GENETICS AND PLANT BREEDING

Introduction to Plant Biotechnology

Dr. Rhitu Rai

Scientist

National Research Centre on Plant Biotechnology

Lal Bhadur Shastri Building

Pusa Campus

New Delhi-110012

(16-07- 2007)

CONTENTS

Keywords

Totipotency, haploid production, cryopreservation, somaclonal variation, somatic hybrids, Agrobacterium, recombinant DNA, transgenic plants, molecular breeding History of Plant Tissue Culture and Biotechnology Scope and Importance of Biotechnology Tissue and Cell Culture Micropropagation Anther Culture Production of Secondary Metabolites Protoplasts Isolation and Fusion Somatic Hybrids Somaclonal Variation Germplasm Conservation Genetic Engineering and Gene Technology Methods for Gene Transfer Generation of Transgenic Plants and their identification Molecular Markers Role of Biotechnology in Crop improvement

History of Plant Tissue Culture and Biotechnology

Biotechnology is name given to the methods and techniques that involve the use of living organisms like bacteria, yeast, plant cells etc or their parts or products as tools (for example, genes and enzymes). They are used in a number of fields: food processing, agriculture, pharmaceutics, and medicine, among others. Plant tissue culture can be defined as culture of plant seeds, organs, explants, tissues, cells, or protoplasts on nutrient media under sterile conditions.

The science of plant tissue culture takes its roots from path breaking research in botany like discovery of cell followed by propounding of cell theory. In 1839, Schleiden and Schwann proposed that cell is the basic unit of organisms. They visualized that cell is capable of autonomy and therefore it should be possible for each cell if given an environment to regenerate into whole plant. Based on this premise, in 1902, a German physiologist, Gottlieb Haberlandt developed the concept of in vitro cell culture. He isolated single fully differentiated individual plant cells from different plant species like palisade cells from leaves of Laminum purpureum, glandular hair of Pulmonaria and pith cells from petioles of Eicchornia crassiples etc and was first to culture them in Knop's salt solution enriched with glucose. In his cultures, cells increased in size, accumulated starch but failed to divide. Therefore, Haberlandt's prediction failed that the cultured plant cells could grow, divide and develop into embryo and then to whole plant. This potential of a cell is known as totipotency, a term coined by Steward in 1968. Despite lack of success, Haberlandt made several predictions about the requirements in media in experimental conditions which could possibly induce cell division, proliferation and embryo induction. G Haberlandt is thus regarded as father of tissue culture.

Taking cue from Haberlandt's failure, Hannig (1904) chose embryogenic tissue to culture. He excised nearly mature embryos from seeds of several species of crucifers and successfully grew them to maturity on mineral salts and sugar solution. In 1908, Simon regenerated callus, buds and roots from Poplar stem segments and established the basis for callus culture. For about next 30 years (upto 1934), there was very little further progress in cell culture research. Within this period, an innovative approach to tissue culture using meristematic cells like root and stem tips was reported by Kolte (1922) and Robbins (1922) working independently.

All these research attempts involving culture of isolated cells, root tips or stem tips ended in development of calluses. There were two objectives to be achieved before putting Haberlandt's prediction to fruition. First, to make the callus obtained from the explants to proliferate endlessly and second to induce these regenerated calluses to undergo organogenesis and form whole plants. It was in 1930s, when progress in plant tissue culture accelerated rapidly owing to an important discovery that vitamin B and natural auxins were necessary for the growth of isolated tissues containing meristems. This breakthrough came from White (1934) who reported that not only could cultured tomato root tips grow but could be repeatedly subcultured to fresh medium of inorganic salts supplemented with yeast extract. He later (1937) replaced YE by vitamin B namely pyridoxine, thiamine and proved their growth promoting effect.

In 1926, Fritz Went discovered first plant growth regulator (PGR), indoleacetic acid (IAA). IAA is a naturally occurring member of a class of PGRs termed 'auxins'. Roger J Gautheret (1934) reported the successful culture of cambium cells of several tree species to produce callus and

addition of auxin enhanced the proliferation of his cambial cultures. Further research by Nobecourt (1937), who could successful grow continuous callus cultures of carrot slices and White (1939) who obtained similar results from tumour tissues of hybrid Nicotiana glauca x N langsdorffii. Thus, the possibility of cultivating plant tissues for an unlimited period was independently endorsed by Gautheret, White and Nobecourt in 1939.

Adding to the ongoing improvements in the culture media, Johannes Van Overbeek (1941) reported growth of seedlings from heart shaped embryos by enriching culture media with coconut milk besides the usual salts, vitamins and other nutrients. This provided tremendous impetus for further work in embryo culture. Stem tip cultures yielded success when Ernest Ball (1946) devised a method to identify the exact part of shoot meristem that gives rise to whole plant.

After 1950, there was an immense advancement in knowledge of effect of PGRs on plant development. The fact that coconut milk (embryo sac fluid) is nutritional requirement for tobacco callus besides auxin, indicated the non auxinic nature of milk. This prompted further research and so other classes of PGRs were recognized. Skoog and Tsui (1957) demonstrated induction of cell division and bud formation in tobacco by adenine. This led to further investigations by Skoog and Miller (1955) who isolated 'kinetin'- a derivative of adenine (6-furyl aminopurine). Kinetin and many such other compounds which show bud promoting activities are collectively called cytokinins, a cell division promoter in cells of highly mature and differentiated tissues. Skoog and Miller worked further to propose the concept of hormonal control of organ formation (1957). Their experiment on tobacco pith cultures showed that high concentration of auxin prmoted rooting and high kinetin induces bud formation or shooting. However, now the concept is altered to multiple factors like source of plant tissue, environmental factors, composition of media, polarity, growth substances being responsible for determination of organogenesis. Besides PGRs, scientists tried to improve culture media by differing essentially in mineral content. In this direction, Murashige and Skoog (1962) prepared a medium by increasing the concentration of salts twenty-five times higher than Knops. This media enhanced the growth of tobacco tissues by five times. Even today MS medium has immense commercial application in tissue culture.

Having achieved success and expertise in growth of callus cultures from explants under in vitro conditions, focus now shifted to preparation of single cell cultures. Muir (1953-54) demonstrated that when callus tissues were transferred to liquid medium and subjected to shaking, callus tissues broke into single cells. Bergmann (1960) developed a technique for cloning of these single cells by filtering suspension cultures. This technique called Plating technique is widely used for cloning isolated single protoplasts.

Next step for realization of Haberlandt's objectives was development of whole plant from the proliferated tissue of these cells. Vasil and Hilderbrandt were first to regenerate plantlets from colonies of isolated cells of hybrid Nicotiana glutinosa x N tabacum. In 1966, the classical work of Steward on induction of somatic embryos from free cells in carrot suspension cultures brought an important breakthrough by finally demonstrating totipotency of somatic cells, thereby validating the ideas of Haberlandt. This ability of regenerating plants from single somatic cells through normal developmental process had great applications in both plant propagation and also

genetic engineering. For e.g. micropropagation where small amounts of tissue can be used to continuously raise thousand more plants. Morel utilized this application for rapid propagation of orchids and Dahlias. He was also the first scientist to free the orchid and Dahlia plants from virus by cultivating shoot meristem of infected plants.

The role of tissue culture in plant genetic engineering was first exemplified by Kanta and Maheshwari (1962). They developed a technique of test tube fertlization which involved growing of excised ovules and pollen grains in the same medium thus overcoming the incompatibility barriers at sexual level. In 1966, Guha and Maheshwari cultured anthers of Datura and raised embryos which developed into haploid plants initiating androgenesis. This discovery received significant attention since plants recovered from doubled haploid cells are homozygous and express all recessive genes thus making them ideal for pure breeding lines.

Next breakthrough in application of tissue culture came with isolation and regeneration of protoplasts first demonstrated by Prof. Edward C Cocking in 1960. Plant protoplasts are naked cells from which cell wall has been removed. Cocking produced large quantities of protoplasts by using cell wall degrading enzymes. After success in regeneration of protoplasts, Carlson (1972) isolated protoplasts from Nicotiana glauca x N. langsdorfii and fused them to produce first somatic hybrid. Since then many divergent somatic hybrids have been produced.

With the advent of restriction enzymes in early 1970s, tissue culture headed towards a new research area. The totipotent plant cells could now be altered by insertion of specific foreign genes giving rise to genetically modified crops. In 1970, Smith and Nathans isolated first restriction enzyme from Haemophillus influenzae which was later purified and named Hind III. Same year witnessed other nobel prize winning discovery by Baltimore who isolated Reverse transcriptase from RNA tumor viruses. This is a useful enzyme in genetic engineering which functions to convert RNA to DNA and hence useful in construction of complementary DNA from messenger RNA. Another pathbreaking discovery establishing potential of genetic engineering came in 1972 when Paul Berg working at Stanford University produced first recombinant DNA in vitro by combining DNA from SV40 virus with that of lambda virus. This led to construction of first recombinant organism by Cohen and Boyer in 1973. Genetic engineering's potential was first exploited when a man made insulin gene was used to manufacture a human protein in bacteria.

Agrobacterium tumefaciens plays a crucial role in plant genetic engineering. The involvement of this bacterium in crown gall disease in plants was recognized as early as 1907 by Smith and Townsend. However, it was in 1974, that Zaenen et aldiscovered that Ti plasmid is the tumor inducing principle of Agrobacterium. This was followed by its successful integration in plants by Chilton et al in 1977. Zambryski et al in 1980 isolated and studied the detailed structure of T-DNA and its border sequences. Soon thereafter in 1984, transformation of tobacco with Agrobacterium was accomplished to develop transgenic plants. Simultaneously, there was an upsurge in development of techniques of genetic engineering in mid 1970s. Sanger et al(1977) and Maxam and Gilbert (1977) reported techniques for large scale DNA sequencing. This was followed by complete genome sequencing projects on many prokaryotes and eukaryotes like Haemophilus influenzae in 1995, E coli in 1997. Human genome was sequenced successfully in 2001, thus laying foundation of genomics which is the main focus of present day biotechnology.

Some of the landmark discoveries are tabulated below:

1902

Haberlandt proposed concept of in vitro cell culture

1966

Guha and Maheshwari produced first haploid plants from pollen grains of Datura

1904

Hannig cultured embryos from several cruciferous species

1970

Smith and Nathans discovered first restriction enzyme from Haemophilus influenza (HindIII)

1922

Kolte and Robbins successfully cultured root and stem tips respectively

1970

Baltimore isolated Reverse transcriptase from RNA tumour virus

1926

Went discovered first plant growth hormone -Indole acetic acid

1972

Carlson produced first interspecific hybrid of Nicotianaby protoplast fusion

1934

White introduced vitamin B as growth supplement in tissue culture media for tomota root tip

1972

Berg produced first recombinant DNA , combining SV40 virus and λ virus

1939

Gautheret, White and Nobecourt established endless proliferation of callus cultures

1974

Zaenen et al discovered Ti plasmid is tumour inducing principle of agrobacterium

1941

Overbeek was first to add coconut milk for cell division in Datura

1975

O'Farrel developed high resolution two dimensional gel electrophoresis system

1946

Ball raised whole plants of Lupinus by shoot tip culture

1977

Chilton et al successfully integrated Ti plasmid DNA from Agrobacterium tumefaciens in plants

1954

Muir was first to break callus tissues into single cells

1977

Sanger, Maxam-Gilbert gave technologies for DNA sequencing

1955

Skoog and Miller discovered kinetin as cell division hormone

1980

Zambryski detailed structure of T-DNA and border sequences

1957

Skoog and Miller gave concept of hormonal control (auxin: cytokinin) of organ formation

1983

Kary Mullis invented Polymerase chain reaction (PCR), for amplification of DNA.

1959

Reinert and Steward regenerated embryos from callus clumps and cell suspension of Daucus carota

1984

Horsh et al developed transgenic tobacco by transformation with Agrobacterium

1960

Cocking was first to isolate protoplast by enzymatic degradation of cell wall

1987

Klien et al developed biolistic gene transfer method for plant transformation

1960

Bergmann filtered cell suspension and isolated single cells by plating

1995

Fleischmann et al sequenced Haemophilus influenzae

1962

Murashige and Skoog developed MS medium with higher salt concentration

1997

Blattner et al sequenced E coli genome

1962

Kanta and Maheshwari developed test tube fertilization technique

2001

Human genome Project consortium and Venter et alsequenced human genome successfully

1966

Steward demonstrated totipotency by regenerating carrot plants from single cells of tomato

2005

Rice genome sequenced under International Rice Genome Sequencing Project

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