Breeding and Biotechnological Approaches for Quality Improvement In Cr

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Breeding and Biotechnological Approaches for Quality Improvement In Crop Plants : 

AK Chhabra &Jitender 2009A48M Breeding and Biotechnological Approaches for Quality Improvement In Crop Plants

Introduction : 

Introduction Designer crop : possess specific characters specified before their development eg. Specific protein, fat or starch quality , vitamin content, elimination of antinutritional factor, colour, flavour, taste etc.of grain/fruit; keeping quality or produce specific novel biochemical or pharmaceutical/industrial value. First transgenic variety: FlavrSavr of Tomato Showed delayed fruit softening.

Considerations for designer crops : 

Considerations for designer crops Modification of starch quality Modification oil quality Modification protein quality Suppression of endogenous genes Male sterility Biochemical production plant derived vaccines Problems in gene transfer Safety regulations for transgenic plants

1. Modification of starch quality : 

1. Modification of starch quality

Applications of modified forms of starch : 

Applications of modified forms of starch

Starch Modification by Genetic Engineering : 

Starch Modification by Genetic Engineering Reduces cost, effort and environmental damage. Offers possibilities for creation of novel starch with novel properties. Can be achieved by modifying the level and properties of enzymes of starch biosynthesis.

It should be kept in mind : 

It should be kept in mind

Genes encoding enzymes of starch biosynthesis and used for production of transgenic lines : 

Genes encoding enzymes of starch biosynthesis and used for production of transgenic lines

Starch yield : 

Starch yield

Starch quality modifying enzymes : 

Starch quality modifying enzymes Enzyme for amylose biosynthesis. Suppression of GBSS I activity in potato (by antisense RNA technology) yielded an amylose free or “Waxy” starch in potato in 1991. GBSS I

Starch quality modifying enzymes : 

Starch quality modifying enzymes Main enzyme of amylopectin biosynthesis; it has two isoforms SSS I and SSS II , Down-regulated activity of both enzymes in potato by antisense RNA technology resulted in shift from longer chain to shorter chain in amylopectin. A freeze –thaw stable potato has been created by simultaneous regulation of all the three starch enzymes viz., GBSS I and SSS I and SSS II genes by antisense RNA technology SSSs

Slide 12: 

This manipulation yielded an amylose free short chain amylopectin. The use of this starch has potential for environment and consumer benefits as its production requires no chemical modification.

Starch quality modifying enzymes : 

Starch quality modifying enzymes Activity required for amylopectin biosynthesis. An increased SBE activity can increase branching while its suppression can reduce amylopectin. In potato SBE A and SBE B activities were suppressed to 1% of the wild type by using antisense construct of the genes encoding them. This resulted in very high amylose starch containing an insignificant level of highly branched amylopectin. This unique starch (high amylose, low amylopectin, and high phosphorous levels) has novel food and industrial applications. SBEs

Starch quality modifying enzymes : 

Starch quality modifying enzymes These enzymes reduce the number and size of branches of amylopectin molecules. The α-amylose type SDBEs reduce the branch size, while pullulnaase type SDBEs reduce number of branches. A suitable combination of both enzyme activities may be used to produce starch molecules of specified branch number and size. SDBEs

2. Modification of oil quality : 

2. Modification of oil quality Oil quality may be defined as types and proportion of different fatty acids present in given oil, which determine the use to which this oil can be put to. The properties of fatty acid depend on its chain length and number of double bonds present in its carbon chain.

Enzymes of fatty acid biosynthesis; targets for genetic modification : 

Enzymes of fatty acid biosynthesis; targets for genetic modification

Strategies used for modification : 

Strategies used for modification Introduction of novel enzyme e.g., an acyl transferase or acyl-ACP thioesterase, acyl-ACP desaturase. Suppression of an enzyme activity. e.g., α- acyl-ACP desaturase. Site directed mutagenesis to alter the specificity of an enzyme e.g., α- acyl-ACP desaturase. Creation of hybrid genes to generate novel enzyme activities

Examples of fatty acid modification by genetic engineering : 

Examples of fatty acid modification by genetic engineering

Modification of Seed Protein Quality : 

Modification of Seed Protein Quality Cereal seed proteins are deficient in lysine Pulses are deficient in sulphur containing amino acids e.g., methionine and tryptophan. This limits the nutritional value for man since these two amino acids are essential for man

Approaches to Improve Storage Protein Quality : 

Approaches to Improve Storage Protein Quality

Introduction of Appropriate Transgene : 

Introduction of Appropriate Transgene A new gene encoding a storage protein (rich in deficient amino acid) is introduced in the crop to correct its amino acid deficiency. The transgene is linked to a seed-specific promoter to ensure its expression only in seeds.

Slide 22: 

e.g. in pea vicilin is major seed storage protein ; it contains 7% lysine but no sulphur containing amino acids (methionine and cystine). In sunflower seed storage protein sunflower albumin 8 (SFA8), contains 235 methionine plus cystine. This gene has been isolated. The presence of introns in transgene construct enhances expression of SFA8. The SFA8 gene has been fused with viciln gene promoter. This promoter results in correct development and tissue (seeds) specific expression and accumulation of the protein in seeds of transgenic tobacco.

Slide 23: 

Attempts are being made to transfer vicilin gene promoter SFA8 gene construct into pea. Estimates are made that accumulation in pea seeds of SFA8 to a level of 4% of extractable seed proteins would result in a 40% increase in sulphur containing amino acids content of pea seed protein.

Slide 24: 

The 2S seed protein of Amaranthus is rich in methionine. Gene ama I has been transferred in potato, and the protein Ama I was found to constitute about 0.3% of the total soluble protein. The Braazil nut (Bertholittia excelsa) seed protein, 2S albumin, is rich in methionine , but transgenic soyabean expressing this protein n was found to be allergenic . Soyabean gene encoding glycinin has bean transferred into rice to increase its protein and lysine contents, and digestibility.

Modification of Endogenous Gene : 

Modification of Endogenous Gene This approach is based on isolation and modification of concerned protein encoding gene sequence by:-

Slide 26: 

e.g., prolamine storage proteins ,e.g. zein ,of cereals are deficient in essential amino acids lysine and tryptophan. Single lysine replacements in the N-terminal coding sequence as well as within and b/w the peptide repeats, and double lysine replacement constructs of prolamine genes have been prepared. In addition, short oligonucliotides encoding lysine and tryptophan –rich peptides were separately several different points in the coding sequence. These constructs were shown to express well in xenopus oocytes and their polypeptides were able to form normal aggregate of protein bodies. Further , high level of mRNA transcripts were produced from these chimaeric constructs in transgenic petunia and tobacco seeds, butein protein were barley detectable.

Slide 27: 

However , both normal and lysine-containing zein polypeptides were found to be unstable in these plants. It is possible that beta, gamma and possibly zeta –zeins are required for alpha –zeins to form protein bodies, or it may be that an accumulation of alpha –zeins is deleterious to dicot cotyledonary cells. Major problems of this approach are as follows: The maintenance of open reading frames in the genes Stability of the resultant protein .

Some successful examples : 

Some successful examples In recent study, the 7S legume seed storage protein, beta-phaseolin, gene promoter was transferred in rice. Transgenic rice plants expressed the gene in their endosperm, and some plants showed upto 4% of their total protein to be beta-phaseolin. The 11S legumin protein gene driven by gt1 promoter has also been transferred and expressed in rice endosperm. The CP3-5 gene was coupled with seed-specific promoters and transferred into maize; the gene was expressed by tissue culture cells. Rice gt1 gene encodes the major rice seed storage protein. It has been modified to encode higher levels of lysine, tryptophan and methionine . The modified gt1 gene driven by its own promoter, was transferred into rice protoplsts; the resulting transgenic rice plants expressed the modified gene in their developing endosperm.

Golden Rice : 

Golden Rice Vitamin A-deficiency is estimated to cause deaths of one million children every year, and as many as 230 million children are at the risk of clinical or subclinical vit.A-deficiency.in addition vit.A-deficiency is the single most important cause of blindness among children in developing countries; it afflicts 500,000 children/year. Vit.A-deficiency often occurs where rice is the staple food, since rice grain does not contain provitamin A,i.e., beta carotene. Rice endosperm, however does synthesize geranyl -2 pyrophosphate, which can be converted into beta –carotene by the following four enzymes activities: Phytoene synthase phytoene desaturase - carotene desaturase Lycopene cyclases. The transgenes providing these enzymes activities were transferred into rice using Agrobacterium mediated transformation .

Slide 30: 

Table: Enzyme activities provided by transgene for β- carotene production and to generate high iron rice

Slide 31: 

The resulting transgenic rice, popularly called ‘golden rice’ contains good quantities of beta –carotene, which gives the grains a golden colour. In one transgenic line, the beta –carotene content was as high as 85% of the total carotenoids present in the grain. Iron-deficiency anaemia is the most common nutritional disorder in the world; it is mainly caused by inadequate dietary iron intake . Fe –deficiency affects an estimated 3.7 billion people of which about two billion are anemic; a vast majority of these are women . Rice grain has the lowest iron content among crops; it also has phytate, which reduces iron absorption in human intestine by up to 98%. In addition , iron absorption from a vegetarian diet is rather poor. Phytate is used for phosphate storage in seeds, and it is used during seed germination.

Slide 32: 

1.A sink for iron was created in rice endosperm by expressing a ferritin gene phaseolus., this led to a 2.5-fold increase in the endosperm iron content. 2.Iron absorption from the rice grain was sought to be enhanced by increasing cystein content of the endosperm because cystein –rich polypeptides are known to enhance iron absorption by human intestine. A metallothionine-like gene from oryza was expressed in rice endosperm; this resulted in a 7-fold increase in the cystein content. 3.Iron absorption was sought to be improved by removing phytate by expressing a mutant gene encoding a thermostable phytase; the gene was obtained from aspergilluus fumigatus. One transgenic line expressed phytase at a level 700-fold higher than that in the nontransgenic control. This mutant phytase refolds to80%after 20 min at 100 C. The enzyme is excreted into the extracellular space so that it digests phytate only after the rice grains are cooked; this ensures that the ectopic phytase does not interfere with seed germination.

Slide 33: 

The high iron rice was produced by resolving these problems as fallows; A high iron- high provitamin A rice was produced by crossing the golden rice and high iron transgenic lines of rice. It is proposed to distribute this rice line free to subsistence farmer of the developing world with a view to alleviate vitamin A and iron deficiencies. A high beta carotene producing transgenic tomato has been developed by increasing its destruction activity. This was achieved by introducing into it a bacterial phytoene desaturase ζcarotene desaturase gene. There was a three-fold increase in beta – carotene content, although the total amount of carotenoids was somewhat reduced. Unexpectedly, other enzymes of carotenoid biosynthesis, especially the endogenous zeta-carotene desaturase and the lycopene β-cyclase , were upregulated as evidenced by elevated trancript levels.

Slide 34: 

Suppression of endogenous gene expression

Slide 35: 

Antisense was used to describe inhibition of mRNA translation by hybridization of an oligonucleotide to a selected region of the mRNA. Since mRNA represent the sense sequence, the oligonucleotide complementary to the mRNA was called antisense oligonucleotide. In any gene , the DNA strand oriented as3’→5’ in relation to a promoter is transcribed; this strand is called the antisense strand . The mRNA base sequence, therefore, is complementary to that of the antisense strand. The remaining DNA strand of the gene, called sense strand, in naturally complementary to the antisense strand of the gene. The base sequence of sense strand of a gene is the same as that the mRNA produced by it. Hence, the hnRNA/mRNA produced by a gene in normal orientation is known as sense RNA. Antisense gene approach

Slide 37: 

An antisense gene is produced by inverting, i.e., reverting the orientation of ,the protein encoding region of a gene in relation to its promoter. As a result, the natural sense strand of the gene becomes oriented in the 3’→5’ direction with reference to its promoter and is transcribed. The RNA produced by this gene has the same sequence as the antisense strand of the normal gene ,and is therefore known as antisense mRNA, or sometimes ,as RNA. When an antisense gene is present in the same nucleus as the normal endogenous gene, transcription of the two genes yields and antisense RNA transcripts, respectively. Since the sense and the antisense RNAs are complementary to each other , they would pair to produce double-stranded RNA molecule.

Slide 38: 

In tomato enzymes poly galacturonase degrades pectin which is the major component of fruit cell wall. This leads to the softening of fruit and a deterioration in fruit quality. Transgenic tomatoes have been produced , which contain antisense construct of the gene encoding PG. These transgenics shows a drastically reduced expression of PG and markedly slower fruit softening; these tomatoes have about 2 weeks longer shelf-life than normal tomatoes. Such tomatoes were approved for marketing in U.S.A. under the name FlavrSavr. Slow Fruit Softening Tomato

Slide 39: 

The enzymes stearoyl-ACP desaturase catalyzes the conversion of steroyal-ACP to oleoyl-ACP, which is the first desaturation steps in seed fatty acid biosynthesis. Transgenic Brassica rapa and napus plants containing the antisense gene construct of B.campestris stearoyal-ACP desaturase encoding gene have been produced. The antisense construct was linked was linked to a seed specific promoter to ensure its only in seeds. The transgenic plants showed highly reduced stearoyl-ACP desaturase activity , a dramatic increase in the level of stearic acid and an associated decline in oleic acid levels in their seeds. These findings demonstrate the potential of antisense RNA technology in modifying the fatty acid composition of modification are aimed at generating alternative source of saturated fatty acids now obtained from cocoa butter. Changed Fatty Acid Composition of Brassica Oil

Male Sterility : : 

Male Sterility : Flavonoids are essential for normal pollen development and function, and flavonoid deficiency prevents pollen maturation. Chalcone synthase is a key enzyme of flavonoid biosynthesis. In petunia, antisense construct of the gene encoding CHS has been transferred, and transgenic plants carrying this construct have been regenerated. These plants shows negligible chalcone sythase activity, white flowers and nonfunctional pollen. However these pollen grains become functional when certain flavonoids are either applied to the stigmas or mixed with the pollen. Antisense approach has also been used to restore fertility in the case of MS induced by the rolC gene of A. rhizogenes.

Ribozyme Approach. : 

Ribozyme Approach. A ribozymes is an RNA molecule, which has enzymatic activity, usually, concerned with RNA degradation, E.G., the satellite RNA of tobacco ringspot virus has sequences that have endoribonuclease activity. In the ribozymes approach, a DNA sequence specifying an enzymatic RNA sequence is fused with a sequence of the gene against which the ribozymes is aimed; the gene sequence is as a rule, in the antisense orientation. Therefore the RNA product of this gene construct has a sequence complementary to the sense RNA produced by the target gene. The complementary sequence of this RNA pairs with the sense RNA produced by the target gene, and the ribosome sequence linked to it degrades the sense RNA.

Disruption of endogenous genes : 

Disruption of endogenous genes The feasibility of transgene integration into the plant genome by homologous recombination has been demonstrated. This has raised the possibility of targeted integration of transgenes into specified sites on plant genomes. Once the techniques for targeted gene transfer are refined it should become feasible to abolish the function of a specified endogenous gene by gene disruption techniques used for the production of transgenic ;Knockout ‘ mice Random gene disruptions are readily produced by an integration, within the gene of either; 1.A mobile genetic element like transposon 2.Agrobacterium T-DNA.

Co-suppression of Genes : 

Co-suppression of Genes In case of many gene endogenous plant gene, an over expression of the sense RNA leads, surprisingly, to a drastic reduction of expression of the genes concerned; this is called Co-Suppression. The mechanism of co suppression is not understood , but several models have been proposed. Acc. To a threshold model, when RNA transcripts of a gene accumulate beyond a critical threshold level, they are selectively degraded by RNAases. The specificity in RNA degradation may be due to; 1. at the site of transgene integration an endogenous promoter may be located at the 3’-end of the transgene.transcription sponsored by this endogenous promoter will yield antisense RNA transcript of the transgene. 2.aberrent sense RNA transcripts of the transgene may be produced due to one of the several reason,e.g., accumulation of high levels of the transcript, delayed RNA processing , delay in the transcription of the 3’-end of the transgene.

RNA-Mediated Interference : 

RNA-Mediated Interference Silencing of homologous gene expression triggered by double-stranded RNA is called RNA-mediated interference. RNAi is a phenomenon that has been conserved during evolution and is found in plants invertebrates as well as mammals. The long dsRNA molecules are cleaved by an RNAase III enzymes called Dicer; this generates small 21-23 nucleotide long dsRNA molecule called small interfering RNAs.

Slide 45: 

Mechanisms by which short synthetic oligonucleotides can be used to modulate gene expression

RNAase dependent mechanism : 

RNAase dependent mechanism Rnaase H is a ubiquitously expressed endonuclease that recognizes a DNA-RNA heteroduplex and hydrolyzes the RNA strand. Antisense oligonuleotides that contain atleast 5 consecutive deoxynuleotides generate substrates for RNAase H action. Phosphorothiote oligonuleotides were the first generation antisense agents these oligodeoxy- nucleotides are dramatically more resistant to endonuclease action then modified oligodeoxynuleotides. The second generation oligodeoxynucleotides are 2’-o-methyl modification of the phosphorothiote oligodeoxynucleotides. Of these 2’-o- methyl phosphorothiote oligodeoxy –nucleotides are preferred since they are more potent and nuclease resistant, less toxic and have a higher oral bio availability.

siRNA mechanisms : 

siRNA mechanisms The SiRNA molecules bind to a protein complex called RNA-induced silencing complex; this complex contains a helicase activity that unwinds the 2 strands of RNA molecules. The antisense RNA strands so generated pairs with the target RNA molecules and an endonuclease activity then hydrolyzes the target RNA at the site where the antisense strand is bound. It is not known if the antisense strand is also hydrolyzed or it recycles and pairs with other target RNA molecules . Both SiRNA and RNAase H’ dependent mechanism are similar in the following respect; 1) not all the sites on the target RNA are good sites for there action 2) there activity is effected by the secondary structure of the target RNA 3) they both have a comparable duration of action of 4-6 days.

Synthesis of SiRNA : 

Synthesis of SiRNA A DNA vector based SiRNA production has been developed for mammalian cells, the vector has an RNA polymerase III promoter , e.g., U6 promoter , which drives the transcription of the DNA template that will yield SiRNA . The DNA template consist of the following sequences ; 1) a 21 bases long sequences of the coding region of the target gene. 2) a followed by a 6 base long spacer sequence 3) the same 21 bases long sequence but in the reverse orientation 4) 5 Ts to signal transcription termination.

Designing of siRNA : 

Designing of siRNA An siRNA molecule must have in its antisense strand an overhang ; the overhang usually 3-4 nucleotides long. The nucleotide sequence of siRNA must correspond to a segment of the coding region of the target gene, but the selection of this sequence is empirical. While selecting a sequence, the following point should be kept in mind. The regions within the first 50-100 bases of the start codon and the termination codon should be avoided. G-C content should be limited to 30-55 %. Region having 4 or more tandem repeats of same base should be avoided A region having 7 or more G/C in a row should be avoided. A region with high secondary structure should not be used. CpG motifs should be eliminated from siRNA.

Slide 50: 

spacer U UU spacer SiRNA A A TTTTT U6 Promoter Transcription Start Point +1 Transcription terminator sequence Fig; Template design of the vector used for generating SiRNA molecule

Introduction of siRNA into cells : 

Introduction of siRNA into cells Chemically synthesized siRNA molecule must be introduced into the cells using a suitable strategy. In case of animal cells calcium phosphate precipitation, electrophoration and lipofection using cationic lipids have been successfully used. A DNA vector based siRNA technology has been developed. Further, defective viral genomes, e.g. of HIV , have also been used as vehicles for delivering siRNA molecules.

Limitations : 

Limitations

Male sterility : 

Male sterility Can be produced by transferring certain genes from other species (e.g., rolB and rolC gens from Agrobacterium rhizogenes, Barnase gene from Bacillus amyloliquefaciens etc. Gene barnase is the first transgene that was used to produce male sterility by Mariani and coworkers in 1990; it has an efective fertility restoration system in barstar (another gene from B. amyloliquefaciens) . This system have performed satisfactory in crops like maize and oilseed rape.

Barnasse-barstar system : 

Barnasse-barstar system Barnase-bar/male sterile Phosphinothricin resistant -/-Normal fertile Phosphinothricin sensitive 50% Barnase-bar/- 50% -/- Barnase-bar Barstar/Barstar 50%Barnase-Bar/Barstar 50% -/Barstar Male sterile herbicide resistant Male fertile herbicide sensitive Phosphinothricin spray kills male fertile plants Male sterile Male fertile restorer Male fertile Male fertile BARSTAR product inhibits BARNASE RNase

Features for commercial value dominant genetic male sterility system : 

Features for commercial value dominant genetic male sterility system

Hormone-inducible Male sterility based on Bcp1 : 

Hormone-inducible Male sterility based on Bcp1 Most attractive system of transgenic male sterility based on antisense construct of B.campesteris gene BCP1 driven by BCP1 promoter linked with a hormone-inducible enhancer sequence. This system is ready for use in hybrid seed production in Brassica oleracea

Slide 57: 

Anti-Bcp1 seed Hormone inducible Male fertile plants Anti-Bcp1 seed Hormone inducible Male fertile plants Maintenance of Anti-Bcp1 male sterile line No hormone spray No hormone spray Anti-Bcp1 seed Hormone spray Male sterile plants (female parent) Any Male fertile line(male parent) Hybrid seed Male fertile hybrid crop No hormone spray Hybrid seed production

Biochemical Production : 

Biochemical Production Plants are chief source of carbohydrates. Transgenes have introduced novel branches in the biosynthetic pathways of plants and to generate valuable proteins. All cases are promising and in developmental stages except for thrombin inhibitor protein hirudin.

Hirudin, A polypeptide : 

Hirudin, A polypeptide Recombinant hirudin is encoded by synthetic gene. Expressed in fusion with the oil body protein olesin. Helps in purification of polypeptides. Successful example of transgene expression in plants for isolation of polypeptide. E.g. -transgenic B.napus produces hirudin at commercial scale.

Phytase , an enzyme : 

Phytase , an enzyme

Polyhydroxybutyrate, : 

Polyhydroxybutyrate, It is a Biodegradable plastic substrate Synthesized from acetyl-CoA . At present being produced by bacterial fermentation which makes it expensive. Attempts being made to produce PHAs in transgenic plants.

Continued… : 

Continued… Gene coding for two enzyme, aceto-acetyl-CoA reductase and PHB synthase and gene phbA involve in PHB synthesis have been transferred form bacterium Ralstonia eutropha and expressed in Arabidopsis thaliana.

Continued… : 

Continued… Enzymes synthesized and located in cytoplasm had a low level of PHB due to the synthesis of biosynthesis of fatty acids occurs in plastids, acetyl-CoA will be available in plastids mainly and in cytoplasm its supply will be low and limiting to PHB biosynthesis, so targeted into plastids and production increased 100 fold. Eg- Transgenic tree Poplar accumulates PHB in their leaves.

Cyclodextrins from starch : 

Cyclodextrins from starch Are cyclic oligosaccharides having 6(∞),7(ß)1,4-linked glucopyranose units. Applications: Drug delivery, flavour and odour enhancement, and removal of undesired compound like caffeine from foods.

Production of cyclodextrins : 

Production of cyclodextrins Starch is hydrolyzated to increase its solubility for better enzyme access and action, and then treated with enzyme cyclodextrin glucosyltransferase from bacterium Klebsiella pneumoniae.

Slide 67: 

The bacterial gene encoding CGT was transferred into potato and expressed in tubers The chimaeric gene construct consisted of the following: patatin gene promoter The sequence encoding the transit peptide of ribulose-bis-phosphate carboxylase. The CGT gene 3’ sequence of nopaline synthase gene of Agrobacterium.

Research Reagents : 

Research Reagents The protein Avidin is traditionally isolated from chicken egg and has been expressed in maize. Avidin pied from transgenic maize seed is being marketed by Sigma, U.S.A as a research reagent.

Downstream processing costs : 

Downstream processing costs Purification of the desired biochemical from the biomass or the culture medium is called downstream processing. It is difficult in plants and easy in micro-organism,due to the low concentration of recombinant protein in total biomass.

Steps to overcome downstream processing in plants: : 

Steps to overcome downstream processing in plants: Concentrating the recombinant protein by suitable process, e.g.-affinity chromatography. Expressing the recombinant protein as a fusion product with the structural oilbody protein olesin. Rhizosecretion Phyllosecretion

Upstream production costs : 

Upstream production costs Production of the biomass from which the desired biochemical is to be purified is called upstream production. Production cost in plant is much lower than microorganism.

Post-Translational Modification : 

Post-Translational Modification Microbes do not provide a good cellular environment for transitional modification. Suitable environment is provided by plants for modification.

Storage costs : 

Storage costs Microbial biomass has to be immediately processed and purified proteins to be stored under cold storage.

Ethical consideration : 

Ethical consideration Biochemical production from transgenic animal raises several ethical concerns while no such problem is associated with plants. “Molecular farming", "Gene farming” or “Molecular farming” are synonym to one another. These terms are used to describe production of pharmaceuticals from transgenic plants/animals.

Detrimental effects on plant performance : 

Detrimental effects on plant performance Accumulation of transgene product may affect the performance of plants, This could be resolved by targeting the transgene expression into the proper cellular compartment.

Advantages of recombinant proteins : 

Advantages of recombinant proteins

Slide 78: 

Molecular Farming Chemical Nutraceutical Pharmaceutical Proteomics Metanomics Transformation Technology Regulation Marketing Processing genomics Relationship of molecular farming with industries, products, technologies and business factors

Plant derived vaccines : 

Plant derived vaccines Conventional vaccines consists of attenuated or inactivated pathogens. Small part of a polypeptide may be used as a vaccine and are known as subunit vaccines. This are produced using rDNA technology. Storage and transport of vaccines in developing countries present many problems like cold storage etc. So plants are being developed as an alternative vaccine production and delivery systems.

Objectives of Plant Derived Vaccines: : 

Objectives of Plant Derived Vaccines:

Table : 

Table

Edible vaccines : 

Edible vaccines Antigens of several pathogens produce immunogenic response when delivered orally, such antigens are good candidates for edible vaccines. Orally active antigenic protein is isolated from pathogen, and suitable construct is prepared. The gene construct is introduced and integrated into the genome of selected plant species to produce the antigen.

Continued… : 

Continued… Edible vaccines are produced from transgenic plants where orally active antigen of the target pathogen is expressed and accumulated. An edible vaccine is provided by the E.coli heat labile enterotoxin and subunit expressed in potato. Palatable approach for such vaccines would be to deliver the immunogenic proteins through honey. The proteins appear to be concentrated two-fold in honey as compared to that in the nectar. A nectar specific promoter has been isolated and honey made from the nectar of such transgenic plants is expected to contain the immunogenic protein and will serve as vaccine when consumed.

Recombinant and subunit vaccines : 

Recombinant and subunit vaccines Plants serve as efficient production system for antigenic recombinant subunit proteins when purified and used as vaccines The vaccine against hepatitis B virus is commercially prepared by expressing its surface antigen in yeast. The HBsAg gene has been integrated into tobacco genome, and the HBsAg is produced up to 0.01% of the total soluble leaf protein. The tobacco derived HBsAg was present as 22 nm average size virus like particle.

Continued… : 

Continued… The recombinant virus is used to infect plants where the virus multiplies and spread systemically in plants. Cowpea mosaic virus gives high yield 1-2 g virus/kg of host tissues and thermostable and is easily purified.

Status of plant derived vaccines : 

Status of plant derived vaccines Three antigens : LT-B, HbsAg and NVCP(norwalk virus capsid protein) have been successfully expressed in transgenic potato and are currently undergoing human clinical trials. The result of the first human clinical trial suggest that LT-B can induce mucosal and systemic immune responses. To produce the concerned autoantigen in plants and to use them to induce autoimmune tolerance.eg- insulin and glutamic acid decarboxylase, play a major role in eliciting autoimmunity in insulin-independent diabetes mellitus. These two proteins have been expressed in potato & feeding of the tubers from these plants to nonobese diabetic mice delayed the progression of clinical diabetes.

Ornamental plants: Its modification : 

Ornamental plants: Its modification Specific gene can be engineered into ornamental plant to modify flower colour, foliage colour and even its morphology. Blue colour in flowers has three prerequisites: production of 3’,5’-hydroxylated anthocyanins; presence of flavanol co-pigments and relatively high vacuolar pH.

Continued… : 

Continued… The gene encoding flavonoid3’,5’-hydroxylase has been cloned from petunia. And was introduced into carnation, it showed high expression in petals and produced violet flowers. These transgenic carnation is being marketed in Australia and Japan under the trade name “Moondust”. 6 genes are involved in the intracellular pH control in petunia.

Continued… : 

Continued… Modification of flower colour has been achieved by suppression of endogenous genes. Suppression of chs gene produces white and pink flowers, while that of fls enhances colour intensity since flavonol tends to make them colourless

Alteration in size & shape of plants : 

Alteration in size & shape of plants By altering the cytokinin auxin ratio by introducing and expressing specific transgenes. Expression of rolC gene of Agrobacterium rhizogenes imparted potato and rose plants buds phenotypes. Over expression of the gene encoding phytochrome A in chrysanthemum and pelargonium made these plants dwarf, delayed their leaf senescence and increased chlorophyll pigmentation.

Phytoremediation : 

Phytoremediation It consists of the use of green plants and their associated microorganism for soil amendments and agronomic practices to remove or to covert environmental contaminants into harmless compounds. Green plants degrade the contaminant, but wild plants used are slow growing and accumulates specific elements, and degrade slowly

Improved phytoremediation by transgenic plant : 

Improved phytoremediation by transgenic plant Animal metallothioneins have been transferred into plants and it showed unhanced tolerance to metals, a promising approach appears to be engineered into plants specific vacuolar membrane transport pumps A gene for such pump has been cloned and expressed in fission yeast and enhances Cd tolerance. Bacterial gene merB encodes organomercurical lyase, which cleaves the carbon-mercury bond to release the less mobile Hg species.

Continued… : 

Continued… Gene merB has been transferred and expressed in Arabidopsis with the aim of degrading highly toxic organomercuric contaminants. Bacterial gene merA encodes Hg reductase that reduces toxic ionic Hg to elementary Hg. Gene merA has been transferred and expressed in Poplar Transgenic Arabidopsis and Poplar could be used together to remediate Hg-contaminated soils.

Targetting transgene product into chloroplast and mitochondria : 

Targetting transgene product into chloroplast and mitochondria Technique for introduction of transgene into chloroplasts/mitochondria is not available So approach to integrate a transgene into the nuclear genome and the transgene product into chloroplast or mitochondria is in progress. This targetting is achieved by fusing DNA sequences encoding specific signal peptides or transit peptides with the transgenes concerned.

Continued… : 

Continued… Chloroplast targetting has been used to develop glyphosate resistant transgenic tomato and tobacco. Glyphosate inhibits the chloroplast enzymes EPSPS, which is involved in aromatic amino acid biosynthesis. The gene aroA of Salmonella typhimurium and E.coli encodes a glyphosate insensitive EPSPS enzyme The bacterial gene aroA was fused with 5’-transit peptide sequence from the cDNA of petunia EPSPS and the gene construct was transferred into tobacco and tomato.

Problems in gene transfer : 

Problems in gene transfer Low level of transgene expression. Gene silencing Undesirable features of transgenic plants Low transformation frequency Regeneration problems Contamination by Agrobacterium

Low levels of transgene expression : 

Low levels of transgene expression The highest levels of expression of the truncated versions of the cry genes of B. thuringiensis are 0.02-0.04% of the total soluble leaf protein. Reason for low level of expression may be : inadequate promoter activity Length of transgene The sequence of transgene

Transgene Silencing : 

Transgene Silencing Expression of transgenes becomes suppressed in transgenic plants after they have been grown for one or more generation is transgene silencing. It is often accompanied by DNA methylation of transgenes. May be due to integration of the transgene into a hypermethylated chromosomal region. It may of two types.

Types of transgene silencing : 

Types of transgene silencing Transcriptional silencing- it is due to promoter methylation Post-transcriptional silencing-this involve methylation of the coding sequence. Gene silencing occurs most likely due to RNA transcript degradation.

Transcriptional silencing : 

Transcriptional silencing It denotes a suppression of transcription of the transgenes, and is the result of methylation of the promoter region of transgenes.

Post –transcriptional silencing : 

Post –transcriptional silencing Gene silencing is mediated by double stranded RNA, this phenomenon is a multistep process The dsRNA is processed by Dicer into short molecules The short siRNAs most likely lead to mRNA degradation

Problems due to gene silencing : 

Problems due to gene silencing Evaluation of transgenic lines may be done in two stages: Prescreening of transformants for elimination of nontransformants and transformants having multiple copies Selection from large transformant population for stable expression of the characters.

Continued… : 

Continued… Both the evaluation steps require : Additional resources. Time Delay the release of transgenic lines as varieties Instability would cause problems in the registration of the varieties as they required to be distinct, uniform, and stable.

Associated Undesirable features : 

Associated Undesirable features Features like necrosis, reduced growth ,sterility may be due to the integration of transgene within an essential gene leading to its inactivation. Can be overcome by screening a large number of independnt transformants and selecting normal looking transgenic plants.

Low Transformation Frequency : 

Low Transformation Frequency Some DNA delivery techniques like electroporation,microinjection, do show relatively high >1% transformation frequency. But they require reliable regeneration protocols from chloroplast, high skill and expensive equipment.

Random Integration : 

Random Integration Gene integration occurs at random sites and often in multiple copies, which may often lead to gene silencing .

Contamination by Agrobacterium : 

Contamination by Agrobacterium Selection methods may not always be able to completely eliminate the bacterium. They give false positive signal in dot blot assay Can be detected as follows: Assays for transgene expression Southern hybridization using different restriction enzymes Using a sequence of Ti/Ri plasmid outside the T-region as a probe for hybridization in dot blot assay

Yield Penalty : 

Yield Penalty The reduction of yield as a consequence of the expression of a transgene transferred to achieve another objective is referred to as yield penalty Reduction in yield penalty can be achieved by: Using tissue or stimulus-specific promters By transferring the transgene in different genetic backgrounds.

Status of Transgenic Research in India : 

Status of Transgenic Research in India The technology for development of transgenic varieties is being effectively used in several laboratories. Progress is limited due to the non availability of useful genes. So genes are imported from various sources.

Continued… : 

Continued… Transgenics have been produced in crops like- Brassica sp., brinjal, chickpea, cotton. At present field trials are carried out for tobacco, rice, potato, tomato, brinjal, cauliflower, cabbage and chillies A gene amal isolated in India from Amaranthus encodes a protein that has well balanced amino acid content.

Continued… : 

Continued… It is expected that protein rich potato, drought resistant mustard and salt tolerant rice transgenic lines developed using genes isolated in India will enter field trials. A stable male sterility restorer system based on barnase/barstar gene has been developed in India, and been used in hybrid seed production of Brassica.

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Genes isolated in ICAR ,CSIR and other institutes

Safety Regulators for transgenic plants : 

Safety Regulators for transgenic plants Following points to be kept in mind: 1)Their becoming persistent weeds. 2)Gene transfers from them to other plants making the latter more persistent or invasive 3)Being detrimental to the enviroment In India, DBT, new Delhi is concerned with regulation of field testing of transgenic plants.

Terminator seed and technology : 

Terminator seed and technology Are produced by transgenic plants Normal seeds are set by this crop. Seeds when treated with a specific chemical, like tetracycline produce a otherwise normal crop, but the seeds obtained in this crop can not germinate as they contain nonviable embryos. This technology employs three specific genes: gene A, gene B and gene C.

Fig. 16.13 : 

Fig. 16.13

Strategy of terminator seed technology : 

Strategy of terminator seed technology Transgenic line R X Transgenic line S (contain gene system I) (contain gene system II) Hybrid Variety T (contain both gene systems I and II) Seed of hybrid variety T treated with tetracycline before sale Good crop and good harvest (seeds produced are nonviable) Seeds do not germinate

Conclusion and future prospects : 

Conclusion and future prospects rDNA technology has made the transfer of genes from any organism to any other organism a technical feasibility. An interesting plant gene used in such a transfer is that producing thaumatin isolated from the African shrub Thaumatococcus daniellii It is a protein that is 100,000 times sweeter than sugar on a molar basis. this gene has been transferred and expressed in E.coli, yeast to obtain thaumatin comercially.

Continued… : 

Continued… Also gene transfer concerned with the mobilization of genes from prokaryotes and eukaryotes into plants to afford protection from various biotic and abiotic stress. The current phase aims at placing genes in plants with a view to produce valuable biochemicals. Also transgenic plants offer several unique advantages in biochemical production and storage.

Continued… : 

Continued… Transgenic plants play an important role in the world of agriculture and in industrial world. The world has so far witnessed two technological revolutions: The machine based industrial revolution. Information technology revolution

Third revolution : 

Third revolution Based on biotechnology and genomics… And expected to revolutionize the diagnosis, treatment and prevention of human disorders and provide alternative ways to produce nutrition supplements ,fuel, medicines and their high value products from genetically modified plants.

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