Plant Tissue Culture Techniques

Huseyin Tombuloglu, Phd.GBE 304 Spring 2015

Definition

the culture of plant seeds, organs, tissues, cells, or protoplasts on nutrient media under sterile conditions.

Basis for Plant Tissue Culture

Two Hormones Affect Plant Differentiation: Auxin: Stimulates Root Development Cytokinin: Stimulates Shoot Development

Generally, the ratio of these two hormones can determine plant development: Auxin ↓Cytokinin = Root Development Cytokinin ↓Auxin = Shoot Development Auxin = Cytokinin = Callus Development

Control of in vitro cultureCytokinin

Auxin

Leaf strip

AdventitiousShoot

Root

Callus

Three Fundamental Abilities of Plants

Totipotencythe potential or inherent capacity of a plant cell to develop into an entire plant if suitably stimulated.It implies that all the information necessary for growth and reproduction of the organism is contained in the cell

DedifferentiationCapacity of mature cells to return to meristematic condition and development of a new growing point, follow by redifferentiation which is the ability to reorganise into new organ

Competencythe endogenous potential of a given cells or tissue to develop in a particular way

Totipotency: ability of a cell or tissue or organ to grow and develop into a fully differentiated organism.

Plant cells are totipotent

Tissue Culture: De-differentiation to Regeneration

seeds

Wheat inflorescence

+ auxinCallus- auxin/ +cytokinin

embryos

Culture Media:

Callus (mass of parenchymatous cells)

1. Minerals2. Carbon source3. Plant Growth Regulators

Explant: any living tissue: leaf, root, zygotic embryos

organogenesis Somatic embryogenesis

Plant regeneration

Organogenesis

Unique to plants. Plant tissue in vitro may produce (de novo) many types of primordia such as shoot and root

Explant Callus meristemoid organ primordia

Explant meristemoid organ primordia

Explant de-differentiation induction differentiation organ

Non-zygotic embryogenesis or somatic embryogenesis

Explant callus embryogenic callus somatic embryo plant

2,4-D BA, zeatin

Some direct applications of tissue culture:1. Synthetic seed technology: encapsulation of somatic embryos

2. Seedless fruits: plants regenerated from triploid endosperm are unable to undergo meiosis

ProtoplastsLandmark:1960: E. C. Cocking (Univ Nottingham) isolated protoplasts by treating explants with concentrated cellulase isolated from a fungus. [Commercial cellulase and macerozyme were not available till 1968]. Tobacco protoplasts

Protoplast fusion1. Somatic hybrids2. Cybrids A cytoplasmic hybrid (or cybrid a portmanteau of the two words

Inter-specific fusions

Datura innoxia X D. stramonium = D. straubii (O. Schieder)

Tomato X Potato = Tomoffel (G. Melchers)

Cybrid Technology

Mixing two cytoplasm without hybrid formation.

2n x 2n4n Synkaryon

2nNuclei fusion

Nuclei separateHeterokaryon

Fusion of haploid protoplasts (derived from anther cultures)n + n= 2n

Somatic hybrids

TomTato plant tomoffel

Haploid Culture

Haploid plant (n) = recessive mutations displayedn+n= double haploid

Later: Microspore cultures.

Occur spontaneously in inter-specific cross or induced byirradiating pollen prior to pollination. Extremely poor efficiency.

Protoplast fusion: gametic hybridization

Haploid cells Protoplast (n)

n X n 2n (synkaryon)

Anther culture techniques/ fusion has been extensively used in rice breeding program

Applications of tissue culture to plant breeding

1. Haploid production (rice, wheat and barley)2. Triploid production (fruits and poplar)3. Embryo Rescue/ Wide hybridization (numerous examples) 4. Somatic hybridization (scientific examples, few commercial products)5. Somaclonal Variations (Tomato with altered color, taste and texture

by Fresh World Farms; Imidazolinone resistant maize, American Cyanimid; Bermuda grass (Brazos R-3) with increased resistance to fall armyworm etc.)

6. Production of disease free plants.7. Clonal propagation8. Secondary metabolite production (eg. Taxol production from cell

cultures derived from the bark cuttings of pacific yew tree)9. Germplasm conservation (cryopreservation)

Seedless watermelon triploid that produces excellent fruit with a dark green skin, consistent round shape, sweet taste (with an average Brix sugar content level of 11 degrees), deep red color, density and texture. In addition, it has intermediate resistance* to both Anthracnose and Fusarium wilt race 1. 

The available genetic resources for citrus fruit are predominantly diploid. Nonetheless, polyploidy can give citrus fruit interesting characteristics. Thus, the genetic breeding programmes aim to create triploid varieties, which are sterile and produce seedless fruit.

Food ProspectsMalthus’s 1798 book: Essay on population: population growth will soon outpace food production.

Marx Das Kapital: Agric will follow the experience of manufacturing, becoming an increasingly concentrated sector with many workers per farm with each worker specializing in small fraction of the tasks involved in farm operation. The USSR and China tried to implement this vision.

Ecologist Paul Ehrlich’s 1968 book: The Population Bomb predicted that the world will undergo famines in 1970s, hundreds of millions of people will starve to death in spite of any crash programs embarked upon now. It is too late!!!

William and Paul Paddock’s 1967 book Famine 1975! America’s Decision: Who will survive? advocated a triage approach to foreign aid. The “can’t be saved group” that included India and Philippines should not receive any aid.

Biologist Garrent Hardin became famous for coining the term “the tragedy of the commons” to describe the problems that can arise from conflicts of interest when there is open access to exploitation to natural resources. In 1977 he published The Limits of Altruism in support of “tough-minded” approach recognizing that countries such as India had exceeded their “carrying” capacity.

Yet over the past century growth in productivity of both land and labor has enabled world food supplies to outpace the unprecedented increase in food demand caused by jumps in the growth rate of world income and by doubling and redoubling of population.

All these theorists were wrong!!

What contributed to this phenomenal increase in ag productivity in last 50 years?

1. Selection of plant varieties: sophisticated genetics based breeding technique.

2. Crop management.3. Improvement of animal breeds.4. New methods of controlling pests and diseases.

Despite doubling and redoubling of crop yields seen in some developing countries, any absolute yield ceiling seems far off.

Scientists have estimated yields that can be generated if a plant is given all the inputs it needs. For most cereals, potential yields are several multiples of the present average US yield.

How far from a yield ceiling?

Year1866 1936 1956 1996

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Yield of a crop is a function of biomass x harvest index (HI). Hence yield can be improved by increasing biomass or HI or both. Since HI of many crops is approaching a ceiling value, so to increase yield potential we have to increase crop biomass, i.e. there will have to be more photosynthesis. The theoretical limits of solar energy utilization efficiency in photosynthesis and the efficiency attained by crop plants provide possibilities and scope for improvement of photosynthetic productivity.

What role might biotechnology play in sustainable agriculture?

"Sustainable agriculture" is both a term and a concept whose definition has varied a great deal. As articulated in the 1990 "Farm Bill" Food, Agriculture, Conservation, and Trade Act of 1990, P.L. 101-624, Title XVI, Subtitle A, Section 1603) sustainable agriculture means "an integrated system of plant and animal production practices having a site-specific application that will, over the long term: (A) satisfy human food and fiber needs; (B) enhance environmental quality and the natural resource base upon which the agricultural economy depends; (C) make the most efficient use of nonrenewable resources and on-farm resources and integrate, where appropriate, natural biological cycles and controls; (D) sustain the economic viability of farm operations; and (E) enhance the quality of life for farmers and society as a whole."

Biotechnology has the potential to assist farmers in reducing on-farm chemical inputs and produce value-added commodities. Conversely, there are concerns about the use of biotechnology in agricultural systems including the possibility that it may lead to greater farmer dependence on the providers of the new technology. Where these two new developments will lead agriculture is open for debate.