Protoplast fusion and its implication, Importance of GM plants · the somatic cells and are...

Protoplast fusion and its implication, Importance of GM plants

Mitesh Shrestha

Protoplast fusion

• Protoplasts are the cells of which cell walls are removed and cytoplasmic membrane is the outermost layer in such cells.

• The protoplast includes the plasmalemma and everything contained within i.e. the entire cell without its inherent cellulosic cell wall.

• Two genetically different protoplast isolated from the somatic cells and are experimentally fused to obtain parasexual hybrid protoplasts

Protoplast fusion

• Protoplast fusion becomes an important tool of gene manipulation because it breakdown the barriers to genetic exchange imposed by conventional mating systems.

• Protoplast fusion technique has a great potential for genetic analysis and for strain improvement

Development of hybrid plants through

the fusion of somatic protoplasts of two

different plant species/varieties is called

somatic hybridization

LEAF-DERIVED CITRUS PROTOPLASTS

TYPICAL SUSPENSION PROTOPLAST + LEAF

PROTOPLAST PEG-INDUCED FUSION

Comparison of somatic hybridization with Sexual crossing

Items Somatic fusion Sexual cross

Time No season limit Only bloom season

Phylogeny Can circumvent graft/sexual

incompatibility Can not

Results

nuclear, cytoplasmic genomes

recombined

Ploidy level increaed

Double fertilization, no

recombination and ploidy

level unchanged

Isolation of Protoplast

(Separartion of protoplasts from plant tissue)

1. Mechanical Method 2. Enzymatic Method

1. Mechanical Method

Plant Tissue

Collection of protoplasm

Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knife

Release of protoplasm

1. Mechanical Method

• Used for vacuolated cells like onion bulb scale, radish and beet root tissues

• Low yield of protoplast

• Laborious and tedious process

• Low protoplast viability

Enzymatic Method

Leaf sterlization, removal of

epidermis

Plasmolysed cells

Plasmolysed cells

Pectinase +cellulase Pectinase

Protoplasm released Release of isolated cells

cellulase

Protoplasm released

Isolated Protoplasm

Enzymatic Method

• Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores

• Mesophyll tissue - most suitable source

• High yield of protoplast

• Easy to perform

• More protoplast viability

Protoplast Fusion (Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion 2. Induced Fusion

Intraspecific Intergeneric Electrofusion Mechanical Fusion

Chemofusion

Spontaneous Fusion

• Protoplast fuse spontaneously during isolation process mainly due to physical contact

• Intraspecific produce homokaryones

• Intergeneric have no importance

Induced Fusion

• Chemofusion- fusion induced by chemicals

• Types of fusogens • PEG

• NaNo3

• Ca 2+ ions

• Polyvinyl alcohal

1. PEG-mediated fusion Characteristics: 1) Merits include: low input, high frequency of hetero-

karyons, no species limit; 2) Defects include: tedious fusion procedure, toxicity to

cells

Induced Fusion

• Mechanical Fusion- Physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette

• Electrofusion- Fusion induced by electrical stimulation

• Pearl chain of protoplasts is formed by low strength electric field (10kv m-1)

• Fusion of protoplasts of pearl chain is induced by the application of high strength electric field (100kv m-1) for few microseco

2．Electrofusion, Electrically induced fusion

Three advantages compared with PEG fusion

No toxicity to protoplasts

Higher fusion frequency

Simpler manipulation

Electro-fusion: A high frequency AC field is applied between 2

electrodes immersed in the suspension of protoplasts- this induces charges on

the protoplasts and causes them to arrange themselves in lines between the

electrodes to form pearl-chain. They are then subject to a high voltage

discharge which causes their membranes to fuse where they are in contact.

The process of electrically induced fusion

Fusion process

Membrane fusion

Cytoplasm fusion

Nucleus fusion

Factors affecting PP fusion

PP quality (intact, viability etc)

Fusion method (PEG or electro-fusion)

Fusion parameters

Possible Fusion Results of Two Genetically Different Protoplasts

= chloroplast

= nucleus

Fusion

heterokaryon

cybrid cybrid hybrid hybrid

= mitochondria

Identification and Selection of somatic hybrid cells

• Hybrid identification- Based on difference between the parental cells and hybrid cell with respect to

• Pigmentation

• Cytoplasmic markers • Fluorochromes like FITC (fluoroscein isothiocyanate) and RITC

(Rhodamine isothiocyanate) are used for labelling of hybrid cells

• Presence of chloroplast

• Nuclear staining • Heterokaryon is stained by carbol-fuschin, aceto-carmine or aceto-

orcein stain

Hybrid Selection

(Several markers are used )

• Genetic complementation

• Phytotoxins

• Specific amino acid

• Auxin autotrophy

• Antibiotics

• Auxotrophic and metabolic mutants

• Chromosomal analysis

• Herbicides

Culture of the hybrid cells

Hybrid cells are cultured on suitable medium provided with the appropriate

culture conditions.

Regeneration of hybrid plants

• Plants are induced to regenerate from hybrid calli

• These hybrid plants must be at least partially fertile, in addition to having some useful property, to be of any use in breeding schemes.

Advantages of somatic hybridization

• Production of novel interspecific and intergenic hybrid – Pomato (Hybrid of potato and tomato)

• Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids

• Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters

Advantages of somatic hybridization

• Production of heterozygous lines in the single species which cannot be propagated by vegetative means

• Studies on the fate of plasma genes

• Production of unique hybrids of nucleus and cytoplasm

Protoplast fusion and its implications

Applications of protoplast fusion • Production of novel interspecific and intergeneric hybrids • Pomato (Hybrid of potato and tomato) •"wide crosses" where even embryo culture won't work

•Citopsis gilletiana (wild) x Citrus sinensis •citrus sexually incompatible spp. •wild relative has disease/nematode resistance •somatic hybrid used as a rootstock

• Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids

•Solanum somatic hybrids •S tuberosum dihaploids fused with wild diploid (S. chacoense) •resulting somatic hybrid (4n) is backcrossed to S. tuberosum cultivars (also 4n) •overcomes sterility due to ploidy differences between somatic and sexual hybrids

Protoplast fusion and its implications

Applications of protoplast fusion…/2 • Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters

• Production of heterozygous lines in the single species which cannot be propagated by vegetative means

• Studies on the fate of plasma genes

• Production of unique hybrids of nucleus and cytoplasm

Limitations of Somatic hybridization

• Poor regeneration of hybrid plants

• Non-viability of fused products

• Not successful in all plants.

• Production of unfavorable hybrids

• Lack of an efficient method for selection of hybrids

• No confirmation of expression of particular trait in somatic hybrids

Future prospects of GM crops

• GM technology is the technology with immense

potential to solve many of the problems the humans face these days • But there has been significant opposition from the so called green or anti-GM groups due to some problems associated with GM crops • Many of these problems are much exaggerated and are based on rhetoric rather than on proven scientific facts • Some of the areas in which this technology can be used for the benefit of humanity are described in following slides


Bioremediation/Phytoremediation: Detoxification of mercury can be done by conversion of Hg2+ ions to metallic mercury which then evaporates from soil. Expression of MerA gene from E. coli coding for mercuric ion reductase has been found to confer resistance to HgCl2 as high as 350 μM in various plants like tobacco (He et al., 2001). These plants could remove 5-8 times more mercury by volatilisation than respective controls.


Bioremediation…/2

Detoxification of chlorinated hydrocarbons by using mammalian cytochrome P450 2E1 in transgenic plants (Doty et al., 2001) and that of explosives like TNT(Esteve-Nunez et al., 2001) by using nitroreductase are some of the possibilities that can be used for bioremediation. Re


• Engineering for plant based fibres

Presence of lignin in plants acts as contaminant during the making of paper pulp. It also decreases the digestibility during its use as animal fodder. Engineering woody trees or grasses for low lignin content would add to increased digestibility as well as cellulose content. Similarly, improved cellulose yield in fibre plants like cotton (Haigler and Holaday, 2001) by using sucrose phosphate synthase gene or production of novel fibres like the expression of spider silk protein gene (Scheller et al., 2001) in transgenic plants are other possibilities.


• Biodegradable plastic

Transgenic plants have been found to have potential to serve as production system for plastic polymers like PHB (poly beta-hydroxybutyrate ) and PHV (poly beta-hydroxyvalerate) by causing co-ordinated expression of genes coding for three bacterial enzymes like beta ketothiolase, acetoacyl CoA reductase and PHB synthase in leucoplast of mature seeds of transgenic plants. This system has potential to produce PHB as high as 8% of total fresh weight of the seed (Liebergesell et al., 2001; Madison et al., 2001).


• Ornamental plants Change in coloration of plants using alterations in flavonoid, carotenoid and betalain metabolism are some of the prospects that may have far reaching economic benefits. One of such prospects has materialized in producing transgenic chrysanthemums, gerberas, roses and carnations having flower colour in the blue (Mauve-violet-blue) spectrum. Normally these plants lack the anthocyanin delphinidin due to absence of the enzyme Flavonoid 3’,5’-oxidase. Cloning of this genes into those plants enabled them to produce flower colour in the blue spectrum. Similar approaches can be used in various ornamental plants to produce flowers with novel colors.

Future prospects of GM crops • Ornamental plants…/2 Increased vase/shelf life is another attribute that is being modified using biotechnological approaches. Production of cytokinins associated with wounding or senescence inducible promoters is being pursued to increase shelf/vase life in various horticultural plants like petunia, lettuce, broccoli, etc.

NOS Wound inducible Promoter IPT or KN1

CYTOKININS SENESCENCE


• Yield increment Attempts are underway at IRRI philippines to convert rice to C-4 plant by cloning the enzyme PEP carboxylase that would enable rice plant to photsynthesize more in tropical conditions without undergoing photorespiration. Similarly yield increment is sought in tobacco plants by utilising shade avoidance mechanisms by making use of phytochrome A. Similarly, yield increment by cloning genes encoding for two bisphosphate enzymes namely, Fructose 1,2 bisphosphate and Seduheptulose 1, 7- bisphosphate is also underway in tobacco.


• Yield increment…/2

Attempts are underway to introduce dwarfism that would allow more of the assimilates to be used for reproductive growth and avoid loss due to lodging. Similarly, productivity increase due to delayed senescence using either ipt (Gan and Amasino, 1995)or knotted1 (Vollbrecht et al., 1994) gene in various plants like tobacco, rice, maize, turfgrass, etc will be the reality in the near future.


• Improved nutritional quality/ health benefits

Rice and Tomatoes rich in pro-vitamin A will be available in the market in near future. These plants are the outcomes of plant biotechnolgy that enhanced the expression of phytoene synthase, phytoene desaturase, lycopene synthase, etc. and led to increased provitamin A in these plants. When these products reach the population they have potential to reduce the chances of night blindness, especially in the third world.


Improved nutritional quality/ health benefits

Similarly the plants rich in iron, antioxidants, seed albumin, methionine, lycopene and various other products are in various stages of development and will reach the marked in future.

Plant based vaccines and medicines are also the matter of future

Hurdles to Development of GM crops

• Due to strong opposition from the so called green anti-biotech groups, the term GM itself is a taboo. Until and unless we succeed in bringing this subject to day to day discussion it will remain so and may hinder the further development in this field.

• Fear factor: Here also the culprits are the so called green groups that maintain their existence by disseminating fear. According to Gardner Radon, a radioactive inert gas causes death of some 41000 people each year in europe and USA compared to zero body count in case of GM crops. But still there is huge outcry against GM crops.

Hurdles to Future of GM crops../2

• Method of dissemination of information by the scientific community- instead of using statistical data the scientists should try disseminating facts in simple terms. For example data regarding human figures should be expressed as body counts rather than as a percentage (Nine out of every ten people instead of 90%). (Daniel Gardner, 2008, The Science of Fear) • Regulatory processes – Lax regulatory processes will minimise cost of development and harassment to the scientists. For this reason future GM crops will appear more in Asia mainly India and China than USA.

Hurdles to Future of GM crops../3 • Government policies on investment in Biotech industries/R & D institutes: Governments with positive attitudes towards the development of biotechnology like India, China, Brazil are pouring heavy investment in this sector. The regulatory processes are also relatively lax and they are at the forefront of the biotech research these days and will lead the development in this field in the near future. Out of 120 commercial transgenic events expected in the world by 2015, half will be from Asia alone (30 from india, 20 from china, 5 from rest of Asia) and another half from rest of the Europe and America. The Zero tolerance policy of EU regarding the contamination of food by GM products is hindering the development in plant biotechnology in that part of the world.

Environmental Impact Assessment (EIA) •Environmental impact assessment (EIA) can be defined as an activity designed to identify and predict the effect of an action specifically on bio-geophysical environment and on man’s health and well being and to interpret and communicate information about the impacts (Munn, 1979)

• It is used to identify, predict and describe in appropriate terms the pros and cons of a proposed development (i.e., to assess all relevant environmental and resulting social effects which would result from a project)

• It provides decision makers an account of the implications of the proposed course of action before a decision is made (Clark, 1983)

EIA

There are four aspects of EIA

•It sees the environment as an aggregate of things and conditions that surround or envelope everything including non living things

•Value of EIA is realized more likely in timely communication of information between individuals conducting assessment and individuals planning a proposed project

• Many environmental components, processes and attributes may or maynot be quantified by currently available methods

• Mitigation of significant impacts must be assessed for all possible impacts (both desirable/undesirable)

EIA

•Can be done prior to initiation of research, i.e., ex- ante or after the completion of the research work, i.e., ex-poste

•The former type of EIA helps in planning and priority setting, help study the likely effects of the proposed project and formulate research priorities by examining the relative benefits of different research programs. It also assists to identify the optimal combination of research programs, and also provides framework for gathering information to carry out ex-poste evaluation

•Ex-poste EIA on the other hand provides feedback for policy makers and researchers, and also helps improve decision making

Principles involved in EIA of GM crops There are two principles involved in potential risk assessment • The Principle of Substantial equivalence applies to the safety of novel foods and food products is to look into whether the product in question is substantially equivalent to an existing analogous food or not (Based on phenotypic and agronomic characters of GM crop and its conventional counterpart) •The precautionary principle concerns the potential risks, if any the GM crop may have on the environment. Appropriate measures designed to prevent harm to the nature should be adopted. Currently in practice in the EU.

EIA of GM crops A well executed impact assessment could provide following outcomes (Porter et al., 1980) • Modify the project • Stimulate research and technology, particularly to deal with the adverse effects of the technology •Stimulate research to specify or define risks •Identify regulatory and legal changes to promote or control the technology •Define intervention experiments to reduce negative or enhance positive consequences •Stop the technology •Provide a reliable base of information to parties at interest

How to proceed? (Nickson, 2008)?

Step 1. Problem formulation (Nickson, 2008)

How to proceed?

Step 2. Formulating and integrating assessment end point (Nickson, 2008)

How to proceed?

Step 3. Formulation of conceptual model and analysis plan (Nickson, 2008)

Step 4. Problem formulation (Nickson, 2008; Plant Physiology

147:494-502)

1. Gene flow to related wild or cultivated relatives: These risks are same for the transgenic species as that for conventionally bred species and depends upon various factors as •Reproductive barriers •Modes of pollination •Methods of seed dispersal (natural and through agricultural activity) •Genetic stability, fitness, and fertility of hybrids •Overlap of flowering times •Proximity and size of populations •The use of go-between organisms such as fungi, viruses, and aphids

Environmental effects of GMOs


Potential for gene flow in GMOs

2. Effects on non target species: Some transgenic traits like pesticidal effects of Bt genes, or those of snowdrop lectins are difficult to prove scientifically. Losey et al., (1999) published that pollen from Bt corn killed monarch butterflies. Several follow up reports published later in PNAS in 2001 found negligible effects of Bt pollen on monarch butterfiles compared to other threats (Conner et al., 2003)


3. Indirect environmental effects due to changing or agricultural practices associated with new variety

• GMOs have changed chemical and land use patterns and farming practices, but no consensus of opinions regarding the net effects (negative or positive effects)

• Reduced the volume and frequency of pesticide use due to introduction of Bt maize, cotton and soybean, but the most significant being that for cotton. Beneficial to in crop biodiversity in comparison to conventional crops with regular high doses of broad spectrum of insecticides. Less health hazards for farmers.


• Herbicide tolerant crops have increased the herbicide use but have shifted to less harmful herbicides. At the same time it has encouraged low till crops with benefits to soil conservation. However there have been significant effects in the UK of GM crops compared to their conventional counterparts in terms of indirect effect on herbivores, pollinators and other populations that feed on these weeds. The effect on farmland biodiversity were positive in HT maize and negative in HT sugarbeet, but none on oilseed rape (Royal Society, 2003).

3. Indirect environmental effects…

• Development of resistance against herbicides/ pesticides (glyphosate, glufosinate or Bt toxins) . These chances are equal to the conventionally bred crops as well. There are 120 weed species worldwide with reported tolerance to herbicides used for HT crops. But these resistances are not due to escape of transgenes.

• Spread of antibiotic resistance genes to soil micro-organisms. (groundless thinking: no scientific basis)

3. Indirect environmental effects…

• Allergnicity or toxicity of GM foods (Equal chances as that of conventionally bred plants). But less likely as the GM foods have to pass through much rigorous testing procedures than their conventional counterparts

4. Effects on human/animal health

5. Control by big MNCs

• True, but can be mitigated provided there is enough public fund availed to research in the this field and for institutional capacity building in the third world. An example of such humanitarian face of GM technology is the development of golden rice. 6. Ethical issues

• In USA Animal and Plant Health Inspection service (APHIS) looks into the regulation of release of GM plants after a thorough EIA. There is involvement of four different government bodies in decision making process regarding the effects of GM plants. USDA and EPA look into the effects on environment while FDA and NIH look into matters concerning safety of food or other products derived from GM plants. Generally the regulatory mechanisms are based on principle of substantial equivalence and are relatively lax. • In the EU, the process is much more complex and lengthy, should satisfy the requirements of all the member states. It is based on precautionary principles and the organisation intending to launch a GM product have to pass through more stringent and hypothetical impact assessment process. •In Nepal, no regulation so far. There is not even a mechanism to detect the adulteration of foodstuff with GM products.

Regulation of Release of GM plants

1. Tryptophan death of 40 EMS (eosinophilia-myalgia syndrome) patients in USA 1989 due to consumption of tryptophan produced by a japanese firm using genetically modified bacteria. GM technology was not the culprit but it was the change of bacterial strain and compromise in some of the purification steps that left to about 60 impurities. One of these might have caused death but not proven conclusively.

Reported negative effects of GMOs

2. Puzstai affair (1989): Rats fed with potatoes engineered with snowdrop lectin to reduced insect damage caused lesions on their stomach. They proposed that these lesions were due to genetic engineering and not due to lectin. But data were inconclusive; effects might have arisen due to nutritional differences between GM and control potatoes and not due to the GM process.

Reported negative effects of GMOs..

3. Allergenicity of Starlink™ corn: Engineered to express BT Cry9C protein did not get EPA approval as human food due to potential allergenic effects (owing to their similarities, mainly heat stability and slow digestibility, with food allergens). But was approved as feed for cattle as they do not have allergenicity. Already withdrawn from market in 2000. The studies were not conclusive, but based on molecular structure the protein has moderate chances of being allergnic.


4. Risk of producing a virus: There is a possibility of transcapsidation or heterocapsidation between the coat protein expressed by a trangenic plant and RNA from invading plant viruses, and there have been scientifically proven incidences of mixing of coat proteins of unrelated viruses like alfalfa mosaic alfamovirus and cucumber mosaic cucumovirus. Other reports have debated these findings as they failed to show transcapsidation. It may occur at very low frequency if it occurs at all in nature

Similar problems may also be possible due to use of RNA technology to control plant viruses.


•GMOs have been unduly subjected to stringent risk assessment measures compared to their conventional counterparts (costing additional 8-10 million dollars on regulatory approval alone) •None of the GM plants are proven to be unsafe for human or animal consumption •Risk assessments of GM plants are being influenced by societal and economic concerns rather than biosafety considerations • Anti GM pressure groups are getting their upper hand in disseminating misinformation regarding the effects of GM crops on human health, environment. Their arguments are based on rhetoric rather than scientific facts, and use any means (vandalism, arson, misuse of media, etc) to achieve their goals

To sum up…..

Protoplast fusion and its implication, Importance of GM plants · the somatic cells and are...

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