplemented with Z (0.1, 0.5, 1.0 and 2.0 mg/l), KN (0.5 and 1.0 mg/l) and BA (0.1 mg/l) separately was high, the maximum number was observed on MS fortified with Z (0.5 mg/l) and KN (0.5 mg/l), respectively, which clearly depicts that there is not much difference comparatively with a variation in hormone concentration in case of Z. High cytokinin concentrations resulted in retardation of shoot growth. Rooting was best achieved on MS basal medium. This protocol could be useful for production of large biomass within 6 weeks for plumbagin bioprospection and long term in vitro conservation.     

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.