The biotechnological intervention

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Abstract: The plants used in the phyto-pharmaceutical preparations are obtained mainly from the natural growing areas. With the increased in the demand for the crude drugs, the plants are being overexploited, threatening the survival of many rare species. In addition, agriculture land decreasing day by day due to the real estate, industrialization and roads for the betterment of human beings. To maintain the required demand of the important secondary metabolites and their sources; several research institutions and pharmaceutical industries using advanced biotechnological tools, which includes culturing of plant cells, genetic manipulation aiming to restore the germplasm, insertion of interest of genes for the production of important active principle. The present review article covering the in vitro micropropagation and production of selected secondary metabolites through biotechnological intervention viz. Alliin, Artimisnin, Podophyllotoxin, and Taxol.

Keywords: Plant Tissue Culture, Secondary metabolites, Alliin, Artimisnin, Podophyllotoxin, Taxol

Introduction

In modern medicine, plants are used as sources of direct therapeutic agents, as models for new synthetic compounds, and as a taxonomic marker for discovery of new compounds. They serve as a raw material base for the elaboration of more complex semisynthetic chemical compounds (Akerele, 1992; Anonymous, 2001). The synthesis of bioactive compounds chemically is difficult because of their complex structure and high cost (Anonymous, 2001). Wide variations in medicinal quality and content in phytopharmaceutical preparations have been observed. They are influenced mainly by cultivation period, season of collection (Abdin et al., 2003). Generally, herbal preparations are produced from field-grown plants (Murch et al., 2000). It was difficult to ensure the quality control as the medicinal preparations are multi-herb preparations and it is difficult to identify and quantify the active constituents. An efficient and most suited alternative solution to the problems faced by the phytopharmaceutical industry is the development of in vitro systems for the production of medicinal plants and their extracts.

Plant tissue culture proved an important technology being used for the conservation of important plants either through organogenesis, somatic embryogenesis and genetic transformation (Sajc et al., 2000; Mujib and Samaj, 2006). The major advantages of cell cultures includes (i) synthesis of bioactive secondary metabolites independently from climatic and soil conditions; (ii) negative biological influences that affect secondary metabolites production in the nature are eliminated (microorganisms and insects) (iii) to select cultivars with higher production of secondary metabolites; (iv) with automatization of cell growth control and metabolic processes regulation, cost price can decrease and production increase ( Jha et al., 1998; ; Abdin et al. 2003; Junaid et al., 2009; Junaid et al.,2010). The objectives of many industries are to develop plant cell culture techniques to the stage where they yield secondary products (Table 1), more cheaply than extracting either the whole plant grown under natural conditions or synthesizing the product. Although the production of pharmaceuticals using plant cell cultures have been highlighted, other applications have also been suggested as a new route for the synthesis, products from plants difficult to grow, or in short supply, as a source of novel chemicals and as biotransformation systems. It is expected that the use, production of market price and structure would bring some of the other compounds to a commercial scale more rapidly and in vitro culture products may see further commercialization. (Doran, 2000; Ramachandra Rao and Ravishankar, 2002; Junaid et al., 2009, Nasim et al.,2009).

Production and accumulation of selected secondary metabolites from cell cultures

Plant cell culture holds much promise as a method for producing complex secondary metabolites in vitro (Ravishankar and Venkataraman, 1993; Junaid et al.,2009; Nasim et al., 2010; Junaid et al., 2010). The sources, medicinal significant and in vitro production has been reviewed here in, Alliin, Artemisinin, Podophyllotoxin and Taxol secondary metabolites.

ALLIIN

Sources

Garlic (Allium sativum) is the main sources of Alliin (Figure 1A). It is a member of the lily family. It may be divided into two subspecies: Allium ophioscorodon (bolting or hard-neck cultivars) and Allium sativum (non-bolting or soft-neck cultivars). Allium ophioscorodon produces elongated flower stalks, often referred to as scapes, and flower-like bulbils at the top of the stalk. Soft-neck garlic does not produce bulbils except in times of stress. While both bulbils and individual cloves can be propagated vegetatively, bulbils take longer up to two seasons to produce mature bulbs, and require special care because the young plants are very small and fragile. (Anonymous, 2001).

Medicinal importance

Garlic (Allium sativum) is an important culinary and medicinal plant used worldwide. Garlic, like many other members of Alliums, contains high organic sulphur compounds in the form of alkylcysteine sulphoxides and ?-glutamyl peptides. On tissue damage and with alliinase enzyme's activity, the alkyl cysteine sulphoxide releases compounds that give unique Allium's odour and flavour. It shows several biological activities such as antibiotic, antitumour, antiatherosclerotic (Chanprame, 1998; Campbell, 2001, Nasim et al., 2009; Nasim et al.,2010), cholesterol-lowering effect (Yeh and Liu, 2001) and also prevent cardiovascular disorders (Rahman, 2001).

Micropropagation and in vitro Alliin production

Cultivated garlic is sexually sterile crop and exclusively propagated vegetatively (Novak et al., 1990). Conventionally the use of seed bulb is the only way for cultivation of garlic. For each plant one seed bulb is needed. The lack of the availability of seed bulbs is the limitation for its large scale propagation. In addition, Garlic is one of the major spice crops of Bangladesh. It is being cultivated on an area of 13077 hectare with a total production of 42805 tons, the average yield is 3.74 t/ha (BBS 1998). The yield of garlic in Bangladesh is very low in compare to other garlic growing countries, like China (7.9 t/ha), Thailand (7.8 t/ha) and Korea (5.0 t/ha). The local cultivars of Bangladesh are infected by viruses causing low yield (Anonymous., 2001). As garlic is propagated vegetatively; viruses are transmitted to the next generation. Propagation of garlic is mainly accomplished by vegetative methods, which demonstrate a low coefficient of multiplication (Novak, 1990; Nagakubo et al., 1993); therefore it takes many years to produce sufficient number of seed bulbs for practical cultivation of new elite variety (Nagakubo et al., 1993). Similarly, the crop improvement by cross fertilization is limited as garlic shows sexual incompatibility (Masanori, 1995).

There are reports of using in vitro methods for propagation of garlic (Novak, 1990; Nagakubo et al., 1993; Seabrook, 1994; Zel et al., 1997, Nasim et al., 2009). However, a few work reported using meristem for its micropropagation (Moriconi et al., 1990). In Allium, callus culture and in vitro morphogenesis have been achieved from various plant parts (Barandiaran, 1998; Myers and Simon, 1998; Robledo-Paz, et al., 2000; Sata, et al., 2001) but the rate of multiplication and the number of plantlets regenerated per explants were not always significantly high. The formation of multiple bulblets from single explant is the most desirable one. In vitro bulblet formation of garlic has also been reported (Moriconi et al., 1990). Multiple bulblet formation was induced by using in vitro developed plantlets, which were acclimatized in out door candition (Roksana et al., 2002).

Khar et al. (2005) studied on the effect of different plasmids and suitability of explants towards Agrobacterium transformation using three genotypes of Allium. There were no significant differences among genotypes, however; the two plasmids showed significant variables response in transient Gus assays. Plant regeneration through somatic embryogenesis is rare but is not uncommon in Allium (Sata et al., 2001). It has several advantages over organogenesis and appears to be the most promising technique for fast propagation of plants (Ignacimuthu, 1995). The developmental protocols to establish embryonic cultures with synchronous embryo forming ability may able to eliminate many of the problems associated with zygotic embryo development.

A simple high frequency direct somatic embryogenesis system is reported from basal part of clove in Allium sativum cv. Yamuna Safed in which we investigated (Nasim et al., 2009) the role of auxins and cytokinins in somatic embryogenesis.Attention has also been paid to identify the biochemical differences that existed between callus and embryogenic tissues in Allium sativum during plant regeneration. In addition Nasim et al. (2010) also reported the effect of sulphur supplementation on Alliin production in different plant organs viz; leaf, root, plantlet, non-embryogenic and embryogenic callus, proliferated, matured and germinated embryos grown under in-vitro conditions. Evaluation of alliin content of in-vitro grown tissues both in normal (control) and sulphur supplemented conditions showed that sulphur treatment at supply of 16 mg l-1 gypsum (CaSO4) significantly enhanced the production of alliin content in all in-vitro grown tissues and organs. The maximum alliin content was recorded in leaves (Nasim et al., 2010).

ARTEMISININ

Sources

The genus Artemisia belongs to the family Compositae. The leaves of the many species of Artemisia having the madicinal properties (Abdin et al.,2003); being used in the treatment of maleria due to which more than 275 million people worldwide effecting and is the cause of at least 1 million deaths every year (Butler, 1997).

Medicinal importance

As one of the world's most serious parasitic diseases, malaria, caused by Plasmodium, causes at least 500 million cases globally every year, resulting in more than one million deaths. The biggest challenge facing in the fighting against malaria is the multi-drug resistance of Plasmodium strains to the widely used antimalarials such as chloroquine, mefloquine and sulfadoxine-pyrimethamine (Greenwood and Mutabingwa, 2002; Liu et al., 2006), is known for the drug artemisinin; an effective antimalarial drug against chloroquinine-resistant and chloroquinine-sensitive strains of Plasmodium falciparum and against cerebral malaria. Likewise, its effectiveness has been demonstrated in the treatment of skin diseases and it is also a natural herbicide.Artemisinin (Figure 1B) is a sesquiterpenoid isolated from the Chinese herb 'qing hao' (Artemisia annua). It is effective against both chloroquinine-resistant and chloroquinine-sensitive strains of Plasmodium falciparum and similarly against the cerebral malaria. It may have some hallucinogenic properties. A. absinthium L. is traditionally used because of its antiheliminthic, insecticidal, antiseptic and febrifuge properties Abdin et al. (2003). At present, the commercial source of the drug is the leaves and flowering tops of field-grown A. annua plants, which are subject to seasonal and somatic variations (Abdin et al. 2003).

Micropropagation and in vitro Artemisinin production

The only commercial source of the drug is extracted from fieldgrown leaves and flowering tops of Artemisia annua L., which are subject to seasonal and somatic variation (Paniego and Giulietti, 1994). Artemisinin content in A. annua is very low (0.01 - 1% dry weight, DW), and the demand for artemisinin is increasing along with the increasing number of people suffering from malaria. Various approaches have been attempted to increase artemisinin production including chemical synthesis. Using shoot tips Nin et al. (1996) established a high regeneration protocol for A. absinthium. In addition, Zia et. al. (2007) evaluate the effect of different combinations of auxins and cytokinins on callogenesis and organogenesis in A. absinthium.

Artemisinin production has been extensively studied in shoot and hairy root cultures. (Liu, 1998; Xie, 1995). An internal-loopmist bioreactor has been devised and applied to the shoot and hairy root cultures of A. annua, achieving an artemisinin yield of 46.9mgl-1 in 25 days, much higher than that in the shake-flasks (Liu, 1998). Hairy root cultures exposed to red light at 660 nm achieved a higher growth rate and artemisinin content compared to those exposed to green, blue, yellow or white light Wang (2001). Climatic condition together with the way and time of planting and harvesting of A. annua can influence artemisinin production in A. annua (Wallaart et al., 2000; Abdim et al., 2003).

The genetic engineering of the pathway genes involved in artemisinin biosynthesis in A. annua (Vergauwe et al., 1996; Chen et al., 2000; Xie et al., 2001; Martin et al., 2003; Ro DK et al., 2006), but not much success has been recorded because of the high cost or complex nature of the gene regulation and expression in artemisinin biosynthesis. New approaches, cheaper and more convenient, are needed for improving artemisinin production.

Plant hormone such as GA3, BA and kinetin may also influence artemisinin production (Whipkey et al., 1992; Fulzele et al., 1995; Smith et al., 1997; Weathers et al., 2005). In addition, stress conditions such as light, temperature and watering may have effects on artemisinin production too (Guo et al., 2004; Wallaart et al., 2000). HPLC analysis was carried out for each level (different developmental stages) and it was found that the plant seeding to salinity stress had higher contents of artemisinin (2-3% DW) compared to those without treatment (1.0-1.5% DW). The result analyzed with two-side T test suggested that the enhancement of artemisinin content caused by 2 g-l NaCl stress was not significant compared to the control, but the enhancement caused by 4 and 6 g/l NaCl stresses was extremely significant (P<0.01) compared to the control. Various approaches have been previously tried to enhance artemisinin production (Vergauwe et al., 1996; Chen et al., 2000; Wang et al., 2001; Xie et al., 2001; Liu et al., 2002. Wang et al., 2001) and it was found that light spectrum would influence biomass and artemisinin content of transformed hairy roots. The highest biomass (5.73 g DW-l) and artemisinin content (31 mg-g) were obtained under red light at 660 nm which were 17 and 67% higher than those obtained under white light, respectively. Liu et al. (2002) found that light irradiation influenced the growth and production of artemisinin in transformed hairy root cultures of A. annua too. When hairy roots were cultured under illumination of 3,000 Lux for 16 h using several cool-white fluorescent lamps, the dry weight and artemisinin concentration reached 13.8 and 244.5 mg-l, respectively.

Wang et al. (2002) regulated the ratio of NO3/NH4 and total initial nitrogen concentration in the culture of hairy roots, and successfully increased artemisinin concentration by 57% compared to the control. Xie et al. (2001) infected A. annua leaf pieces and petiole segments with A. rhizogenes and obtained a clone of hairy root with artemisinin content of 0.12% DW. Vergauwe et al. (1996) transformed A. annua plants mediated by Agrobacterium tumefaciens and slightly higher artemisinin content (0.17% DW) in the leaves of regenerated plant than normally cultured plant (0.11% DW) was achieved. Chen et al. (2000) transformed a cDNA encoding cotton FDS (farnesyl diphosphate synthase) under the control of CaMV 35S promoter into A. annua via A. tumefaciens or A. rhizogenes. By overexpressing FDS, a key enzyme in the biosynthesis of artemisinin, in transgenic plants the artemisinin content could reach 2 - 3% DW, the highest artemisinin content in A. annua reported so far, by the procedure of treating plants with suitable concentrations of NaCl.

Rita et al. (2007) established a regeneration protocol A. annua L. and quantified the production of artemisinin and flavonoids in different aerial parts of in vitro raised plantlets. Artemisinin content in A. annua was enhanced through salinity stress Qian et al. (2007).

Sujatha Govindaraj et al. (2008) reported mass propagation through in vitro liquid culture technology fortified with 6-benzyl adenine (BA). The plantlets were then acclimatized under standard laboratory conditions and later under greenhouse conditions. Sharaf and Elkholy (2009) reported a high regeneration protocol. Shoot cultures of Artemisia annua L. were cultivated in three different micropropagation systems: an ultrasonic nutrient mist bioreactor (UNMB), a modified ultrasonic nutrient mist bioreactor (MUNMB) and solid culture in Magenta boxes. The shoots cultivated in the UNMB and MUNMB showed excellent growth. The dry weight increase (35 times) of shoot cultures in the MUNMB was higher than those (25 times and 19 times) in both the UNMB and the Magenta boxes. Additionally, artemisinin content of shoot cultures in the MUNMB was 1.2-2.0-fold higher than those in both the UNMB and the Magenta boxes, respectively. The modified ultrasonic nutrient mist bioreactor was found to be advantageous for A. annua L. shoot cultures and artemisinin production (Sharaf and Elkholy (2009).

PODOPHYLLOTOXIN

Source

Podophyllotoxin is the most abundant lignan isolated from Podophyllin, a resin produced by species of the genera Podophyllum (Berberidaceae).

Medicinal importance

Podophyllotoxin (Figure 1C) is an antitumor aryltetralin lignan, commonly used in the treatment of numbers of cancers (Issell et al., 1984).

Micropropagation and in vitro Podophyllotoxin production

The genus Podophyllum is an important anticancerous plant, growing sexually, but due to the seed dormancy growth rate is very noticeable, which limiting the podophyllotoxin production. The alternative way to overcome the problem is in vitro cell and tissue culture. Using, in vitro technique, the first time podophyllotoxin was quantified by the Kadkade et al., (1981, 1982). A number of researchers used various explants, different types of elicitors and precursor to enhance the level of podophyllotoxin (Hyenga et al., 1990; Kim et al., 2007). A podophyllotoxin precursor (Coniferyl alcohol, and b-cyclodextrin) was added in the P. hexandrum suspension culture and a remarkable variation was noticed in yield, when compared with the non added precursor in the medium (Woerdenberg et al., 1990). Kim et al. (2007) reported the establishment of plantlet regeneration of P. peltatum via somatic embryogenesis. Somatic embryos differentiated directly from cotyledon explants of zygotic embryos. The germinated embryos grow into plantlets with well developed roots. Rooted plantlets were acclimatized. Anbazhagan, et al. (2008) induced somatic embryogenesis and quantified podophyllotoxin in P. peltatum and used elicitor which strongly enhanced the production of podophyllotoxin in vitro raised culture.

TAXOL

Source

The genus Taxus belongs to the family Taxaceae having the seven species. These species are slow growing evergreen trees that occurs in various geographical areas and accumulate taxol to a higher or lower extent. Taxol (plaxitaxol) (Figure 1D), a complex diterpene alkaloid originally, the main source of taxol is bark, but it is also extracted from different parts.

Medicinal importance

In 1983, Food and drug administration had approved taxol for the treatment of ovarian and breast cancer, lung cancer, malignant melanoma, as well as AIDS etc. (Wickremesinhe and Arteca, 1993; 1994, Cragg et al., 1993), because it played a potent role on microtubular cell system (Jordan and Wilson, 1995).

Micropropagation and in vitro Taxol production

The main source of the taxol being yew trees. Wani and his colleagues for the first time discovered a novel anticancer diterpene amide,"taxol" from the Pacific yew (Taxus brevifolia) extract (Wani et al., 1971). The high demand for the drug cannot be met by extraction from the trees due to the scarcity and slow growth of Taxus yew trees and the low Taxol content (Zhong, 1995). Chemically, taxol is a very complex in structure; therefore, the synthesis at industrial level is not economic friendly. To over come the scarcity of the Taxus tree, and to reduced the synthetic cost, in vitro micropropagation being a good technology to produced the plantlets in masses and its production in several ways viz; forestry, and taxol production. In vitro micropropagation of Taxus spp. is recommended as one of the approach available to produced taxus plantlets, continious supply of taxol and other related t derivatives (Slichenmyer and Von Horf, 1991).

The first time, in vitro taxol production was carried out by the Christen et al. (1989), thereafter, similar approached has been adopted in severeral research laboratories throughtout the world (Flores and Sgrignoli, 1991; Ma et al., 1994; Lee et al., 1995; Yukimune et al., 1996; Jha et al. 1998; Nguyen et al., 2001; Wu et al., 2001; Linden et al., 2001; Parc et al., 2002; Abbasin et al., 2010).

The seeds of the taxus species undergoes for a lengthy dormancy period which can be overcome using in vitro method. Viable embryos were excised from seeds of Taxus brevifolia and four cultivars and were cultured on Whites', Gamborg's B5 and Murashige and Skoog's medium under dark or light conditions. Embryos excised from green seeds with undeveloped arils showed the highest germination rates, as the seeds approached maturity, in vitro germination rates of the excised embryos declined dramatically (Flores and Sgrignoli, 1991).

Various types of medium supplements have been studied aiming to enhance the taxol production. Fett- Neto et al. (1994) used different amino acids and phenylalanine to the culture medium of T. cuspidata and reported a significant increased in taxol production. Lee et al., (1995) reported remarkable differences in taxol content in bark and leaf tissues of in vitro developed taxus culture. (Lee et al., 1995).

Biomass accumulation and the taxol production has been studied by Srinivasan et al. (1995) in cell suspension culture of T. baccata. In addition, an aquivalent amount (Kim et al.,1995) of paclitaxel was also reported from T. brevifolia cell suspension cultures. Effect of media compositions and other factors were evaluated on the production (Fett-Neto et al. 1995) of paclitaxel production in T. cuspidata . Morever, addition of different carban sources increased the paclitaxel production (Ketchum and Gibson, 1996). Use of the biotic and abiotic elicitors to improve taxol production has been studied (Strobel et al., 1992; Ciddi et al., 1995; Yukimune et al., 1996; Jha et al. 1998 ). Ellis et al., (1996) established cultures using nodal segment in seven Taxus cultivars and screened the taxol production. Similarly, various Taxus species has been widely explored as an alternative for the production of Taxol and other useful taxane compounds in the world (Abdinet et. al., 2003).

Majada et al. (2000) reported a high yielding procedure for the in vitro propagation of juvenile material of T. baccata with respect to the taxane contents. A positive correlation was found between growth and secondary metabolites yield. Tsay et al. (2001) reported a high regeneration protocol for Taxus mariei using bud explants derived from approximately 1,000-year-old field-grown trees, and a comparison was made with bud explants derived from 1-year-old stecklings raised from rooted cuttings of these trees. The steckling-derived cultures performed better than mature tree-derived cultures in terms of shoot multiplication and rooting ability. Taxus genotypes were cultured and screened for the taxol production by Parc et al. (2002). Bud explants and the embryos were used as experimental material, and placed on the different auxins type and cytokines. And it was noticed that micro propagation was auxins, cytokinin concentrations and genotype dependent. Plantlets were successfully acclimatized and established in outdoor conditions (Abbasin et al., 2010).

Conclusion

Since long human being used and still continiously using plants in the form of carbohydrates, fat, food, protein, and shelter etc. Morever, its also a sources of variety of secondary metabolites, which being used in the production of several valuable products (agrochemicals, biopestisides, colours, flavours, fragrances, food additives and pharmaceuticals). The commercial values of plant secondary metabolites have been the main impetus for the enormous research effort put into understanding and manipulating their biosynthesis using various chemical, physiological and biotechnological pathways. The information scored in the present communication would be highly valuable to understand the role of biotechnological intervention to enhance the level to meet the required demand of selected secondary metabolites.

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