The study deals with recombinant manipulations of small plants gooseberry (phyllanthus emblica ) which are helpful in production of food. More explicitly, it affects fruits, vegetables and seeds with improved sugariness formed by transgenic plants which displays controlled appearance of genes programming sweet-tasting proteins, as well as the monellin genes (Ogata, C., et al 1987)
Introducing the monellin gene (Dioscoreophyllum cumminsii) into the callus of gooseberry by means of agrobacterium mediated gene transfer. By means of this process the gooseberry can be changed from sour to sugary taste.
Ultimately the sour suppression progresses the taste and essence content into fresh and new gooseberry plant.
Rationale:
This project is all about to apply the gooseberry as medicine plant and it has lot of useful stuffs and vitamin C is a major role in gooseberry. As per medical records the gooseberry is helping to cure the several diseases cancer, Thyroid problems, paralysis, Diabetic, blood circulation, Anemia and high blood pressure. By improving the flavour and sweetness into gooseberry, people are willing to take it regularly and their health is in good condition without any medicine (AK Roye, 1991).
Introduction
In recent years, the amazing development is appeared in the transgenic plant by genetic engineering. The new genetic material is inserted into the plant and that new genes are entirely diverse species from the plant. The new genetic enhancement is termed as transgenic (Hails., 2000 and Goodman et al., 1987).
The first genetically modified plant is tomato in 1992 and the U.S Food and Drug Administration has announce that genetic modification plant is new achievement in the world and it�s not endanger us (Larissa Parsley., 2011).
Now day�s biotechnologist are developing edible vaccines food to defend against disease by genetic engineering technology. These methods are very helpful for disease control than usual immunization and it�s not expensive. (Larissa Parsley., 2011)
The plant transformation steps are construction of vector take specific genetic material and transfer into the host plant with help of promoter. Agrobacterium tumefaciens is a pathogen of plant and segment of DNA (T-DNA) infect plant by Ti plasmid. The particular genetic material is carried out from the bacteria to include into the Gooseberry plant. Gel electrophoresis and Polymerase chain reaction technique is to determine the success of specific modified gene transformation. (Lorence and Verpoorte., 2004)
Gooseberry:
Gooseberry has plentiful source of vitamin C and is equivalent to 20 times of orange. Which is growing at cariable agro climatic and soil conditions. The gooseberry can be abided at 45�C and freezing too.
The Gooseberry has approximately 80% of water as well as protein. (Vitamines minerals, fiber, minerals, majority include calcium, phosphorus, carotene, iron, Vitamin C and B complex and also has gallic acid and ascorbic acid (AK Roye, 1991)
The Indian gooseberry (Phyllanthus emblica, syn. Emblica officinalis) is a determined tree of the Euphorbiaceae family. The size of the tree is 8 to 18 m in height, it has a curved trunk and widely spreaded brachess. The branchlets are finely pubescent, sized about 10�20 cm in length, and are mostly deciduous; the leaves are easy, subsetting beside with the branchlets, light green, resembling pinnate leaves. Greenish � yellow are the colour of the flowers normally.
Indian gooseberries is also known as Aonla, Nelli, Amla, Amlika, Dhotri, Emblica and Usuri. Aonla (Phyllanthus emblica L.) belongs to the family Euphorbiceae is one of the important small fruit crops of our country. In the recent cultivation of advanced varieties of aonla is highly remunerative.
The flavour of Indian gooseberry is sour, bitter and astringent, and is very rich in fibre. Indian gooseberries are liked to be eaten when soaked in turmeric water and salt because it suppresses the sourness flavour in the fruit.
REGION OF CULTIVATION
Since ancient times, In India, Aonla is under cultivation, also indigenous to tropical South-east Asia, particularly central and southern India .The cultivation is very common in the eastern districts of Uttar Pradesh particularly Pratapgarh and Varanasi. It is also grown in states like Haryana, Himachal Pradesh, Maharashtra and some parts of Karnataka (Firminger, 1947).
IMPORTANCE OF INDIAN GOOSBERRIES:
� It is also used as a very good conditioner and straightener for human hair.
� Resists body heat and used as a cooling agent.
� Used as a medication for treatment of biles n phlegm.
� Increase in the production of semen and help in urinary and gynecological problems
� Redution of fat in the body and also clear vision.
It is one of the most nutritious and is a very richest source of vitamin C (400-1300 mg/100 g) amongst other the fruits like Barbados cherry as per medical records. It is also the richest source of pectin which is mostly useful in making jam and jellies
MONELLIN
Monellin contain sweet protein of two noncovalently linked polypeptide chains, an A chain of 44 amino acid residues and a B chain of 50 amino acid residues. The protein can be purified from the fruit of Dioscoreophyllum cumminsii grown in West Africa. Monellin is roughly 100,000 times sweeter than sugar on a molar basis and numerous thousand times sweeter on a weight basis. A Single-chain monellin (SCM) is an engineered 94-residue polypeptide.
The invention is directed to plants that are regenerated from or containing these cells, to edible portions of these plants, and to foodstuffs prepared from them. Thus the invention is therefore converting the methods to produce fruits, seeds and vegetables with enhanced sweetness wherein,the procedure comprises cultivation of the transgenic plants of the invention followed by recovery of the desired edible portions.
Overconsumption of sugar always leads to many health related issues. Several attempts are progressed to substitute low caloric sweetners for sugar. But due to its side effects, it is prohibited recently. Currently the presence of natural sweeteners with low calorie are identified from several plants. Advances associated with molecular biology enables the incorporation of structural genes that codes for specific protein to the host cell of the microbes for the expression of that particular protein molecule (Schestibratov and Dolgov., 2005).
Methods and Materials:
This methods covers to regenerate a new gooseberry plant with sweet gene. The first step is to isolate the sweet gene (monellin) from Dioscoreophyllum cumminsii and inserted into the invitro callus of gooseberry
Isolation of monellin:
The twigs of berries were taken from the plant of Dioscoreophyllum cumminsii Diels from Ghana in West Africa. It is deionized with water twice. This extraction method was stored at 15 degree C.
Dry (NH 4 ) 2 SO 4 added for basic extract to 40% saturation and centrifuge it. We can observe the solid materials were settled and supernatant. The yellow supernatant will show at 60% saturation for further analysis. The precipitate solution is melt in buffer phosphate. DEAE cellulose is detect the sweetness. (Robert C. H et al. 1976)
Synthetic Monellin Gene:
A single chain protein of amino acid sequence is obtain by direct fusion of B and A chain and determined by synthetic DNA sequence constructed. A synthetic gene is arranged from oligomers (Ncol at 5� end and Sall at 3� end). Oligomers was isolated and purified by sep-pak c18 column with reaction mixture to find the synthetic gene. (Robert C. H et al. 1976)
Synthetic monellin DNA fragment was set up with combination of XbalSall using M13mp19FR and incubated at 4�C overnight to provide M13mp19 MON-1RF. By adding 55 �l of the ligation mixture to 200 �l of E. coli JM101 competent cells in Enzymology into the hosts, the sequence is verified by dideoxy sequencing. (Robert C. H et al. 1976)
Monellin gene expression
E8 promoter was isolated from the pE8mutRN2.0 by cleave Ncol filled with DNA polymerase and plasmid was absorbed with EcoRI. Then ligated plasmid has pass on into E.coli HB101 and DNA isolated. pE8mon, enclosed the E8 5' regulatory sequences. Finally E8 monellin introduced into pMLJ1 and released with EcoRI. Agrobacterium nopaline terminate the sequence and ATG begin the translation with combined monellin gene. (Robert C. H et al. 1976)
Incorporation of Monellin Gene Vectors
Taken plasmid of pMLJ1 and monellin gene to improve by vector under the control of promoter pGV3850 and were allow to clone in plasmid. A.tumefaciens have the cointegrative plant transformation vector pGV3850 and E coli straing of pGJ23 as a helper. Finally the cointegration of construction happened into the pGV3850 and to incorporated with fused the monellin gene into the genome of gooseberry. (Zambryski et al., EMBO J (1983) and Tommaso et al., 1992 and (Robert C. H et al. 1976)
Agrobactrium Gene transform:
The Agrobacterium mediated gene transfer is not pact with protoplast, its effectiveness transformation of DNA. Agrobacterium tumefaciens T-DNA transfer process involves necessary steps. 1.Bacterial colonization is the first step when A. Tumefaciens attached to plant cell surface. 2.induction of bacterial virulence system is next step of bacterial colonization and The T-DNA transfer is mediated by 30-40 kb vir region of the Ti plasmid. The Vir region is togethered by important 6 operons (vir A, vir B, vir C, vir D, vir E, virG ) and two non-essential (virF, virH). (Nixon, 1986, Iuchi, 1993)., (3)generation of T-DNA transfer complex and Vir genes produce the ss molecules if any of molecules put between the T-DNA borders and it is transferring to the plant cell (4) T-DNA transfer and its plays a transferring vehicle to the plant. (5)integration of T-DNA into plant genome
Transformation of Gooseberry with fused monellin Gene construction:
Plasmid of pMLJ1 promoter E8-monellin was taken for DNA transfer of fused monellin gene into gooseberry. Neomycin phosphotransferase gene is capable of present kanamycin resistance in transgenic plants. When set up the host plant for further transformation. The callus is transmitted to feeder plates and cocultivated by the bacteria. Finally it is placed for regeneration medium. The callus is transmitted to medium to stimulate root formation.
Shoot and root procedure
The callus will be clearly visualized within 8 to 10 days, After three weeks, the callus was transmitted to shooting medium (except the concentration of zeating is condensed at .1 mgml), meanwhile transfer the callus to fresh plates in every weeks.
Transplantation
Remove the callus gently from agar with help of spatula and transferred into soil. The callus rinsed by water to take away much of agar. The callus place into GA7 boxes and opened it after several days and watered it half of strength Hoagland�s solution. After 15 days they moved into greenhouse.
Finally the transgenic gooseberry has improved the sweet taste because of integrated monellin gene.
The callus of gooseberry is transmitted to soil
Preparation and explants of Gooseberry
phyllanthus emblica is grow in short period of days. The flower bud of gooseberry 5mm long and tender stems with 2mm diameter has taken from gooseberry plant. The stem and bud are set aside in Petridis for the preparation of callus. The stem and bud surface is purified with alcohol (70%) and 0.1 mercuric chloride solution for few minutes. Wash the stem with distilled water for few more minutes and the removal filament appeared. (Trehan., 1996). The explants was transferred to bacterial suspension for 30 minutes. The bacterial cells are crop in various growth stages. This bacterial suspension and explants are incubated in room temperature for infection process. Now transfer the explants to new petri dish to remove the unwanted bacteria (P.C. Kutty et al. 2011)
Take 15 ml of co cultivation MS medium (Murashige &Skoog�s medium) in a tube and inoculate anthers with stem. The stem was sterilized for 20 minutes at 1.06 kgsq cm. This MS medium provided as basal medium and including 2 % of sucrose and 0.8 % of agar and equipped with IAA NAA, 2,4-dichlorophenoxyacetic acid(2,4-D) 0.5mg�l-1 and benzyladenine (BA) 2.0mg%. This trial was done two times and culture must be maintained at 25� C for 48 hours in chamber. After 2 days the wash with sterile distilled water to remove excessive agar (sehgal, c.b. and khurana, s., 1985). After A subcultrable callus was examined after 6 weeks and carried out into the new MS supplement medium containing a mixture of TDZ;005, IAA, NAA and minerals. Adjust the ph to 5.8 before continue with autoclave. This culture requires to maintain at 15� C in dark for 6 days in the growth chamber. Callus was visualized on the medium after 60 days of period. (Cortina et al., 2004)
Transformant regeneration is proceeded by 1.explant�s cell- appearance of transformance 1st via the 2nd month of selection 2 by intermediate callus stage- 3rd via the 4th month of cultivation on the selected medium. Transformation is confirmed by the expression of induced enzyme expression. Susceptibility of explants is indicated by the expression of GUS activity at cut ends (Tattersall et al., 1997)
GUS histachemical analysis were taken on explants after 2 days to verify the successful treatment
RESULT:
This experiment
The expected result by carring out the incorporation of thaumatin gene into the in vitro developing callus of bitter melon, induce the production of bitter-free bitter melon with improved flavour content. Improper incorporation or failure of this gene transfer does not induce adverse effects to health and also to environment as the transfer mechanism are carried out only between plant genomes.
OUTCOMES AND CONCLUSION:
The understanding of this project is how they callus recognize the gene and they control. The successful gene transformation and continuoes growth of callus. The GUS is very important for the transformation protocol in plants. effectiveness of T DNA expression are estimated via GUS expression to know the exact genetic transformation mediated by A. Tumefaciens. Recently proved that the high concentration of A. Tumefaciens are capable for transforming plant. Bacterial cells are taken by the high transient high GUS activity. Sometime undiluted culture caused the problem of overgrowth of bacteria. The sweetness will be destroyed if the native monellin is heat at 90 � C and pH or 50 � C and then cooled. The chain is separated while heating of protein and not able to reform into conformation (Kim et al., 1993)
By this methods, the people are uptake the fruits regularly. If this method get failure and it�s not harm to any one and society as the proteins and genetic materials are relies on the plant genome. US Food and Drug Administration and ethical issues are not too cruel in this transformation.
Additional work also carried out in this modification by improving other transformation and incorporation with several disease.
Ethics
Before a genetically modified product can be released onto the public market, it must pass a test of �substantial equivalence,� a term that refers to the relative safety of the new product as compared to its traditional counterpart. If the new product is found to be as safe as or safer than the traditional product, then it is declared to be �substantially equivalent� and can be commercialized without any special regulation. For the consumer whose main concerns about GMPs lie in safety and health, this equivalence test may provide some comfort.
However, most consumers have doubts about GMPs on a less easily defined level. In addition to the substantial equivalence test, companies and regulatory agencies consider three main criteria when evaluating a new genetically modified product: efficacy, quality, and safety. It has been often noted that a fourth criterion unofficially exists: morality. Ironically, this criterion, more so than the other three combined, often dictates what action the company or agency will take with regards to marketing the product.
Many consumers are hesitant to support a science that they believe is �playing God� with nature. Other consumers, either for religious or personal reasons, may want to avoid eating plants which contain bacterial, animal, or human genes and believe that such plants should not be allowed to enter the human food chain.
Although many consumers do make informed decisions concerning GMPs from a health, economic, or even an ethical standpoint, at times a lack of familiarity with the process of genetic modifications and its affects leads to a suspicion which makes an objective decision difficult. Consumers� doubts about GMPs may simply arise from a fear of the unknown and a distrust of the scientific community which does not always clearly communicate its objectives and methods to society.
So, what will it take to alleviate health and safety worries in the minds of the consumers? One possible solution, which is, ironically, a new controversy in itself, is being explored.
Hails, R.S. (2000), Genetically modified plants � the debate continues. Trends in Ecology & Evolution, 15, 14-18.
Goodman, R.M., Hauptli, H., Crossway, A. and Kn, V.C. (1987), Gene transfer in crop improvement. Science, 236, 48-54.
Larissa Parsley. (2011), Transgenic Plants: A Budding Controversy Stems from Consumer Concerns 21, 4
Lorence, A. and Verpoorte, R. (2004), Gene Transfer and Expression in Plants. Recombinant Gene Expression Methods in Molecular Biology, 267, 329-350.
Nixon, B.T., Ronson, C.W. and Ausubel, F.M. (1986). Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proceedings of National Academy of Sciences USA 83: 7850-7854.
If possible try to make this paragraph into ur own sentence, but should not come the 3 same words continuesly...
.Recombinant plasmid and E.coli cells:
E.coli cells with plasmid pUR520 pUR 530, pUR 540 between the mature thaumatin gene and regulon with approprate reading frame and direction are allow to grow in a appropriate condition along with the anti-biotic presence to enhance selective pressure. Under these state, either of these plasmid will produce significant quantity of mature thaumatin.
Later, the thaumatin protein�s presence is clearly identified using S.D,S electrophoresis and ELISA and also by several physiological tests over its sweetness. E.coli cell strains K12(294) enclosing plasmids are taken for further process.
Ethics
In Hinduism, amla is regarded as a sacred tree attributed to Lakshmi http://webapps.uni-koeln.de/cgi-bin/tamil/recherche.
Kutty, P.C., G.K.A. Parveez and F. Huyop, 2010. An easy method for Agrobacterium tumefaciens-mediated gene transfer to Nicotiana tabacum cv. TAPM26. J. Bioliogy Science, 10: 480-489.
Ogata, C., et al.(1987). Nature 328, 739-742
Roye, A. K., Dhir, H., and Sharm. A. (1991). Pharmaceutical Biology
Robert C. H., Morley K. R. And James M. A. Monellin, a sweet polypeptide derived from fruit of dioscoreophyllum cumminsii.United States Patent. 3998798.
Zambryski, P., Joos, H., Genetello, C., Leemans, J. M., Montagu, V and Schell, J. 1983, EMBO J. 2(12): 2143�2150
Tommaso, E., Mugnozza, S., Porceddu, E., M. A. Pagnotta, M.A., Gian,C.(1992), Genetics and Breeding for Crop Quality and Resistance: Proceedings of the XV., 23, 166.
Kim, S, H. Cho, J. M. 1993. Single-chain monellin analog as a low calorie protein sweetner United State Patent. 5264558
