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The plant mediated biogenic synthesis of nanoparticles is of magnificent concern due to its eco-benign and single pot nature. Here, Cinnamomum tamala (C. tamala) aqueous leaf extract was utilised for the silver nanoparticles’ (Ag NPs) synthesis. The phytoconstituents in the leaf extract were analysed by standard methods. These metabolites, especially carbohydrate polymers reduce Ag ions to Ag NPs accompanied by a reddish-brown coloration of the reaction mixture. The visual observation of intense brown colour is the first indication of the formation of Ag NPs. Various spectro-analytical techniques further characterise the Ag NPs. The green synthesised spherical Ag NPs were crystalline with an average size of 38 nm. The Ag NPs were scrutinised for antioxidant, antimicrobial and cytotoxic activity and obtained good results. The free radical scavenging was studied by 2, 2-Diphenyl-l-picrylhydrazyl (DPPH) assay. The antibacterial activity of Ag NPs was assessed against human pathogens, and it shown to have good antibacterial potency against a wide spectrum of bacteria. The cytotoxic activity against HEK-293T (human embryonic kidney) cell line was evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. These potent biological activities enable C. tamala capped Ag NPs to be suitable candidates for the future applications in various fields, predominantly clinical and biomedical.

For large scale production through shoot organogenesis from callus, seeds of Gymnema sylvestre were cultured on MS containing various concentrations of 2,4-D. An optimum callusing (62.40%) was observed on MS with 1.5 mg/l 2,4-D. Upon transfer to multiplication medium, the calli multiplied 2.5 times in 45 days. The calli were then tested for shoot induction and an optimum result in terms of both per cent response (52.04%) and an average number of shoots (47.2) were observed on MS supplemented with 1.0 mg/l BAP. Maximum shoot elongation was obtained on 0.5 mg/l Kn and best rooting took place on half strength MS with 0.5 mg/l IBA. This protocol can be followed for the large scale in vitro multiplication of G. sylvestre. Plant Tissue Cult. & Biotech. 30(1): 27-32, 2020 (June)

Climbers are plants with weak stems that require support to grow upright. This group of plants develops various climbing mechanisms, classified as tendrillar, twiners, scramblers, clinging, adhesive and woody climbers (lianas). Although climbers are predominantly angiosperms, some are ferns, and one gymnosperm member Gnetum is included in this category. The angiosperm families mainly composed of climbers include Cucurbitaceae, Convolvulaceae and Dioscoreaceae. India’s biodiversity includes two regions, the Western Ghats and the Eastern Himalayas. According to the Botanical Survey of India, of approximately 18,000 species of angiosperms in India, 8000 of them are medicinal plants and several of them are climbers. Many climbers are medicinally important and used as herbal remedies in traditional systems of Indian medicine, including Ayurveda, Sidha and Unani. Several active phytochemicals from different parts of these plants are isolated and used as medicine either alone or in combination with other compounds. Unfortunately, many of these plants are under the threat of extinction due to habitat depletion and overexploitation and conservation efforts are required to ensure their long-term stability. In this review, we discuss in vitro propagation and conservation strategies for Indian medicinal climbers from diverse families including Fabaceae, Aristolochiaceae, Asparagaceae, Caesalpiniaceae, Sapindaceae, Asclepiadaceae, Vitaceae, Ranunculaceae, Menispermaceae, Colchicaceae, Apocynaceae, Rubiaceae, Passifloraceae, Stemonaceae, Euphorbiaceae and Cucurbitaceae due to their high medicinal value.

Over 80% of world population relies on plant-based traditional medicine for health care. The genetic diversity of medicinal plants is declining due to excessive and unsustainable harvesting. Hence there is strong need to develop protocols for effective and reproducible micropropagation of these plants. Micropropagation is undoubtedly the most reliable method for the production of a large number of plants in a short time. The technique also has immense value in plant genetic engineering. The efficiency of micropropagation depends on the composition of culture medium, especially the type and concentration of plant growth regulators (PGRs) added to the medium. N6-(3-hydroxybenzyl)adenine (meta-topolin, mT) is a natural aromatic cytokinin first isolated from the leaves of Populus robusta. Since its discovery, especially in the past two decades, there are many studies conducted worldwide using mT and its derivatives to develop new protocols for the micropropagation of a variety of plant species, resolve defects due to in vitro propagation, and compare its properties and efficiency with other cytokinins. Various studies show that mT is more effective in morphogenesis than other cytokinins. It has superior regeneration capacity, reduced hyperhydricity, and no root inhibition in culture compared with commonly used cytokinins, thus gaining global interest, even though its efficacy is not universally applicable to all plant species. This chapter focuses on the use of mT in the micropropagation of plants with special emphases on its effects on the physiology of the plant culture and morphogenesis in vitro.

Tylophora indica, a medicinal climber, belongs to the family, Asclepiadaceae. Roots and leaves of the plant contain several alkaloids including tylophorine, tylophorinine and tylophrinidine. The major alkaloid, tylophorine found in T. indica possesses several properties, such as immunosuppressive, antitumour, antifeedant, antibacterial, antifungal, antiamoebic, diuretic and hepatoprotective activities. In addition to this, tylophorine provides positive stimulation to adrenal cortex. Biotechnological production of tylophorine was fulfilled by inducing hairy roots mediated by Agrobacterium rhizogenes (A4 strain). It was followed by its growth in liquid suspension culture that could yield maximum biomass and tylophorine production. This type of liquid suspension culture yielded 9.8 ± 0.21 mgL−1 tylophorine within 4–6 weeks of incubation. Maceration technique employed for the extraction of tylophorine was the most viable and efficient protocol. Although many reports are available regarding the biotechnological production of tylophorine, its competent and economic production still continues as a problematic issue.

Capsaicinoids are a group of molecules, which impart pungency (hotness) to Capsicum fruits. The capsaicinoids are synthesized in the placenta of the Capsicum fruits. During the past few decades, there is an increasing interest in elucidation of the biosynthetic pathway of capsaicinoids. The whole genome sequencing of the highly pungent species, Capsicum chinense has revealed several important genes that are responsible for the biosynthesis of capsaicin and dihydrocapsaicin, the two major active constituents of the pungency complex, also known as capsaicinoid complex. The capsaicinoids have recently attracted a wide attention, because of its huge pharmacotherapeutic potential in treating several diseases, such as diabetes, cancer, obesity, cardiovascular, respiratory, gastric diseases and urological disorders. Further, capsaicin has been increasingly used as a molecule of choice for pain relief and many other diseases. In this chapter, we have presented briefly the sources and types of capsaicinoids, its biosynthesis, quantification of pungency and the genes responsible for its biosynthesis. Further, we have highlighted the candidate genes that are important for the biosynthesis of capsaicin and can be used for further manipulation experiments. At the end, this chapter focuses on the therapeutic potential of capsaicin in treating several diseases, such as diabetes, cancer, obesity, cardiovascular, respiratory, gastric diseases and urological disorders and its increasing role in pain relief. Finally, we have briefly discussed the mode of action of capsaicin against cancer, cardiovascular diseases, obesity management, pain relief and urological disorders.

An efficient protocol for the mass propagation of Torenia bicolor using internode derived callus organogenesis has been described. The optimum callusing was observed when internode explants were cultured on MS medium augmented with 4.0 μM 2,4-dichlorophenoxy acetic acid (2, 4-D) and 0.5 μM kinetin (Kn). The calli were subcultured on MS medium supplemented with various concentrations of N 6 -benzylaminopurine (BA; 1.0-5.0 μM) or Kn (1.0-5.0 μM) alone or in combination with naphthalene acetic acid (NAA; 0.5-1.5 μM) for shoot regeneration. The highest shoot regeneration in terms of percent response (100%) and number of shoots (89.3) was observed on MS medium supplemented with 4.0 μM BA and 1.0 μM NAA. The best rooting was observed on half strength MS medium augmented with 0.6 μM indole-3-butyric acid (IBA). On this medium, 100% shoots rooted with an average number of 6.8 roots per shoot. The rooted shoots were transplanted to soil and acclimatized successfully. Of the 80 plants transplanted to soil 72 survived. Inter simple sequence repeat (ISSR) analysis clearly indicated that callus derived plants of T. bicolor resembled the parent plant at the genetic level. The protocol described here could be used for the mass multiplication and conservation of this endemic plant.

Curcuma caesia Roxb., commonly called “Black turmeric”, is an important medicinal plant. Normally this plant is micropropagated via rhizome or rhizome bud explant culture. However, the shoot induction from rhizome explants is low and thus, alternate methods could be adopted to induce maximum shoots. In this report, we subjected the rhizome segments to abiotic stresses such as warm water, hot air, cold water and sodium chloride (NaCl) at various time intervals for testing their effect on shoot induction. The best shoot induction medium (control) in terms of percent explants producing shoots (66 %) and number of shoots (11.2 per explant) was Murashige and Skoog medium supplemented with 6-thidiazuron (6.8 µM) and indole-3-butyric acid (1.5 µM). The application of various stresses significantly increased both percent response and number of shoots. Rhizome explants exposed to 1 or 2 h at 30–36 °C warm water significantly improved the response. Optimum response (94 % cultures responding with 22.4 shoots per explants) was observed when rhizomes were exposed at 33 °C for 1 h. In hot air as well as cold water treatments the percent cultures responding and number of shoots showed moderate increase. The increase brought about by 300 and 500 mM NaCl for 1–3 h was significantly higher than control. The rhizomes pretreated with 500 mM NaCl for 3 h showed highest response in contrast to other treatments. Here, 96 % rhizomes responded with an average number of 23.4 shoots per explant. These observations supported the positive role of abiotic stresses in inducing shoots from rhizome segments of C. caesia in cultures.

Rhinacanthus nasutus (L.) Kurz (family Acanthaceae) produces several important bioactive molecules. Its leaves and roots were widely used in traditional systems of medicine for the treatment of cancer and other serious diseases. In the present study, the contents of three potent natural naphthoquinone esters, rhinacanthin (RC)-C, RC-D, and RC-N, were evaluated in R. nasutus hairy root cultures. Hairy roots were induced from leaves, stems, and cotyledons following transformation with Agrobacterium rhizogenes (MTCC Strain No. 532). The highest frequency (73%) of hairy root induction was observed with cotyledons. Four media (½ Murashige and Skoog [MS], MS, Schenk and Hildebrandt medium, and Woody Plant Medium) were then compared for their effects on hairy root growth and RC production. The transgenic status of hairy roots was confirmed by PCR analysis and Southern hybridization analysis. A time course study revealed that the highest hairy root biomass (0.89 g/flask dry weight [DW]) and RC contents (RC-C, 3.8 mg/g DW; RC-D, 0.43 mg/g DW; and RC-N, 0.18 mg/g DW) were observed following culture on MS medium 6 wk after culture initiation. The root biomass (1.41 g DW/flask) and RC contents (RC-C, 4.4 mg/g DW; RC-D, 0.69 mg/g DW; and RC-N, 0.21 mg/g DW) were further increased by the use of 4% sucrose. The growth and RC production from hairy roots of R. nasutus demonstrated in the present study may offer an effective and reliable alternative for the exploitation of this medicinal plant.

An efficient in vitro propagation protocol has been standardized for Curcuma caesia Roxb., an important source of camphor, using bud- and leaf-derived callus. The optimum response of 84 and 71 % callus induction was obtained when bud and leaf segment explants were cultured on Murashige and Skoog (MS) medium supplemented with 6.7 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 2.7 µM naphthalene acetic acid (NAA). The white, friable, organogenic calli were subcultured on MS medium supplemented with 6.8 µM thidiazuron (TDZ) and 1.6 µM NAA for shoot induction. On this medium, 90 % of the bud-derived calli responded with an average number of 16.2 shoots per culture. Comparatively, bud-derived calli demonstrated a better regeneration response than leaf calli. In vitro rooting of shoots was also obtained on the regeneration medium. The rooted shoots were successfully hardened and transferred to field conditions. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed no evidence of genetic variation in all 22 plants established from the callus with the parental plant, suggesting this protocol could be used for large-scale true-to-type propagation and multiplication of elite clones of C. caesia.

An efficient protocol for the rapid micropropagation of medicinally important Elephantopus scaber has been standardized using cotyledonary node explants. Direct multiple shoot induction was observed when the cotyledonary node explants at various age groups were cultured on MS medium supplemented with various plant growth regulators. The highest shoot induction was obtained when the cotyledonary node explants from 20-day-old seedlings were cultured on MS medium supplemented with 1.5 mg L-1 TDZ and 0.5 mg L-1 NAA. On this medium, 98% of the cultures responded, with an average number of 33.7 shoots per explant. The highest frequency of rooting (100%) and mean number of roots (3.3 per shoot) were observed when the shoots were transferred to MS medium supplemented with 1.0 mg L-1 IBA. The plantlets raised in vitro were acclimatized and transferred to soil with a 92% success rate. The protocol described here may be utilized for multiplication and conservation of elite clones of E. scaber.

Thidiazuron (TDZ; N-phenyl-1, 2, 3-thidiazole-5ylurea) is a cytokinin-like compound routinely used for in vitro culture studies including shoot proliferation and regeneration in various plants. Recently, there is an increased use of TDZ in in vitro propagation of plants including medicinal and horticultural crops. TDZ was found to be more effective in micropropagation, multiple shoot induction, somatic embryogenesis, callus induction, and shoot organogenesis as compared to other cytokinins. In some systems the synergistic effect of TDZ with other cytokinin/auxin was found more effective than using alone. The optimum concentration of TDZ may vary based on the plant species, explants, and duration of exposure. In this review we have described the recent trends in the use of TDZ for micropropagation of some medicinal plants. The medicinal plants described in this chapter include Picrorhiza kurroa, Aronia melanocarpa, Scutellaria ocmulgee, Salvia miltiorrhiza, Aphyllorchis montana, Gentiana decumbens, Aconitum balfourii, Pelargonium sidoides, Withania somnifera, Curculigo latifolia, Hedychium coronarium, Mentha arvensis, Clitoria ternatea, Saussurea involucrata, Jatropha curcas, Medicago sativa, Crocus sativus, Zingiber officinale, Cannabis sativa, Arnebia euchroma, and Azadirachta indica.

Plants are the prime life-supporting system on earth. Despite its use as food, it is also utilized as a source of life-saving drugs for majority of the population in the world. Many plants yield phytochemicals known as secondary metabolites, which are pharmaceutically important and are extracted directly from the plants collected from natural habitat. Regardless of conventional methods, biotechnological approaches especially plant tissue culture techniques play a unique role in producing and extracting secondary metabolites at industrial level. This book chapter discusses the various strategies adopted for secondary metabolite production in plants.

Several biologically active secondary metabolites like anthraquinones, sterols, triterpenes, flavonoids and naphthoquinones are present in Rhinacanthus nasutus. Naphthoquinones are important group of compounds generally known as rhinacanthin (RC) consists of 15 derivatives named RC A–D and G–Q of which RC-C, RC-D and RC-N have various medicinal properties. The individual role of two auxins i.e. indole-3-butyric acid (IBA) and α naphthalene acetic acid (NAA) and two elicitors i.e. methyl jasmonate (MJ) and salicylic acid (SA) in Murashige and Skoog medium on hairy root growth and RC (RC-C, RC-D and RC-N) accumulation was investigated in the present study. Time course study revealed that IBA and NAA at 2.5 μM showed maximum fresh weight (FW) and dry weight (DW) 4 weeks after culture. However, RC production was maximum after 6 weeks of culture on both media. A concentration-dependent response was observed when various concentrations of MJ (2.0, 5.0, 10 and 15 μM) and SA (10, 50, 100 and 150 μM) were supplemented in the medium. On MJ and SA media the FW and DW decreased as the concentration of elicitors increased. However, this decrease was more severe in MJ treated cultures. All the MJ and SA treated cultures showed significantly higher amount of RC-C, RC-D and RC-N in hairy roots harvested 7 days after elicitation as compared to control. Of the two elicitors, MJ was more efficient in inducing RC accumulation than SA. The highest RC content (6.3 mg/g DW RC-C; 1.1 mg/g DW RC-D and 0.61 mg/g DW RC-N) was observed after treatment with 10 μM MJ which was about 1.7-, 2.5- and 3.5-fold higher RC-C, RC-D and RC-N respectively than the control.

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.

Hairy root is induced when Agrobacterium rhizogenes , a naturally occurring gram-negative soil bacterium, infects a plant that is susceptible to infection. During the infection process of the A. rhizogenes to a plant, parts of its plasmid DNA (T-DNA) in the Ri plasmid will be transferred and integrated into the nuclear genome of the host plant. This transformation process eventually resulted in a valuable by-product called as hairy root. During infection of A. rhizogenes the plant oncogenes (i.e. rol A, rol B, rol C and rol D) present in bacteria are incorporated into the plant genomes and cause tumour formation resulting in hairy root disease. The characteristic feature of hairy root is that they have the capability to grow fast on basal media, genetic and biosynthetic stability and ability to produce elevated amount of several useful secondary metabolites. This chapter reviews the recent reports on hairy root culture and secondary metabolite production in various plant species.

An efficient plant regeneration protocol was established for an endangered ethnomedicinal plant Desmodium gangeticum (Linn.) DC. Morphogenic calli were produced from 96 % of the cultures comprising the immature leaf explants on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.0 mg l(-1)) in combination with 6-benzylaminopurine (BA; 0.8 mg l(-1)). For callus regeneration, various concentrations of BA (1.0-5.0 mg l(-1)) or thidiazuron (TDZ; 1.0-5.0 mg l(-1)) alone or in combination with indole-3-acetic acid (IAA; 0.2-1.0 mg l(-1)) were used. Highest response of shoot regeneration was observed on MS medium fortified with TDZ (4.0 mg l(-1)) and IAA (0.5 mg l(-1)) combination. Here, 100 % cultures responded with an average number of 22.3 shoots per gram calli. Inclusion of indole-3-butyric acid in half MS medium favored rooting of recovered shoots. Out of 45 rooted plants transferred to soil, 40 survived. Total DNA was extracted from the leaves of the acclimatized plants of D. gangeticum. Analysis of random amplified polymorphic DNA using 13 arbitrary decanucleotide primers showed the genetic homogeneity in all the ten plants regenerated from callus with parental plant, suggesting that shoot regeneration from callus could be used for the true-to-type multiplication of this plant.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl2·2H2O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA3). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.

An efficient plant regeneration protocol via shoot regeneration from callus derived from immature leaf explants was established for Rhinacanthus nasutus (L.) Kurz., a potent ethnomedicinal plant for various ailments including cancer. 5-day old leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA; 1.0–5.0 mg/l) or Kinetin (Kn; 1.0–5.0 mg/l) in combination with 0.5 mg/l IBA for callus induction. Optimum morphogenic callus induction (98.8%) was observed on MS medium supplemented with 4.0 mg/l Kn and 0.5 mg/l IBA. The excised callus subcultured on MS medium supplemented with BA (1.0–5.0 mg/l) or Kn (1.0–5.0 mg/l) with NAA (0.2 and 0.5 mg/l). The highest frequency of shoot regeneration (96.3%) and shoot number (36.5 shoots per 1.0 gm calli) was observed on MS medium supplemented with Kn (3.0 mg/l) in combination with NAA (0.5 mg/l). Half MS medium supplemented with IAA (2.0 mg/l) produced an optimum rooting response of 96.23% with an average number of 6.1 roots per shoots. Out of 50 rooted shoots transferred to soil 47 survived after acclimatization. Clonal fidelity of the regenerated plants with mother plant was evaluated using 12 inter simple sequence repeats (ISSR) primers and genetic uniformity among all the regenerants and mother plant is confirmed.

A protocol has been developed for achieving somatic embryogenesis from callus derived from nodal cuttings and production of synthetic seeds in Hemidesmus indicus L. R. Br. a highly traded ethnomedicinal plant. Proembryogenic, friable, light yellowish callus was induced from the basal cut end of the nodal cuttings on Murashige and Skoog (MS) medium supplemented with 3 μM indole-3-butyric acid (IBA). The highest rate of somatic embryogenesis (92 %) was observed when the callus was subcultured on half strength MS medium supplemented with 2 μM IBA. On induction medium somatic embryos were developed up to the torpedo stage. Further elongation and germination of somatic embryos were obtained in MS medium supplemented with 4 μM 6-benzylaminopurine (BA) in combination with 1.5 μM gibberellic acid (GA3). Somatic embryos were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V) dropped into 75 mM calcium chloride (CaCl2·2H2O) solution for the production of synthetic seeds and later transferred to MS medium for germination. The synthetic seeds were successfully germinated on medium even after 120 days of storage at 4 °C. The plantlets were eventually transferred to soil with 92 % success.

Coelogyne nervosa is an epiphytic orchid endemic to Western Ghats, South India. The mature seeds of C. nervosa were cultured on ½ MS (Murashige and Skoog), MS, Kn (Knudson) and VW (Vacin and Went) media to evaluate the seed germination response. Of the four basal media used, MS medium supported maximum seed germination. Further experiments to enhance seed germination were done on MS medium supplemented with various concentrations (10, 20, 30 and 40 %) of coconut water (CW). Thirty percent CW gave the highest response in terms of percent seed germination (96), fresh weight (7.2 mg/seedling) and protocorm length (15.2 mm). Since CW containing medium did not support further seedling growth, each seedling was isolated and cultured on MS medium supplemented with either BA (6-benzylaminopurine) or Kin (kinetin) alone (1.0-4.0 mg/l each) or in combination with NAA (1-naphthaleneacetic acid; 0.2-1.0 mg/l). Maximum growth was observed on MS medium supplemented with BA (3.0 mg/l) and NAA (0.5 mg/l). On this medium, the seedlings reached an average length of 3.6 cm with 2.8 well expanded green leaves per seedling. Similarly optimum, healthy, white root induction (3.3 roots/seedlings) was also observed on the same medium. The rooted seedlings were successfully transplanted to pots with 91 % success. The 2-year-old tissue culture derived plants produced normal flowers and fruits.

An efficient plant regeneration protocol has been developed from embryogenic callus derived from cotyledonary explants of Clitoria ternatea Linn., an important medicinal climber species. Optimum embryogenic callus (75 %) was induced on Murashige and Skoog (MS) medium supplemented with 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D). On subculturing the callus on MS medium supplemented with 2 mg/l 6-benzyladenine (BA) and 0.5 mg/l α-naphthalene acetic acid (NAA), 61 % of cultures responded with a mean number of 22 somatic embryos per gram callus at different stages of development after 45 days of culture. The addition of higher concentrations of sucrose and abscisic acid (ABA) significantly increased the embryogenic response. MS medium supplemented with 2 mg/l BA, 0.2 mg/l NAA and 4 % of sucrose resulted in 76 % of cultures responding with a mean number of 28 embryos per one gram callus. The highest embryogenic response, frequency of 83 % and mean number of 37 embryos per gram callus, was observed when the MS medium was supplemented with 2 mg/l BA, 0.2 mg/l NAA, and 3 mg/l ABA. Synthetic seeds were produced by encapsulating embryos in calcium alginate gel. The gel contained MS medium with 3 % of sucrose, 1.0 mg/l BA and 0.2 mg/l NAA. The synthetic seeds germinated on MS medium supplemented with BA (1.0-4.0 mg/l) alone or in combination with NAA (0.1–0.7 mg/l) or indole-3-butyric acid (IBA; 0.1–0.7 mg/l). The highest synthetic seed germination (92 %) was observed on MS medium supplemented with 2 mg/l BA and 0.5 mg/l NAA. The synthetic seeds were stored at 4 °C and lab conditions (25 ± 2 °C) up to 5 months. The synthetic seeds kept at 4 °C showed 86 % viability even after 5 months of storage. Both somatic embryos and synthetic seeds germinated and were transferred to soil successfully.

Brachypodium distachyon has been recently proposed as a new model system for monocot functional genomics because of its unique properties such as small size, short generation time, self-fertile, chromosome base number five and small genome size. In this study, we have standardized a protocol to obtain 100% green plant regeneration from embryo and shoot tip explants. The optimum callus induction in terms of percentage of cultures responding and callus growth was observed on Murashige and Skoog (MS) medium supplemented with 4 mg/l 2, 4 dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg/l kinetin (Kn). On this medium, 88% and 100% explants responded from embryo and shoot tip explants, respectively. Comparatively, shoot tip explants produced better response than embryo explants. The callus multiplied and maintained on MS medium supplemented with 2, 4-D (4 mg/l) and Kn (0.5 mg/l) for about 10 months without decline in growth rate. The callus subcultured on MS medium supplemented with thidiazuron (TDZ; 0....

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l−1 2,4-D and 1 mg l−1 BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l−1 BA and 1 mg l−1 indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l−1 indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5 cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96 % callus induction from cotyledonary node explants on MS medium supplemented with 1.5 mgl(-1) 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgl(-1) 1-naphthalene acetic acid (NAA), (2) 97 % shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0 mgl(-1) N6-benzylaminopurine (BA) and 1 mgl(-1) NAA,37 (3) 98 % rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5-4 mgl(-1)) after 45 days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5 %).

Justicia gendarussa is a valuable medicinal plant and various parts of this plant are pharmaceutically used for the treatment of different diseases. In vitro regeneration of shoot buds was obtained from culture of nodal cuttings as well as shoot regeneration from callus. The nodal cuttings differed in shoot proliferation in terms of percentage of explants that responded and average shoot length with various concentrations (4.4, 8.9, 13.3, 17.7, 22.2 μM) of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron. In all treatments, one shoot was invariably present. Optimum 87% of cultures responded with an average shoot length of 4.4 cm on Murashige and Skoog (MS) medium supplemented with 17.7 μM BA. Callus was induced from the mature leaf segments on MS medium supplemented with Kn (4.7, 13.9, 23.2 μM) alone or in combination with 2, 4-dichlorophenoxyacetic acid (2, 4-D; 2.3 μM, 4.5 μM). Optimum callus induction (78%) was obtained on MS medium supplemented with 14 μM Kn and 4.5 μM 2, 4-D. When the callus was subcultured on MS medium fortified with BA (8.9, 17.7, 26.6 μM) or Kn (9.3, 18.6, 27.9 μM) alone or in combination with α naphthalene acetic acid (NAA; 2.7, 5.4 μM), shoot regeneration was obtained. The highest response (92%) was observed on MS medium containing 17.7 μM BA and 5.4 μM NAA. On this medium, an average number of 12.2 shoots were obtained per responding callus. The shoots obtained from callus and nodal cuttings were rooted with a frequency of 73% on MS medium augmented with 9.8 μM indole-3-butyric acid. The rooted shoots were successfully transplanted to soil and sand mixture (1:1) with 90% survival rate. The protocol standardized for shoot proliferation and regeneration in J. gendarussa from nodal cuttings and leaf-derived callus is suitable for micropropagation and conservation of this essential medicinal plant.

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l−1 induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l−1 α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l−1 TDZ and 0.5 mg l−1 NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l−1 TDZ and 0.5 mg l−1 NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l−1 TDZ, 0.5 mg l−1 NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l−1 BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l−1 indole-3-butyric acid (IBA) and 1.5 mg l−1 activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.

An efficient and quick in vitro propagation of Cyclea peltata by repeated subculture of nodal cuttings has been standardized. The nodal cuttings were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (0.5–7 mg/l) of 6-benzylaminopurine (BA) or kinetin (Kn) alone or in combination with indole-3-acetic acid (IAA; 0.5 mg/l) for culture initiation. Optimum response in terms of percent cultures responding, days to bud break and average shoot length was observed on MS medium fortified with 3 mg/l BA and 0.5 mg/l IAA. On this medium 90% cultures responded with 6.1 cm long unbranched vigorous solitary shoots in 45 days. The proliferated primary shoot emerged from the culture initiation step was incised into small nodal explants measuring a size of about 1.5 cm in length containing a single node and subcultured on multiplication medium supplemented with BA (0.5–7.0 mg/l) and IAA (0.5 mg/l). This process was repeated for another three subcultures (each of 45 days) to study the effect of subculturing on shoot multiplication. At the end of third passage 100% of nodal explants produced an average number of 14.2 healthy green shoots on MS medium supplemented with 3 mg/l BA and 0.5 mg/l IAA. The multiplied shoots were harvested and used for rooting on half-strength MS medium containing indole-3-butyric acid (IBA; 1–7 mg/l) or naphthalene acetic acid (NAA; 1–7 mg/l) for 45 days. The best rooting response was achieved on half-strength MS medium supplemented with 5 mg/l IBA. Here 96% cultures responded with an average number of 4.1 roots per shoot. Of the 40 plants transplanted to soil 28 survived (70%). This cost effective protocol will help the mass multiplication of C. peltata for commercial propagation.

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 105 protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 µM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 µmol m−2 S−1 of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.

Bitter melon (Momordica Charantia L.) is an important vegetable crop with nutritional and medicinal qualities. As a member of cucurbitaceae it is monoecious with varying proportions of staminate and pistillate flowers. This study was undertaken to determine the effects of various applications of ethrel and gibberellic acid (GA3) on sex modification in M. charantia. In the first set of experiments, various concentrations of hormones were added to the seed germination medium, in the second, adult plants growing in the field were sprayed with aqueous solutions of ethrel or GA3 three times at three-day intervals. The number and sex of open flowers was recorded daily for 60 days after the first flower opened and total number of staminate and pistillate flowers was calculated at the end of the experiment. The highest frequency (29.5%) of pistillate flowers was observed in plants treated with 500 ppm ethrel at germination. Similarly, spraying of adult plants with 100 ppm GA3 increased the proportion of pistillate flowers to 26% relative to 15% in untreated controls. Both ethrel and GA3 induced significantly higher number of pistillate flowers than control. In vitro hormone application during seed germination was much more successful than spraying of field grown plants.

An efficient and reproducible method for inducing a large number of bulblets from rhizome explants of Curculigo orchioides Gaertn., an endangered medicinal herb, has been developed. The rhizome pieces, measuring about 1 × 1 cm (length × width), were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of the cytokinins 6-benzylaminopurine, kinetin, and thidiazuron (TDZ) alone or in combination with 1-naphthalene acetic acid or indole-3-butyric acid (IBA). Of the three cytokinins used, TDZ at 7 μM gave the maximum response, with 82% of the cultures responding with an average number of 15.4 bulblets per explant. The addition of auxins with cytokinin considerably increased the response. The optimum induction occurred on MS medium supplemented with 7 μM TDZ and 0.5 μM IBA. On this medium, 88% of the cultures responded with an average number of 21.4 bulblets per explant. Experiments were also carried out to investigate the role of the sugars sucrose, mannose, and glucose along with 7 μM TDZ and 0.5 μM IBA. The results indicate that sucrose and mannose at particular concentrations have critical roles in promoting in vitro bulblet induction. The maximum result was observed on MS medium supplemented with 7 μM TDZ, 0.5 μM IBA, and 200 mM mannose. On this medium, 97% of the cultures responded with an average number of 26.8 bulblets per culture. Several secondary bulblets developing from the leaf blades of primary bulblets were produced when the latter were transferred to MS basal medium for further development. Out of the 45 bulblets transferred to soil, 40 survived. This protocol can be used for the rapid micropropagation of this endangered medicinal herb.

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L−1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.

There has been an increase in use of traditional medicine during the last decade due to the reason that medicinal plant-derived drugs are more effective against several diseases as compared to antibiotics and other medicines and the former has less side effects on patients. Moreover, medicinal plants are a rich source of antibacterial compounds. The increased use and harvest of medicinal plants coupled with habitat destruction caused a decrease in natural population of most of the medicinal plants. In vitro conservation strategies can not only offer large number of plants in limited time but also it can decrease tension on natural populations. Similarly, several horticultural plants including vegetable crops need improvements and therefore in vitro conservation strategies could be applied for their further progress. The present review is an attempt to provide information on various in vitro conservation strategies adopted in selected medicinal and horticultural climbers. The important medicinal and horticultural climbers such as Benincasa hispida, Cardiospermum halicacabum, Tylophora indica, Sarcostemma brevistigma, Cyclea peltata, Caesalpinia bonduc, Clitoria ternatea, Hemidesmus indicus, Momordica charantia, and Embelia ribes have been discussed in detail.

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.

Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.

Mulberry (Morus alba L.) is a dioecious plant. In vitro sex modification in mulberry by using two chemicals, viz., ethrel and silver nitrate, is described. 2-Chloroethylphosphonic acid (ethrel) and silver nitrate were filter sterilized and added to Murashige and Skoog (1962) medium supplemented with 5 µM 6-benzyl amino purine (BA). Ethrel applied at different concentrations produced female, male and mixed inflorescences in male mulberry nodal cuttings. Maximum 13.6% female inflorescences were observed on media containing 2000 µg l−1 ethrel. The induction of male, female and mixed inflorescences was observed in female plants after applying silver nitrate to the culture medium. The number of male inflorescences increased with the silver nitrate concentration to a maximum of 22.4% induction on 2500 µg l−1. Bisexual flowers were also observed along with male and female flowers in silver nitrate treated plants.