Biotechnology

Biotechnology is the use of living organisms, or parts of them, to make products and services that people need. It is old — curd, gundruk, jaand and chhurpi are all biotechnology — but since the 1970s the ability to cut and rejoin DNA has turned it into the fastest-moving branch of biology. This unit covers what plant biotechnology can actually do: grow a whole plant from a few cells, breed better crops, build disease resistance, replace chemicals with living fertilisers and pesticides, move a gene from one species to another, and help keep a country's food both sufficient and safe.

5.1 Tissue culture

Totipotency was proposed by Haberlandt in 1902 and first demonstrated by F. C. Steward in 1957, who grew whole carrot plants (Daucus carota) from single phloem cells suspended in coconut water.

The medium

The standard medium is MS medium (Murashige and Skoog, 1962). It contains:

Component	Examples	Role
Macronutrients	N, P, K, Ca, Mg, S salts	bulk nutrition
Micronutrients	Fe, Mn, Zn, B, Cu, Mo	enzyme cofactors
Carbon source	sucrose, 2–3 %	energy (cultures are not photosynthetic)
Vitamins	thiamine, nicotinic acid, pyridoxine, myo-inositol	growth factors
Amino acid	glycine	nitrogen source
Growth regulators	auxin (IAA, NAA, 2,4-D) and cytokinin (kinetin, BAP)	control what the tissue becomes
Gelling agent	agar, 0.8 %	support

The ratio of auxin to cytokinin decides the result (Skoog and Miller, 1957): a high auxin : low cytokinin ratio makes roots, a low auxin : high cytokinin ratio makes shoots, and a roughly equal ratio keeps the tissue as an unorganised mass of dividing cells, the callus.

The steps

1. Choice of explant — a small piece of tissue (shoot tip, node, leaf disc, anther, embryo) from a healthy, disease-free mother plant.
2. Surface sterilisation — 70 % ethanol for a few seconds, then 0.1 % mercuric chloride or 1 % sodium hypochlorite, followed by several rinses in sterile distilled water.
3. Inoculation — the explant is transferred aseptically to the medium inside a laminar air-flow cabinet.
4. Callus formation and multiplication — incubation at about 25 °C with a 16-hour photoperiod; the callus is subcultured every 3–4 weeks.
5. Organogenesis — the callus is shifted to a high-cytokinin medium to make shoots, then to a high-auxin medium to make roots.
6. Hardening (acclimatisation) — the tiny plantlets are moved to a greenhouse with high humidity, then gradually to normal conditions.
7. Transfer to the field.

Kinds of culture and what they are used for

Culture	What is grown	Chief use
Callus culture	unorganised mass on solid medium	starting point for regeneration; somaclonal variation
Suspension culture	free cells in shaken liquid	industrial production of secondary metabolites
Meristem / shoot-tip culture	the 0.2–0.5 mm apical dome	virus-free plants, because viruses do not reach the meristem
Anther / pollen culture	microspores	haploid plants; doubling with colchicine gives instantly homozygous lines
Embryo culture (embryo rescue)	a young hybrid embryo	saves wide-cross hybrids that would abort in the seed
Protoplast culture	wall-less cells made by cellulase and pectinase	somatic hybridisation — the "pomato" from potato + tomato protoplasts

Applications. Micropropagation multiplies elite plants by the thousand — banana, orchid, potato, sugarcane and strawberry are produced this way in commercial labs, including in Kathmandu and Chitwan. Meristem culture gives virus-free seed potato. Cryopreservation in liquid nitrogen at −196 °C conserves germplasm. Cell suspensions make alkaloids and other secondary metabolites. And tissue culture is the essential last step of every genetic engineering project in plants: the transformed cell must be regenerated into a whole plant.

Its limits are real too: it is expensive, it needs skilled staff and absolute asepsis, contamination can destroy a whole batch, and unwanted somaclonal variation can creep in.

5.2 Plant breeding

Objectives: higher yield; better quality (more protein, oil, vitamins); resistance to diseases and pests; tolerance of drought, cold, heat and salinity; early maturity, so that two or three crops fit into one year; dwarf, stiff-strawed habit that does not lodge under heavy fertiliser; and suitability for machinery.

The steps of a breeding programme:

1. Collection of germplasm — gathering landraces, wild relatives and released varieties into a gene bank.
2. Evaluation and selection of parents — screening the collection for the desired character.
3. Hybridisation — the chosen female parent is emasculated (its anthers removed before they dehisce) and bagged; when the stigma is receptive, pollen from the male parent is dusted on and the flower is bagged and tagged again.
4. Selection among the progeny — the F₁ is selfed and superior recombinants are selected from the F₂ onwards, generation after generation.
5. Testing, release and multiplication — yield trials at several sites over several years, then release by the National Seed Board and multiplication of certified seed.

Methods of breeding: introduction of a variety from elsewhere; selection (mass selection, pure-line selection, clonal selection); hybridisation (intervarietal, interspecific, intergeneric); mutation breeding with gamma rays or EMS; polyploidy breeding with colchicine; and, most recently, marker-assisted selection, in which DNA markers linked to a useful gene let the breeder pick the right seedling in the laboratory instead of waiting for the adult plant.

The Green Revolution of the 1960s was plant breeding on a national scale: Norman Borlaug's semi-dwarf, rust-resistant wheats and IRRI's IR8 rice (1966) roughly doubled cereal yields in South Asia. Nepal's own programme, run by the Nepal Agricultural Research Council (NARC), has released wheat varieties such as Bhrikuti (1994) and Gautam, rice varieties such as Sabitri, Radha-4 and Khumal-4, and maize varieties such as Rampur Composite and Manakamana-3.

Two ideas underlie hybrid breeding. Heterosis (hybrid vigour) is the superiority of an F₁ hybrid over both its parents in size, yield and vigour; inbreeding depression is the opposite loss of vigour when a cross-pollinating crop such as maize is repeatedly selfed. Hybrid seed must therefore be bought fresh each season, because the F₂ segregates and the vigour is lost.

Biofortification is the newest objective: breeding varieties that are richer in iron, zinc, protein or vitamins, so that nutrition improves without anyone having to change what they eat.

5.3 Disease-resistant plants

A plant is resistant when it can prevent or limit infection by a pathogen. Resistance is of two kinds:

	Vertical (race-specific) resistance	Horizontal (field) resistance
Genetic basis	one or a few major genes	many genes, each of small effect
Effect	complete immunity to some races	partial protection against all races
Durability	breaks down when a new race appears	durable
Inheritance	simple, easy to breed	difficult to breed

The gene-for-gene hypothesis (H. H. Flor, 1956) explains vertical resistance: for every resistance (R) gene in the host there is a matching avirulence (Avr) gene in the pathogen, and the plant resists only when both are present.

Sources of resistance genes are wild relatives and old landraces, which is why gene banks matter: the terraces of Nepal's mid-hills still hold rice and barley landraces carrying genes that modern varieties have lost.

Methods of producing resistant plants:

• Conventional breeding, usually by the backcross method — the resistance gene is crossed into a good variety, and the hybrid is repeatedly backcrossed to that variety so that only the resistance gene is retained.
• Mutation breeding — mildew-resistant barley (the mlo mutants) came this way.
• Genetic engineering, which can bring in a gene from any organism at all.

The best-known transgenic resistance uses the cry genes of the soil bacterium Bacillus thuringiensis.

Transgene	Source	Protects against	Crop
cry1Ac, cry2Ab	Bacillus thuringiensis	cotton bollworm	Bt cotton
cry1Ab	Bacillus thuringiensis	maize stem borer	Bt maize
cry1Ac	Bacillus thuringiensis	brinjal fruit-and-shoot borer	Bt brinjal
viral coat protein gene	papaya ringspot virus	the virus itself	virus-resistant papaya
chitinase, glucanase	fungi, plants	fungal pathogens	several
RNA interference construct	the nematode's own gene	root-knot nematode Meloidogyne incognita	tobacco

Bt cotton was approved for commercial planting in India by the GEAC in 2002 and now covers roughly 90 % of the cotton area there. Bt brinjal was approved by Bangladesh's National Committee on Biosafety on 30 October 2013 and has been grown there since 2014 — the first genetically engineered food crop released in South Asia.

5.4 Green manure, bio-fertilizer, bio-pesticide

Green manure

The common green-manure crops are dhaincha Sesbania aculeata, sunn hemp Crotalaria juncea, cowpea Vigna unguiculata, guar Cyamopsis tetragonoloba and Trifolium. When the leaves of a tree are cut and carried to a field instead, it is called green-leaf manure — in Nepal the leaves of Azadirachta indica (neem), Gliricidia and Artocarpus are used this way.

Green manuring adds nitrogen (a good dhaincha crop can add 60–90 kg N per hectare), raises the organic-matter content, improves soil structure and water-holding capacity, encourages soil microbes, suppresses weeds and checks erosion between crops.

Bio-fertilizers

A bio-fertilizer is a preparation of living micro-organisms that, when applied to seed, soil or root, increases the supply of nutrients to the plant. It does not itself contain the nutrient; it makes the nutrient available.

Group	Organism	What it does
Symbiotic N-fixers	Rhizobium in legume root nodules	fixes atmospheric N₂
	Frankia in Alnus nepalensis (utis)	fixes N₂ in a non-legume tree
	Anabaena azollae inside the fern Azolla	classic bio-fertilizer of paddy fields
Free-living N-fixers	Azotobacter (aerobic), Clostridium (anaerobic)	fix N₂ in the soil
Associative N-fixer	Azospirillum	lives around the roots of cereals
Blue-green algae	Nostoc, Anabaena, Aulosira	fix N₂ in flooded rice fields
Phosphate solubilisers	Bacillus megaterium, Pseudomonas striata, Aspergillus	release insoluble soil phosphate as acids
Mycorrhiza	vesicular-arbuscular mycorrhiza, Glomus	greatly extends the root surface for phosphorus and water uptake

Bio-pesticides

A bio-pesticide is a living organism, or a substance obtained from one, used to control pests.

Type	Agent	Target
Bacterial	Bacillus thuringiensis	caterpillars (Lepidoptera)
Fungal	Trichoderma viride, T. harzianum	soil-borne fungal diseases
	Beauveria bassiana, Metarhizium anisopliae	many insects
Viral	nuclear polyhedrosis virus (NPV)	Helicoverpa, Spodoptera
Botanical	neem Azadirachta indica (azadirachtin)	broad-spectrum antifeedant
	pyrethrum Chrysanthemum cinerariifolium, Derris (rotenone)	many insects
Predator / parasitoid	Trichogramma wasp, ladybird beetle	eggs of borers, aphids

Compared with chemical pesticides, bio-pesticides are specific (they spare bees, birds and natural enemies), biodegradable so they leave no residue on food, and they do not accumulate along food chains or cause resistance as quickly. Against that, they act slowly, they are sensitive to sunlight and temperature, and they have a short shelf life.

5.5 Genetic engineering and GMOs

The tools

1. Restriction endonucleases — the molecular scissors. These bacterial enzymes recognise a short palindromic sequence of 4–8 base pairs and cut the DNA within it. EcoRI, from Escherichia coli strain RY13, recognises G↓AATTC and cuts between G and A on both strands, leaving four-base single-stranded sticky (cohesive) ends that can base-pair with any other fragment cut by the same enzyme. Some enzymes, such as SmaI, cut straight across and give blunt ends. Arber, Nathans and Smith shared the 1978 Nobel Prize for this discovery.

2. DNA ligase — the molecular glue. It seals the phosphodiester backbone between the joined fragments.

3. Vectors — the carriers. A good vector needs an origin of replication (ori) so that it multiplies in the host, selectable marker genes such as antibiotic resistance so that transformed cells can be picked out, and unique restriction sites where the foreign gene can be slotted in. The commonest are plasmids — small circular extra-chromosomal DNA of bacteria, such as pBR322 — along with bacteriophages, cosmids and, for plants, the Ti plasmid of Agrobacterium tumefaciens, a natural genetic engineer that normally inserts its own T-DNA into plant cells and causes crown gall.

4. Host cells — Escherichia coli, yeast, or the plant cell itself.

Steps of recombinant DNA technology

1. Isolation of DNA. The cells of the donor organism are broken open with lysozyme (bacteria) or cellulase (plant cells), the proteins are digested with protease and the RNA removed with ribonuclease. Chilled ethanol is then added; the DNA separates out as fine white threads that can be spooled on a glass rod.
2. Cutting. The donor DNA and the vector are cut with the same restriction enzyme, so that both carry complementary sticky ends.
3. Ligation. The gene of interest and the cut vector are mixed. Their sticky ends base-pair, and DNA ligase seals the nicks. The product is recombinant DNA (rDNA).
4. Transformation. The rDNA is pushed into a host cell.
5. Selection and screening. Only a few host cells actually take up the rDNA, so the marker genes are used to pick them out (see below).
6. Multiplication and downstream processing. The selected cell is grown in a fermenter or regenerated into a whole plant, and the product — protein, metabolite or transgenic plantlet — is harvested and purified.

Methods of transferring DNA into the host

Method	How it works	Chiefly used for
CaCl₂ + heat shock	Ca²⁺ makes the bacterial wall porous; a 42 °C pulse draws plasmid in	E. coli
Electroporation	A brief high-voltage pulse opens transient pores in the membrane	bacteria, protoplasts
Microinjection	DNA injected into the nucleus with a fine glass needle	animal cells, eggs
Gene gun (biolistics)	Gold or tungsten particles coated with DNA fired at cells	monocots, chloroplasts
Agrobacterium method	T-DNA of the Ti plasmid is transferred naturally into the plant genome	dicots

Selection. In pBR322 the gene is inserted into tetᴿ. Cells carrying the recombinant plasmid have a working ampᴿ but a broken tetᴿ, so they grow on ampicillin but die on tetracycline — this is insertional inactivation. Modern vectors use lacZ instead: recombinant colonies cannot make β-galactosidase and stay white, while non-recombinants turn blue on X-gal. This is called blue–white screening.

The polymerase chain reaction (PCR)

PCR, devised by Kary Mullis in 1983 (Nobel Prize 1993), makes millions of copies of one chosen stretch of DNA in vitro, without any host cell. The reaction tube contains the template DNA, two short primers (about 18–25 nucleotides) that flank the target, the four dNTPs, Mg²⁺ buffer and a heat-stable DNA polymerase — Taq polymerase, isolated from the hot-spring bacterium Thermus aquaticus, which survives 95 °C. A thermal cycler then repeats three temperature steps:

Step	Temperature	What happens
Denaturation	94–95 °C, 30 s	hydrogen bonds break; the duplex separates into two strands
Annealing	50–65 °C, 30 s	primers base-pair with their complementary sites
Extension	72 °C, ~1 min per kb	Taq polymerase adds dNTPs 5′ → 3′ from each primer

Each cycle doubles the number of target molecules, so after n cycles one molecule becomes 2^n copies. Thirty cycles take about two hours and give over a billion copies. PCR is used in disease diagnosis, DNA fingerprinting, prenatal testing, detecting GM material in imported grain, and amplifying a gene before cloning it.

Gel electrophoresis

To check that the right fragment has been cut or amplified, the DNA is separated by agarose gel electrophoresis. A slab of agarose gel is laid in a buffer tank; the samples are loaded into wells at the cathode (−) end. Because its phosphate groups make DNA negatively charged at all pH values, every fragment moves towards the anode (+). The gel acts as a molecular sieve: short fragments thread through the pores quickly and travel far, long fragments lag behind, so distance moved is inversely related to the logarithm of fragment length. The bands are stained with ethidium bromide and seen as orange bands under UV light. Comparing them with a DNA ladder of known sizes run alongside gives the size of each fragment. The wanted band is then cut out of the gel and the DNA recovered — this is called elution.

1Wells where the DNA samples were loaded (cathode end)2Ladder lane — marker fragments of known sizes3A band: fragments of one size, stained with a fluorescent dye4Loading-dye front, running ahead of the smallest DNA

Genetically modified organisms

A genetically modified organism (GMO) is an organism whose genome has been deliberately altered by inserting, deleting or silencing a gene using rDNA technology. A GMO that carries a gene from a different species is called transgenic.

GMO	Gene transferred	Result
E. coli producing humulin (1982)	human insulin genes A and B	cheap, non-allergenic human insulin
Bt cotton (India, 2002)	cry1Ac from Bacillus thuringiensis	resists bollworm; now over 90 % of India's cotton area
Bt brinjal (Bangladesh, approved 2013)	cry1Ac	resists fruit and shoot borer
Golden Rice	psy (daffodil/maize) + crtI (Erwinia)	grain makes β-carotene, a vitamin A precursor
Flavr Savr tomato (1994)	antisense polygalacturonase	slower softening, longer shelf life
Roundup Ready soybean	bacterial EPSPS	tolerates the herbicide glyphosate
Bt maize	cry1Ab	resists stem borer

Benefits. Higher yield with less pesticide, tolerance of drought, salt and herbicides, longer shelf life, biofortified food such as Golden Rice, and cheap medicines (insulin, human growth hormone, hepatitis B vaccine, clotting factor VIII).

Concerns. Possible allergens in new proteins; antibiotic-resistance marker genes; gene flow to wild relatives, creating "superweeds"; harm to non-target insects; loss of local landraces and farmer dependence on bought seed; and the ethical objection to patenting life. Because of these risks, every country that ratified the Cartagena Protocol on Biosafety regulates GM release.

5.6 Bio-engineering

Bio-engineering (biochemical engineering) applies engineering design to living systems so that biological reactions can be run on an industrial scale. The central piece of hardware is the bioreactor or fermenter — a stirred, sterilised stainless-steel vessel in which temperature, pH, dissolved oxygen and nutrient feed are controlled automatically while microbes or cultured cells make the product.

Area	Principle	Example
Industrial fermentation	microbes grown in a bioreactor secrete a product	penicillin from Penicillium chrysogenum, citric acid from Aspergillus niger
Enzyme technology	enzymes immobilised on beads and reused	glucose isomerase for high-fructose syrup
Biofuel	plant biomass or oil converted to fuel	ethanol from sugarcane, biodiesel from Jatropha curcas
Biogas	anaerobic digestion of dung by methanogens	about 450,000 household biogas plants in Nepal
Bioremediation	microbes break down pollutants	Pseudomonas putida ("superbug") degrades oil
Phytoremediation	plants take up heavy metals from soil	Brassica juncea accumulates lead
Biosensors	an enzyme or microbe coupled to an electrode	glucometer for blood sugar
Bioleaching	bacteria release metal from low-grade ore	Thiobacillus ferrooxidans leaches copper
Single-cell protein	microbial biomass used as food or feed	Spirulina, Methylophilus methylotrophus
Biomedical engineering	engineered tissues and devices	artificial skin, dialysis membranes, stents

Nepal's biogas programme, begun in 1992, is the best local example: a fixed dome digester loaded with cattle dung and water produces a gas of roughly 60 % methane for cooking and lighting, and the spent slurry returns to the field as manure. It replaces firewood, cuts indoor smoke and reduces pressure on forests.

5.7 Food safety and food security

These two terms are often confused. Food safety is about the quality of food — that it is free from anything that could harm the eater. Food security is about access — that everyone always has enough of it.

Hazards in food

Type of hazard	Examples
Biological	Salmonella, E. coli O157:H7, Vibrio cholerae, hepatitis A virus, Entamoeba, aflatoxin from Aspergillus flavus on damp maize and groundnut
Chemical	pesticide residue, heavy metals, excess food colour, adulterants such as melamine or chemical ripening agents
Physical	glass, metal, stone, hair, insect parts

How safety is ensured. Good agricultural and hygienic practice in the field; correct drying and storage to keep grain below the moisture level at which moulds grow; pasteurisation, canning, refrigeration and irradiation; HACCP (Hazard Analysis and Critical Control Points), in which every step of processing is examined and the critical points are monitored; and labelling to Codex Alimentarius standards. In Nepal the Food Act 2023 BS (1966) and the Department of Food Technology and Quality Control (DFTQC) enforce these rules, and the Right to Food and Food Sovereignty Act, 2075 (2018) gives every citizen a legal right to food, implementing Article 36 of the Constitution of Nepal.

Where biotechnology helps. Higher-yielding and stress-tolerant varieties raise availability; biofortified crops such as Golden Rice, iron-rich beans and zinc-rich wheat improve utilisation; tissue culture supplies disease-free planting material; bio-pesticides and bio-fertilizers cut chemical residues; and PCR and ELISA allow rapid detection of pathogens, aflatoxin and undeclared GM material in a consignment. Post-harvest technology matters just as much: Nepal loses roughly a fifth of its cereal harvest to poor storage and transport, and saving that loss adds as much food as a large rise in yield.

Answering the 8-mark biotechnology question

Long questions almost always ask you to "describe the steps" of something — tissue culture, rDNA technology, PCR, or plant breeding. Number the steps, give one sentence and the key reagent or temperature for each, and finish with two applications. A labelled sketch of the plasmid or the cycle earns marks even when the question does not ask for a diagram.

Chapter summary

• Tissue culture works because plant cells are totipotent. Explant → surface sterilisation → MS medium → callus → shoots (high cytokinin) → roots (high auxin) → hardening. It gives disease-free, genetically identical clones all year round.
• Plant breeding improves crops by selection, introduction, hybridisation and mutation breeding; the Green Revolution used dwarf, fertiliser-responsive varieties such as wheat Bhrikuti and rice Sabitri in Nepal.
• Disease resistance is vertical (one R gene, complete but easily broken, gene-for-gene) or horizontal (many genes, partial but durable). Bacillus thuringiensis cry genes make a protoxin that becomes active only in the alkaline insect gut, so Bt crops kill the pest but not the eater.
• Green manure adds organic matter and nitrogen by ploughing in a legume; bio-fertilizers are living organisms (Rhizobium, Azotobacter, Azolla–Anabaena, mycorrhiza, blue-green algae) that supply nutrients; bio-pesticides are living or natural agents (Bt, Trichoderma, neem) that control pests without residues.
• Genetic engineering needs four tools: restriction enzymes to cut (EcoRI cuts G↓AATTC and leaves sticky ends), DNA ligase to join, a vector with ori + marker + unique sites, and a host cell. Steps: isolate → cut → ligate → transform → select → multiply.
• PCR copies a target in vitro using primers and Taq polymerase from Thermus aquaticus: denature 94–95 °C, anneal 50–65 °C, extend 72 °C; after n cycles one molecule becomes 2^n copies.
• In gel electrophoresis DNA is negatively charged and moves to the anode; small fragments travel furthest. A linear molecule with n cut sites gives n+1 fragments, a circular one gives n.
• GMOs include Bt cotton, Bt brinjal, Golden Rice and humulin-producing E. coli. Their release is controlled under the Cartagena Protocol; no GM crop is approved for cultivation in Nepal.
• Food safety is freedom from biological, chemical and physical hazards (HACCP, DFTQC); food security is availability, access, utilisation and stability of enough safe food for all.

Practice questions

Group A — Multiple choice (1 mark each)

1. The capacity of a single differentiated plant cell to regenerate a complete plant is called [1]atotipotencybpluripotencycapomixisdsenescence
2. In a tissue culture medium, shoot formation from callus is favoured by [1]ahigh auxin : low cytokininbhigh cytokinin : low auxincauxin alonedgibberellin alone
3. EcoRI recognises and cuts the sequence [1]aGGATCCbAAGCTTcGAATTCdCTGCAG
4. Taq polymerase used in PCR is obtained from [1]aEscherichia colibThermus aquaticuscAgrobacterium tumefaciensdBacillus thuringiensis
5. During agarose gel electrophoresis DNA fragments move towards the anode because DNA is [1]apositively chargedbneutralcnegatively chargeddamphoteric
6. Which one of the following is a bio-fertilizer? [1]aureabRhizobiumcBacillus thuringiensisdneem oil

Answers to Group A

1. (a) — totipotency is the basis of all plant tissue culture.

2. (b) — a high cytokinin to auxin ratio induces shoots; the reverse induces roots.

3. (c) — EcoRI cuts the palindrome G↓AATTC, leaving four-base sticky ends.

4. (b) — T. aquaticus lives in hot springs, so its polymerase survives 95 °C.

5. (c) — the phosphate groups of the backbone carry a negative charge at all pH.

6. (b) — Rhizobium fixes nitrogen; Bt and neem are bio-pesticides, urea is chemical.

Group B — Short answer (4 marks each)

1. What is micropropagation? List its steps in order and give two advantages over conventional propagation. [4]
2. Differentiate between vertical and horizontal resistance in plants, with one example of each. [4]
3. Explain how the Bt toxin kills a caterpillar but is harmless to human beings. [4]
4. A PCR tube contains 8 copies of a target sequence and is run for 25 cycles at full efficiency. Calculate the number of copies formed, and state why the actual yield is lower after about 35 cycles. [4]
5. Distinguish between green manure and bio-fertilizer, giving two examples of each. [4]

Answers to Group B

1. Micropropagation is the rapid multiplication of plants in vitro from small explants using tissue culture. Steps: (i) select and surface-sterilise the explant with 0.1 % HgCl₂ or sodium hypochlorite; (ii) inoculate on MS medium with a high cytokinin : auxin ratio to get shoots (through callus if needed); (iii) transfer the shoots to a high auxin medium for rooting; (iv) harden the plantlets in a greenhouse, then transfer to the field. Advantages: thousands of genetically identical, disease-free plants from one mother plant in a small space, all the year round; it works for species that are sterile or slow to propagate, such as banana and orchids.

2. | Vertical resistance | Horizontal resistance | |---|---| | Governed by one or a few major R genes | Governed by many minor genes (polygenic) | | Complete immunity, but only to certain races | Partial, slows the disease down | | Breaks down when a new race appears | Durable across races | | Easy to transfer by breeding | Difficult, needs many crosses | | Example: Sr stem-rust genes in wheat | Example: field resistance to late blight of potato |

3. Bacillus thuringiensis makes a crystalline protoxin (Cry protein) that is inactive as made. The caterpillar eats leaf tissue containing it. The insect midgut is strongly alkaline (pH about 9–10), so the crystal dissolves there and gut proteases cut it into the active toxin. The toxin binds to specific receptors on the midgut epithelium and makes pores, so the cells swell and burst; the insect stops feeding and dies. The human stomach is strongly acidic (pH 1.5–3), the crystal does not dissolve, the activating proteases are absent and human gut cells carry no receptor for it — so the protein is simply digested like any other protein.

4. Copies = N_0 \times 2^n = 8 \times 2^{25}. 2^{25} = 33{,}554{,}432, so N = 8 \times 33{,}554{,}432 = 268{,}435{,}456 \approx 2.68 \times 10^{8} copies. After about 35 cycles the primers and dNTPs are used up, Taq polymerase loses activity from repeated heating, and the many product strands re-anneal to each other instead of to primers, so the reaction enters the plateau phase and the number of copies stops doubling.

5. | Green manure | Bio-fertilizer | |---|---| | A green crop grown and ploughed back into the same field | A preparation of living micro-organisms applied to seed, soil or root | | Adds organic matter, nitrogen and improves soil structure | Supplies nutrients by fixing N₂ or solubilising phosphate | | Acts after decomposition, over one season | Acts as long as the organism stays alive in the soil | | Examples: Sesbania aculeata (dhaincha), Crotalaria juncea (sunn hemp) | Examples: Rhizobium, Azotobacter, Azolla–Anabaena, mycorrhiza |

Group C — Long answer (8 marks each)

1. Describe the steps of recombinant DNA technology. Mention the role of restriction endonuclease, DNA ligase and a cloning vector, and explain how recombinant cells are selected. [8]
2. Describe plant tissue culture under the headings: principle, medium, steps and applications. [8]
3. Define food safety and food security. Explain the four pillars of food security and describe four ways in which biotechnology can improve food safety and food security in Nepal. [8]

Answers to Group C

1. Outline. (i) Isolation of DNA — lyse cells with lysozyme or cellulase, digest protein with protease and RNA with ribonuclease, precipitate DNA with chilled ethanol. (ii) Cutting — a restriction endonuclease such as EcoRI cuts both the donor DNA and the vector at the same palindromic sequence (G↓AATTC), giving complementary sticky ends. (iii) Ligation — the sticky ends base-pair and DNA ligase seals the phosphodiester bonds, producing recombinant DNA. (iv) Vector — the plasmid (pBR322) or Ti plasmid carries an ori so it replicates in the host, selectable marker genes, and unique restriction sites. (v) Transformation — the rDNA is introduced by CaCl₂ + heat shock, electroporation, gene gun or Agrobacterium. (vi) Selection — insertional inactivation: the insert destroys tetᴿ, so recombinants grow on ampicillin but not on tetracycline; or blue–white screening on X-gal, where recombinant colonies are white. (vii) Culture and downstream processing — the selected clone is grown in a bioreactor or regenerated into a plant and the product purified. Applications: insulin, vaccines, Bt crops, Golden Rice.

2. Principle: totipotency — every living plant cell carries the whole genome and can regenerate a complete plant under the right conditions. Medium: Murashige and Skoog (1962) medium supplying macronutrients, micronutrients, 2–3 % sucrose, vitamins, myo-inositol, agar and growth regulators; autoclaved at 121 °C and kept at pH 5.6–5.8. Steps: choose the explant (shoot tip, node, anther, embryo) → surface sterilise → inoculate aseptically → callus induction with 2,4-D → organogenesis: high cytokinin : auxin gives shoots, high auxin gives roots → subculture → hardening and transfer to soil. Types: callus, suspension, meristem, anther (haploids), embryo rescue and protoplast culture. Applications: rapid clonal multiplication of banana, orchid, potato and sugarcane; virus-free plants from meristem tips; haploids and homozygous lines from anther culture; somatic hybrids by protoplast fusion; germplasm conservation by cryopreservation; secondary metabolites from cell suspension; and it is the essential regeneration step in making any transgenic plant.

3. Food safety means food that is free from biological, chemical and physical hazards and is fit to eat. Food security (FAO, 1996) exists when all people, at all times, have physical, social and economic access to sufficient, safe and nutritious food for an active, healthy life. Four pillars: availability (enough food produced or imported); access (people can afford and reach it); utilisation (the body can use it — safe food, clean water, balanced diet); stability (the first three do not fail in drought, flood or price shock). Contribution of biotechnology in Nepal: (i) tissue culture supplies disease-free potato, banana and orchid planting material, raising yield; (ii) improved and stress-tolerant varieties bred with marker-assisted selection add availability and stability; (iii) bio-fertilizers and bio-pesticides cut chemical residues, so food is safer and cheaper to grow; (iv) PCR and ELISA in the DFTQC and Seed Quality Control Centre laboratories detect pathogens, aflatoxin and unapproved GM material in imports; (v) biofortified crops such as Golden Rice and zinc-rich wheat and better post-harvest storage reduce malnutrition and the large storage losses.