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Advantage of Basillus thurengiensis (Bt) gene engineering in Plant Biotechnology

admin — Fri, 16 Oct 2009 15:48:48 +0000

Advantage of Basillus thurengiensis (Bt) gene engineering in Plant Biotechnology

By:Ali Parsaeimehr

Introduction:

Basillus thurengiensis (Bt) is an aerobic, motile , gram-positive, endospore-forming bacillus initially isolated in Japan by Ishiwata and formally described by Berliner in 1915 ??(20). The insecticidal ,tivity of Bt used commercially thus far comes from included in crystals formed during sporulation, although “vegetative insecticidal proteins” (Vips) from before sporulation (24) are also being developed. The crystals of different strains of most Bts contain varying to different groups of insects. More than 100 Bt toxin genes have been cloned and sequenced, providing an array of proteins which can be expressed in plant or in foliar applications of Bt products (10).

Insecticidal crystal proteins (Cry) of Bacillus thuringiensis (Bt) have been used since long to control insect pest (Tabashnik 1997).In recent years transgenic cultivar expressing the cry gen have been shown to defend insect attack in a wide variety of crops (Cheng et al 1998; Xiang et al 2000).

Insecticidal products containing subspecies of bacterium B.thuringiensis were first commercialized in France in the late 1930s with the product Sporeine.Bt was first introduced into tobacco plants in 1987 (21). However , much more effective plants that use synthetic genes modeled on those from Bt but designed to be more compatible with plant [removed]18 ) were introduced a few years later.of? the $US 8.1 billion spent annually on all insectides world wide, it has been estimated that nearly $2/7 billion could be substituted with Bt biotechnology products (14).Insect targeted for control by Bt plant are primarily Lepidoptera through the production of Cry1Ab, Cry1Ac, and Cry9c proteins, and with some experiment? this proteins don’t have any harm for mankind.

Genetically transformed plant incorporating genes from the soil bacterium Bacillus thuringiensis (Bt) were developed in the 1980` s. Tabacco, cotton and tomatoes were among the first crop plants transformed to produce insecticidal protein to protect the plant from insect attack and consequent yield losses. Bt cotton varietes were developed and field tested by the early 1990`s.

Agrobacterium tumefaciens is a Mediated Gene Transfer this Bacterial plant pathogen found in the soil that results in tumorous growths and/or roots to develop in infected plants (?Agrobacterium tumefaciens? 2001) ,This infection is known as Crown Gall Disease (Deacon 2002) The bacteria transfers a tumor-inducing (Ti) plasmid located in a section of its DNA (known as T-DNA) into the nucleus of an infected plant cell this bacteria could? altered from the bacterial genes of Bacillus thuringiensis (Cry1Ab, Cry1Ac,?) by insertion of the cauliflower mosaic virus (CaMV) promoter and enhancers, the leader sequence. An antibiotic resistance marker nptII, also with the CaMV promoter and the NOS transcription terminator from Agrobacterium tumefacians nopaline synthetase gene could insert into the cotton chromosome along with the Bt-Cry1Ac gene which could expression toxin crystal proteins that has affect on pests of Cotton and the modified Cry1Ac gene has been produced by the modifying amino acid sequence of the wild- type Cry1Ac protein which produced in Bacillus thuringiensis (22, 25) .

2-Review of Literature and Relevant Topics:

In an experiment at the University of Georgia,Zygotic hypocotyls of canola (Brassica napus L.) varieties of cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cry1Ac) under the control of the ?cauliflower ?mosaic virus 35S promoter using Agrobacterium tumefaciens-Mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein (Xiang Y et al,2000).

Helicoverpa armigera is a primary pest in Iran and it could raise it damage’s to 75%. Gharosy et.al transferred Bt cry1Ac ?insecticidal crystal protein gene under the control of the? cauliflower? mosaic virus 35S promoter using Agrobacterium tumefaciens-Mediated strains; LBA4404 and plasmid pBII21 to ?hypocotyls ?explants of Coker,Varamin and Sahil varieties of cotton .PCR analysis of putative transgenic plant showed the integration of? the 1/66 Kb especial band of? cry1Ac gene in the produce transgenic plant by variety of? Varamin (Gharosy et.al,2004).

Conventional breeding methods have not been very successful in producing pest-resistant genotypes of pigeon pea, due to the limited genetic variation in cultivated germplasm. Kiran?K.?Sharma et.al developed an efficient method to produce transgenic plants of pigeon pea by incorporating the cry1Ab gene of Bacillus thuringiensis through Agrobacterium tumefaciens-mediated genetic transformation. The novel tissue culture protocol have been the direct regeneration of adventitious shoot buds in the axillary bud region of in vitro germinating seedlings by suppressing the axillary and primary shoot buds on a medium containing a high concentration of N 6-benzyladenine (22.0?M). The tissue with potential to produce adventitious shoot buds explanted used for co-cultivation with A. tumefaciens carrying the synthetic cry1Ab on a binary vector and driven by a CaMV 35S promoter. Following this protocol, over 75 independently transformed transgenic events of pigeon pea were produced and advanced to T2 generation. Amongst the recovered primary putative transformation events, 60% showed positive gene integration based on initial polymerase chain reaction (PCR) screening. PCR analysis of the progenies from independent transform ants followed gene inheritance in a Mendelian ratio and 65% of the transformants showed the presence of single-copy inserts of the introduced genes. Reverse transcription-polymerase chain reaction analysis showed that the transcripts of the introduced genes were normally transcribed and resulted in the expression of Cry1Ab protein in the tested T2 generation plants. Interestingly, the content of Cry1Ab protein as a percent of total soluble protein varied in different tissues of the whole plant, showing the highest expression in flowers (0.1%) and least in the leaves (0.025%) as estimated by enzyme-linked immunosorbent assay. The transgenic plants produced in this study offer immense potential for the improvement of this important legume of the semi-arid tropics for resistance to insect pests (Kiran?K.?Sharma et.al? ,2005).

In the other experiment Guo, X. et.al? transfered, Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene into four upland cotton cultivars Ekangmian, Emian, Coker and YZ via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect (Guo, X. et.al ,2007 ).

?In an experiment Si-Jun?Zheng et al? use Agrobacterium-mediated genetic transformation for applied to produce beet armyworm (Spodoptera exigua H?bner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot (Si-Jun?Zheng et al ,2005 ).

In another experiment transgenic Japanese lawngrass plants containing a synthetic cryIA(b) gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter were developed by Agrobacterium-mediated transformation. A total of 1540 embryogenic calluses derived from dormancy-removal mature seeds were co-cultured with the disarmed strain EHA105 harbouring the binary vector pKUB. Three days after co-culture with EHA105 in the dark at 21?C, transient ?-glucuronidase (GUS) expression frequency was 74.2%. After selection with 100?mg/l hygromycin B, a total of over 50 independent resistant cell clones and 25 regenerated plants were obtained. The integration and expression of the cryIA(b) gene into the genome was confirmed in 22 regenerated plants by the GUS histochemical assay, PCR amplification, Southern blotting and Western blotting analysis, with a transformation efficiency of 1.4%. The entire process from callus induction of mature seeds to production of transgenic plantlets was 80?100?days. T1 progeny segregation analysis of these transgenic lines demonstrated that 59.1% of the transgenic events were inherited in a typical Mendelian fashion (Lei Zhang,2007).

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1.PhD student at the field of plant Biotechnology at the National Academy of Science of Republic of Armenia

Ali Parsaeimehr

The potential behavior of plants alkaloids in animal cells vitality

admin — Thu, 08 Oct 2009 16:53:25 +0000

The potential behavior of plants alkaloids in animal cells vitality

Ali Parsaeimehr1

1.PhD student at the field of plant Biotechnology at the National Academy Of science institute of G.S.Davtyan Institute of Hydroponics Problems NAS RA and Academician of young researcher organization.

Abstract: it is clear that Plants, as a renewable source with low energy consumption that can offer complex biochemical syntheses even in medicinal purposes , between the synthesis compounds of plants alkaloids play an important but dilemma role at these aims? , alkaloids for plants known as a secondary metabolite which could by? essential for plants ecological surviving but it s a question remaining are these compound?s as a source for medical uses that it s going to world spread are vital for animals cells or not ?, well through this question a disquisition have been done for clearing some of the answers , we know that cells in both kingdom of plants or animals regulate intra from extra environment well these adjustment? and regulations applies by use of? ions channels? (Na+ ,K? , Ca? , Cl?) or the activity of? Na?/K? pump well we can safely assume that? behavior and the mobility? of ions? guarantied the cells vital. Some of secondary metabolites such as alkaloids in plants, known as the inhibitors of Na+, K?, ATPas or some of plant alkaloids like Harmaline, Nitidine, Capsaicin, Soleonopsin could disrupt these act , the molecular? structure of plant alkaloid with some vital compound?s in cells? and their similarity of theme could be the reason of their serious potential of act as an inhibitor or resonancer in animals cells vitality well Amino acids like Phe, Tyr, Try have Many physiologically active, and have limited distributions in the plant kingdom for example the strychnine alkaloids from the dried seeds of Strychnos vux vomica could be play a deadly way in the vitality of animals Kingdome and according to this acaealepsy we can safely assume there are too many targets for alkaloids acts such as: plasma membrane , ribosome?s or even DNA or RNA in animals cells ,? The other location for alkaloids effects are the neurotransmitters? which closely coupled and band with ion channels (Na+ ?K? , Ca? ? Cl?) as a fact the receptors or ion channels both are incipit for nervous signals ?and as a consequences affected? one of them? by inheritors or resonators? could occasion the vitality of animal cells. It?s amazing that the plant alkaloids even could show allergic sign in the animals cell? which its refers to the immunological responds to the? plants alkaloids? for instance alkaloids such as coumarins, furanocoumarine could act in this importance, the Mechanism of Allelochimical Activities in Antiviral, Antimicrobial and Allelopathic Interactions of alkaloids are the other mysterious role of these kind of secondary metabolites in animals vitality There are circumstantial evidence that some alkaloids such as Quinolizidine alkaloids protect the producing plants against viruses, bacteria , fungi, and other plants , relative to alkaloidesanimal interaction , these modes of action have been less well or hardly at all, a number of antimicrobial alkaloids such as sanguinarine, quinine , or berberine intercalate with viral and microbial DNA to bind it well these compounds may thus inhibit process such as DNA replication and RNA transcription which are vital for the microorganisms or animal cells, Well all in all alkaloids augment which is far from complete shows that many plant alkaloids inhibiter or over stimulate central process at the cellular or organ level. In this completion only a limited of structures have been discussed.

?Key word: plants Alkaloids, animals cell vitality, antimicrobial effects

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1.Introduction: In toadies world use of medicines by the base of plants between the nations and countries have been wide spread and the effect of these kinds of drugs are undeniable but of course it?s better that we show the effect of secondary metabolites compounds and between them ,the alkaloids which synthesizes in plants cells and

shows their dilemmas effect of these kind of secondary metabolites, well according to cells biology and their? metabolites , cell s adjust their intra ?from? extra environment in a caution way , these adjustment? and regulations applies by use of? ions channels? (Na+ ,K? , Ca? , Cl?) or the activity of? Na?/K? pump well we can safely assume that ?behavior and the mobility? of ions ?guarantied the cells vital(Alberts et al.1993). Some of secondary metabolites such as cardiac glycoside in plants, insects and a kind of toads from the family of (Bufonidae) have been discovered, these kinds of secondary metabolites known as the inhibitors of Na+, K?, ATPas or some of plant alkaloids like Harmaline, Nitidine, Capsaicin, Soleonopsin which act as the same way and affect at Na+, K?, ?ATPas or ion channels and as it clearly shown these ions plays a vital role in order to cells vitality or even at the animals nervous system (Mann, J. 1992).in animals cells most of the cells activities such as endo- exocytose, cells division? is by base of microtubules or microfilaments activities in these suit some of Alkaloids such as ??Colchicines, Maytansin, Taxol, Vinca Alkaloids ?have the potential of coupling? with theme and inhibiter their activities due the cells progress , these expanded domain of activity of plants alkaloids in animals cell and the reason of it should by search in their molecular structure.

2. Discussion & Deliberation of Alkaloids potential: alkaloids have been extracted from and found in ~20% of vascular plants, their Compounds usually basic (alkaline) ~40,000 compounds currently described and Structurally the most diverse class of secondary metabolites and most of them use for medicinal usage like Morphine (painkiller),Vincristine (anticancer agent) , Cocaine (anesthetic, drug of abuse), Caffeine (stimulant) , well its seems that the best describe of this kind of secondary metabolites have been done at 1963 through their? chemotaxonomy and by this base they remain in 3 group such as:

Proto alkaloids .3??????? Pseudo alkaloids .2????? ?????Proper alkaloids .1

2.1.1. Proper alkaloid: they also known as true alkaloids we can describe them as the fallow: Basic Nitrogen part of a heterocyclic ring system, Chemically complex, Derived biosynthetically from amino acids, especially the cyclic ,Amino acids: Phe, Tyr, Try, Many physiologically active, and have limited distributions in the plant kingdom for example the strychnine alkaloids from the dried seeds of Strychnos vux vomica. Small tree found from India to

Northern Australia. Fruit is a large berry with a hard coat and pulpy interior containing 3-5 fleshy grey seeds ? contain ~1.2% strychnine, 60mg can kill an adult by the way we can say that the highly potential of it s activity is by the base of it molecular structure( Habermehi, G. 1983, Mann, J. 1992.).

And as we can see strychnine, simply coupled with L-tryptophan one of the most important amino acid?? in cells vitality (Albert?s ET al.1993).

2.2.2. Pseudo alkaloid: Nitrogen containing (physiologically active) compounds not derived from amino acids, the purine ring is gradually elaborated by piecing together small components from primary metabolism for example caffeine the alkaloids which extracted from coffee

2.2.3. Proto alkaloids:? they have?? physiologically active, Nitrogen atom is outside the ring system and this is the reason for nominated them as proto alkaloids well the alkaloids such as ephedrine or colchicines are one of the good examples of these kinds of alkaloids which play medicinally rolls in the industry of drugs. COLCHISINE the copesetic alkaloid of the Colchicum autumnale plant the family of Liliaceae which sustainability band through

Tubulin in a compeer of 1:1 (the Antimitotic potential) and thus inhibits the assembly of microtubules and as a consequence the meiotic spindle of dividing cells disappear suddenly after colchicines treatment and the chromatids no longer separated but whereas the animal cells die under this condition the plant cells become polyploidy? and? leads in breeding programs, using this alkaloid as an anticancer because of its Antimitotic potential widespread but after a while because of its damage to cells skeleton colchisin lay away and another alkaloid by the base of it by the name of colcemide use in this aim well this alkaloids because of their

Lipophilicity potential, simply absorb by cells and its clear why the family of colchicum plants are not attack by herbivores (Teuscher E et al.1995).

2.3. The effect of Alkaloids on Neurotransmitter Receptors: the nerve single transduction in the central nervous system and in neuromuscular junction is mediated by receptor proteins residing in the membrane which directly or indirectly coupled with ion channels the neurotransmitters involve include (Alberts et al.1993). among others adrenalin(NA),serotonin, dopamine, histamine, glycine, GABA, and acetylcholine have been deduced to ion channels ,to many plant alkaloids have the similar molecular structure? with these neurotransmitters for? instance acetylcholine? and histamine in sting hairy roots of Urtica or serotonin and dopamine in several species, and the acts can be:

I. The receptor itself through inhibition or overstimulation

II.The enzymes that deactivate neurotransmitters after they have bound to a receptor

III.Transport process, which are important for storage of the neurotransmitters in synaptic

IV.Enzymes involved in the biosynthesis of a neurotransmitter (Mothes, K.et al.1985, Robinson, T. A. 1981).

Table I. some of the alkaloids as inhibitors of Neurotransmitters

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Enzyme????????????????????????????????? Natural Substrate?????????????????????????? ?????????Alkaloid???????????????? ?????occurrence (plant)

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?? Acetylcholine esterase ??????????????????????????Acetylcholine??????????????????????????? Physostigmine (eserine)??? Physostigma veneosum?????????????????????????

??????????????????????????????????????????????????????????????????????????????????????????????????????? Berberine????????????????????? ?????several papaveracea

?????????????????????????????????????????????????????????????????? ?????????????????????????????????????????????????Coptisine?????????????????????? ????several papaveracea

???????????????????????????????????????????????????????????????????????????????????????????????????? ???????????????Galanthamine??????????????? ???several Amaryllidaceae

????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????Chaconine?????????????????????? ?Solanum

?????????????????????????????????????????????????????????????????????????????? ?????????????????????????????????????Solanine???????????????????????? ??Solanum

???????????????????????????????????????????????????????????????????????????????????????????????????????? ???????????Solanidine??????? ????????????????Solanum

????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????Huperzine A???????????????? ????Huperzia srrata

?? Monoamine oxidase????????????????????????? ???NA. dopamine, Serotonin??????????? Harmaline???????????????? ???????Pegamum

????????????????????????????????????????????????????????????????????? histamine?????????????????????????????? Harmine?????????????????????????? Peganum

???????????????????????????????????????????????????????????????????????????????????????? ???????????????????????????Ephedrine??????????????????????? Ephedra

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The other location for alkaloids effects are the neurotransmitters? which closely coupled and band with ion channels (Na+ ?K? , Ca? ? Cl?) as a fact the receptors or ion channels both are incipit for nervous signals? and as a consequences affected? one of them? by inheritors or resonators? could occasion the vitality of animal cells (Rosenthal, et al.1992).

All animals need to transport nutrients, hormones, ions, signals compounds, O2 and CO2 between the different organs of the body well this achieved in higher animals through blood in the circulatory system.Inhibitorrs of its motor, the heart ,were discussed earlier . But the synthesize of red blood cells is also vulnerable and can inhibited by antimiotic alkaloids , such as vinblastin or colchisin and some allelochemicals have hemolytic properties, such as saponins and steroidal alkaloids , these compounds complex membrane sterls and make cells leaky these effected consequently effected the vitality of animal cells(Teuscher E. and Lindequist, U. 1994).

Table II .The Alkaloids which known as inheritors or resonators for nervous signal transmission

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?????????????????? Receptor?s?? ?????????????????????????Ligand???? ???????????????????????????Alkaloids???????????? ???????????????Occurrence(plant)

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??? ???Acetylcholine receptor ???????????????Acetylcholine???????????????? ????????????Nicotine????????????????????? ??????Nicotinana, many other plants

???????? ??????????????????????????????????????????????????????????????????????????????????????????????????C-toxiferine??????????????? ????????strychnos

?????????? ????????????????????????????????????????????????????????????????????????????????????????????????Tubocurarine?????? ??????????????Chondodendron

? ?????????????????????????????????????????????????????????????????????????????????????????????????????????Cytosine and other QA??? ?Several legumes

???????? ??????????????????????????????????????????????????????????????????????????????????????????????????Lobeline???????????????????? ????????Lobelia

????????? ?????????????????????????????????????????????????????????????????????????????????????????????????Anabasine???????????????? ????????Anabasis , Nicotiana

????????? ????????????????????????????????????????????????????????????????????????????????????????????????Hyoscyamine (atropine)???? Atropa, Hysoscyamus, Datura.

????????? ????????????????????????????????????????????????????????????????????????????????????????????????Scopolamine???????????????????? ??Several Solanaceae

????????? ????????????????????????????????????????????????????????????????????????????????????????????????Arecoline????????????????????????????? Areca

????? Adrenergic receptors ????????Noradrenaline/adrenaline???????????? ????Norlaudanosoline?????????????? ??Papaveraceae

???????? ?????????????????????????????????????????????????????????????????????????????????????????????????Ephedrine, norephedrine? ???Ephedra

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Its amazing that the plant alkaloids even could show allergic sign in the animals cell? which its refers to the immunological responds to the? plants alkaloids? for instance alkaloids such as coumarins, furanocoumarine, hypercin , helenalin , Activation or repression of the immune response are certainly targets that were selected during evaluation as an antiherbivore strategy(Luckner et.al.1990).

2.4. Mechanism of Allelochimical Activities in Antiviral, Antimicrobial and Allelopathic Interactions of alkaloids:

There are circumstantial evidence that some alkaloids such as Quinolizidine alkaloids that protect the producing plants against viruses, bacteria , fungi, and other plants , relative to alkaloids animal interaction , these modes of action have been less well or hardly at all, a number of antimicrobial alkaloids such as sanguinarine, quinine , or berberine intercalate with viral and microbial DNA to bind it well these compounds may thus inhibit process such as DNA replication and RNA transcription which are vital for the microorganisms, protein biosynthesis in ribosome?s is another vulnerable target for plant alkaloids even the stability of biomembranes can be disrobed by steroidal alkaloids(Luckner et.al.1990).

? In another view even herbicidal properties or germination inhibition which can be observed in plant-plant interactions, can also proceed via the above mentioned mechanisms but interaction with growth hormones and their metabolites also must be considered (Wagner, H. 1993).

3. Counclusion:

Plants, as a renewable source with low energy consumption that can offer complex biochemical syntheses, will be even more compatible in the future. Well all in all alkaloids augment which is far from complete shows that many plant alkaloids inhibiter or over stimulate central process at the cellular or organ level. in this completion only a limited of structures have been discussed .In many instance , plants contain mixture of related alkaloids , which only differ for particular substitution patterns well this allelochemical properties are requisite for chemical defense compound in an ecological context? for surviving of plants but also constitute the base for their exploitation in medicine or agriculture, well after all of these dilemma situation we should use medicines by the source of plants in a secure and caution way ??but to many experiments demands through this complicated way.

4. References:????????????????

Alberts, B. D., Lewis, J. Raff, M., Roberts, K. and Watson, J. D. 1993. .Molecular Biology of the Cell.3rd ed . Garland. New York. Habermehi, G. 1983. Gifttiere and ihre Waffen. Springer. Berlin. Harborne, J. B. 1993. introduction to Eclogical Biochemistry.4 th ed. Academy press. san Diego. Luckner, M. 1990 Secondery Metabolism in Microorganism.Plants and Animals .Springer, Berlin. Luckner, M. 1990. Secondary Metabolism in Microorganisms, Plants and Animal, Springer, Berlin mann,J..1992, Murder,Magic and Medicine.Oxford University Press. London. Mann, J. 1992. Murder Magic and medicine.Oxfordr University press .London. Mothes, K., Schutte, H. R. and Luckner, M. 1985. Biochemistery of Alkaloides .VCH. Weinheim. Robinson, T. A. 1981. The Biochemistry of alkaloids, Springer. Berlin. Rosenthal, G. A. and Berenbaum, M. R. 1992. Herbivores: Theair Interaction with Secondary Plant Metabolites vol.Academic Press, San Diego. Teuscher E. and Lindequist, U. 1994. Biogene Gifte. Fischer. Stuttgart. Urk H, Schipper D, Breedveld GJ, Paul RM, Scheffers WA, van Dijken JP (1989) Biochim Biophys Acta 992: 78 Scopes RK (1989) In: van Unden N (ed), Alcohol toxicity in yeast and bacteria, CRC Press Inc, p. 89 Wagner, H. 1993. Pharmazeutische Biologie . 2. Drogen and ihre Inhalisstoffe, Fisher, Stuttgart. Zhou, G.M. 1980. Studies on useful compounds of Bai-Zhi for healing Yin- Hsieh Ping. Chung-Chen-Yau Res. 4: 33. (In Chinese). Zhou, G.M., C.G. Yu, Y.C. Han, and C.T. Mun. 1988. Studies on Bai-Zhi. IV. The toxicity test of useful compounds. Med. J. China Hospital 8: 220-221. (In Chinese).

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