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Plant Tissue Culture: Historical Perspectives

Lesson Prepared Under MHRD project “National Mission on

Education through ICT”

Discipline: Botany

Paper: Plant Biotechnology

National Coordinator: Prof. S.C. Bhatla

Lesson: Plant Tissue Culture: Historical Perspectives

Lesson Developer: Kuldeep Sharma, Dr. Siva Prasad Konwar Chetri,

Monika Heikrujam, Prof. Veena Agrawal

Department/College: Department of Botany, University of Delhi,

Lesson Reviewer: Dr. Vinay Kumar Baranwal

Department of Plant Molecular Biology

University of Delhi, South Campus

Language Editor: Namrata Dhaka

Department/College: Department of Genetics, University of Delhi,

South Campus

Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL


Learning outcomes

The students shall learn about the following:

The important discoveries that led to development of modern techniques in tissue

culture.

The scientists who played important role in development of tissue culture methods.


Table of Contents

Chapter: Plant Tissue Culture: Historical Perspectives

Introduction

Historical Developments

Somatic embryogenesis and synthetic seeds

In vitro regeneration of haploids and triploids

In vitro pollination and fertilization

In vitro production of somatic hybrids

Somaclonal variations

In vitro production of secondary metabolites and metabolic

engineering

Genetic engineering: Production of transgenic crops

Summary

Exercise

Glossary

References

Web Links


Introduction

Plant tissue culture is a collective term to grow plant cells, protoplasts, meristems (axillary

or apical), anther, ovary, embryo, tissues, organs, etc or even any plant part axenically

under defined chemical compositions and physical conditions in vitro.

Figure: The cyclic process of plant tissue culture from an explant for regeneration of

complete plantlets.

Source: ILLL inhouse

The concept of plant tissue culture was addressed by a German botanist Gottieleb

Haberlandt in 1902 in his classical paper presented before the Vienna Academy of Sciences

in Berlin. However, the concept of totipotency was put forward by Schleiden (1838) and

Schwann (1839) in their famous cell theory and the term was probably introduced by T. H.

Morgan in 1901.

Now, it is feasible to regenerate a complete plant by culturing a single cell, a tissue or an

organ regardless of their source and ploidy level in vitro.


Figure: Depiction of the various approaches for in vitro regeneration of a complete plant,

viz. embryo culture, organ culture, callus culture and cell culture.

Source: ILLL inhouse

Nonetheless, the plant tissue culture has seen enormous progress over the century leading

to many revolutionary studies around the world and further giving the required momentum

for commercial production of micro propagated plants, somatic embryo and synthetic seed

production, in vitro production of somatic hybrids, in vitro production of haploids, triploids,

in vitro pollination and fertilization, selection of somaclonal variants and mutants, in vitro

production of secondary metabolites and most significantly the incorporation of the desired

gene in the plant genome. Tissue culture serves as an indispensable tool for transgenic

plant production in genetic engineering and metabolic engineering. For nearly any

transformation system an efficient regeneration protocol is imperative.


Figure: Prospects and applications of plant tissue culture.

Source:http://www.eplantscience.com/index/images/Biotechnology/chapter09/079_small.jp

g

Today, the field of plant tissue culture is considered as a unique combo of science and art

being basic and an essential integral to plant biotechnology; enabling the crops amenable

for their genetic and metabolic manipulations as well as providing the experimental

materials to study the growth and developmental processes at physiological, biochemical

and molecular levels.

Historical developments

In the history of plant tissue culture, Gottlieb Haberlandt (1898) was the first person to

culture the isolated leaf mesophyll tissues and hair cells in vitro on an artificial medium.

Figure: Gottlieb Haberlandt- The father of plant tissue culture who conceived the concept of

plant cell culture in 1902.

Source: https://en.wikipedia.org/wiki/Gottlieb_Haberlandt (cc)

Haberlandt (1898) was the pioneer to culture plant cells isolated from the leaves of Lamius

purpureoum, petioles of Eichornia crassipes, epidermis of Ornithogalum, epidermal hairs of

Pulmonaria mollissima, stamen hairs of Tradescantia in Knop‟s (1865) solution with a view

to achieve the division of cells and subsequently raising complete plants from them. The

cultured cells could grow for a month only and no division was seen in the cells. But an

increase in cell shape, thickening of cell walls and deposition of starch in the chloroplasts

http://www.eplantscience.com/index/images/Biotechnology/chapter09/079_small.jpg



https://en.wikipedia.org/wiki/Gottlieb_Haberlandt


were observed in the cultured cells. Unfortunately, he failed to achieve his goals but on the

basis of his observations he established the concept of totipotency and several postulates

and principles of plant tissue culture in 1902. He opined that a cell‟s growth stops after

acquiring the features required by the entire organism retaining its inherent potentiality for

further growth on getting suitable stimulus. He further affirmed that one might consider the

utilization of embryo sac fluids and an extract from vegetative apices or else culture the

cells from vegetative apices. He also postulated the possibility to obtain artificial embryos

from vegetative cells. He also suggested for culturing the pollen tubes and vegetative cells

together in hanging drops to induce the divisions in vegetative cells probably based on the

observations of Winkler (1901) that the pollen tubes stimulate growth in ovules and ovary.

Simultaneously, Hanning in 1904 too initiated the work on embryo culture excised from

seeds of several crucifers for the first time. However, it was Liabach who accomplished the

dream of Hanning later in 1925 and demonstrated first embryo culture in a Linum hybrid (L.

perenne X L. austriacum) with a view to rescue the embryo which gets aborted at

premature stage (because of the incompatibility of the two species) and further to obtain

the complete plantlet from the cultures. Another important initiative was put forward by

Kotte (one of Haberlandt‟s student) from Germany and independently by Robbinson from

USA who succeeded in proliferation of excised root/ shoot tips of pea and maize in vitro in

1922. Later, discovery of an auxin, indole acetic acid (IAA) by F. Kogle and his coworkers,

recognition of significance of vitamins B in the culture medium and culture of isolated roots

by Philip White (1934), callus cultures of tobacco hybrid (Nicotiana glauca X N. langsdorfii)

by P. White and Nobecourt (1939), cultures from root cambium in carrot by Gautheret

(1939), demonstration of stimulatory effect of coconut milk for cell division in Datura by Van

Overbeek (1941) and further their application in tissue culture medium, culture and

proliferation of meristem and shoot tips in Asparagus, Cuscuta, by Loo (1945) are some of

the vital events which have laid down considerable pebbles in the progression of plant tissue

culture. Thereafter, the arena of plant tissue culture has been accompanied by major

breakthroughs and pioneer works including in vitro cultivation of plant cells, tissues, and

organs and further their proliferation and differentiation as well as formulation of several

nutrititive substitutes (culture medium) especially during 1920 to 1960 which have

established remarkable progress in this field. In this progression, most of the postulates and

principles of Haberlandt became true contributing a significant role in understanding plant

biochemistry, plant physiology, cytodiffrentiation, etc. Therefore, Gottilieb Haberlandt

(1854- 1945) has now been recognized as the father of plant tissue culture.


Figure: P. Nobecourt; He along with White and Gautheret achieved in vitro cultivation of

plant tissues for an indefinite time period in 1939.

Source: http://users.ugent.be/~pdebergh/his/his7az1.htm

Figure: P. R. White; In 1934, White was the pioneer to obtain indefinite cultures with root

tips from tomato plants.


In 1946, E. A. Ball, who is probably considered the as the father of micropropagation

exhibited the development of complete plants of Tropeolum and Lupinus from shoot tip

cultures.

http://users.ugent.be/~pdebergh/his/his7az1.htm



Figure: E. Ball; Ball successfully developed an important technique in 1946 to raise

transplantable complete plants of Lupinus and Tropaeolum by culturing their shoot tips with

leaf primordia.


In 1952, the synergistic effect of 2, 4- D and coconut milk was demonstrated in potato

tissue culture by Steward and his coworkers. Morel and Martin (1952) revealed that the

virus free plants can be obtained through meristem culture. Later in 1960, it was Morel who

could obtain first virus free progenies in Cymbidium employing meristem culture technique.



Figure: G. Morel; Morel and Martin in 1952 recovered virus-free Dahlia plants from infected

individuals through in vitro culture of their shoot tips.


Muir (1953) successfully broke the callus tissue into small cell aggregates and accomplished

first suspension culture by transplanting it into liquid medium and subsequently in 1954

Muir established the nurse culture. However, it was L. Bergemen (1960) who introduced

Bergemen plating technique and obtained callus on semisolid medium. He produced callus

by mixing the cells with warm sugar and agar medium just prior to gelation. During this

period, incredible findings by a group (Folke Skoog and his co workers) from University of

Wisconsin too gave an impetus to this field. Carlos Miller, F. Skoog and co workers (1958)

discovered the first cytokinin, 6- furfurylaminopurine (kinetin) from degraded sample of

herring sperm DNA. F. Skoog and C. Miller (1957) postulated that the interplay of

exogenously supplied auxins and cytokinins may regulate the fate of morphogenesis in the

cultures. They demonstrated in tobacco cultures that the high levels of auxin induce rooting

and high levels of cytokinin promote shoot bud formation.

Figure: Effect of variable ratios of Cytokinin and Auxin on regeneration of a calli.

Source: http://vle.du.ac.in/mod/book/view.php?id=12147&chapterid=24629 (cc)

Later, Toshio Murashige and F. Skoog (1962) gave the formulation of Murashige and

Skoog‟s (MS) medium which is one of the most extensively employed basal medium today.

They also revealed that nutritional requirements may vary with respect to species or

explants (tissues excised from different parts of a plant).


http://vle.du.ac.in/mod/book/view.php?id=12147&chapterid=24629


Figure: Folke Skoog; Skoog alongwith Miller in 1957 by manipulating the relative

concentrations of auxin and kinetin put forth the concept of chemical control of

organogenesis (root and shoot differentiation). Furthermore, he alongwith Murashige in

1962 formulated the most widely used plant tissue culture medium (Murashige & Skoog‟s

medium).

Source: http://www.news.wisc.edu/newsphotos/skoog.html

Figure: Toshio Murasighe; T. Murashige alongwith F. Skoog in 1962 formulated the most

widely used plant tissue culture medium (MS) for in vitro plant propagation.

Source: www.ishs.ir

http://www.news.wisc.edu/newsphotos/skoog.html

http://www.ishs.ir/


The nutritional requirements of the explants are fulfilled by supplying essential mineral

elements such as K, Ca, Mg, N, P and S (macro nutrients which are required in the large

quantities) and B, Mn, Cu, Zn, Mo, Fe (micro nutrients which are required in small

quantities). Certain vitamins like nicotinic acid, pyridoxine- HCl, thiamine- HCl, folic acid,

biotin along with amino acid, glycine and some organic constituents, inositol, etc. are also

supplied into the medium. Sugar, chiefly sucrose is added to the medium to serve as the

carbon source. Some other sugars like glucose, galactose, fructose, lactose, maltose,

mannose, etc have also been replaced with sucrose often for this purpose. Indole acetic acid

(IAA), indole butyric acid (IBA), napthalen acetic acid (NAA), 2, 4- diphenoxy acetic acid (2,

4- D), 2, 4, 5- triphenoxy acetic acid (2,4, 5- T), naphthoxy acetic acid (NOA) are some of

the key auxins being used in the medium for the stimulation of cell division in the cultures.

Besides, cytokinins like N6- benzylaminopurine (BA), 6- (furfurylamino)purine (Kinetin), (2-

isopentenyl)- adenine (2- iP), or 6- (γ, γ- enoyl amino)- [purine]- dimethylallyl amino)

purine (2-iP), zeatin and thidiazuron (TDZ), etc. gibberellins, ussually GA3 and abscicic acid

(ABA) are frequently used growth regulators which are supplemented in the culture medium

for evoking desired and differential morphogenic responses. Certain undefined organic

supplements, viz. casein hydolysate (CH), coconut water (CW), corn milk, yeast extract,

banana extract, tomato extract, etc. have also been used in the medium to promote the

growth of the callii or shoots in vitro. Agar (0.8 to 1.0%) usually obtained from red algae

(Gelidium and Gracilaria) is the most extensively used gelling agent to make the media semi

solid in nature. Besides, agarose (0.4- 1.0%) has also been reported to be used as a gelling

agent. However, gellon gum (0.1- 0.2%) has proved a better alternative to agar and

agarose. This is obtained from a bacterium, Pseudomonas elodea which is marketed as

gelrite by CP Kelco and Co. and as phytagel by Sigma Alderich Chemical Co. Since, in vitro

morphogenic responses are vitally regulated by the chemical composition of the culture

media. Therefore, before starting with plant tissue culture it has now been established that

it is mandatory to standardize the differential nutritional or hormonal requirements of

different explant or species.

If we look at the history, the earliest culture medium was formulated by Gautheret (1939)

and White (1943). Gautheret‟s callus culture medium formulation developed for the growth

of higher plants was based on Knop‟s (1865) salt solution. White‟s root culture medium was

derived from Uspenski and Uspenkaia‟s formulation which was developed in 1925 for algal

culture.


Figure: R.J. Gautheret; Gautheret in 1939 obtained continuously growing cultures from

carrot root cambium employing Indole-acetic acid and B vitamins.

Source: http://users.ugent.be

Thereafter, several media compositions have been composed by several legendary tissue

culturists. Some of the earlier and fundamental media being used frequently for in vitro

cultivation of plants even today are Heller‟s by Heller (1953), White‟s by White (1963), ER

by Erricson (1965), LS (Linsmaier and Skoog, 1965), B5 by Gamborg et al. (1968), Nitsch‟s

by Nitsch (1969), Woody plant medium (WPM) by Lloyd and McCown (1981). Furthermore,

large varieties of powdered media formulations are enormously available in the market

today and are being sold commercially.

Somatic embryogenesis and synthetic seeds

Somatic embryogenesis is a process by which a somatic cell undergoes dedifferentiation and

redifferentiation for the formation of embryo either directly (on the explant) or indirectly

(via callus phase) under in vitro conditions. First somatic embryo formation was reported by

J. Reinert (1958) from Germany and F. C. Steward (1958) from USA in cell cultures of

carrot.

http://users.ugent.be/


Figure: F. C. Steward; In 1958, he alongwith Reneirt J. established the process of somatic

embryogenesis.

Source:http://allaboutplantbiotechnology.blogspot.in/2012/03/hrefhttpphpweby.html

Since then, development of the discrete bipolar structures, i.e. embryos having plumular

and radicular ends with no vascular connection to the maternal tissue have been

demonstrated variously in versatile plant systems. It would not be exaggeration to state

that the somatic embryogenesis has now been reported in all major species or plants

employing different explants such as root, leaf, shoot tip, floral parts, etc. Even the zygotic

embryos have been reported to be the best suited explants for the induction of somatic

embryos. Raghvan (1976) revealed that somatic embryos usually are devoid of suspensor.

In 1993, Zimmermann established that similar to zygotic embryogenesis, somatic

embryogenesis too proceeds through globular, heart, torpedo and cotyledonary stages. But

contrary to zygotic embryos, somatic embryos do not go through the dessication and

dormancy; the somatic embryos germinate directly and produce fertile plantlets. Somatic

embryogenesis has now been achieved both on semisolid medium and liquid medium and

the former being the potent alternative for scaling up as well large scale production of

somatic embryos. Backs- Husemann and Reinert (1970) were the first to scale up carrot

somatic embryo production, though their success rate was quite low. Since then, several

attempts have been made for the large scale production of somatic embryos using different

types of bioreactors such as stirred tank bioreactor, airlift or bubble- column bioreactor,

round bottom bioreactor, etc.

http://allaboutplantbiotechnology.blogspot.in/2012/03/hrefhttpphpweby.html


The study of somatic embryogenesis further led to the idea of synthetic seed technology.

The synthetic seeds are defined as an alternative to seeds consisting of somatic embryos

surrounded by artificial coats which is most equivalent to an immature zygotic embryo.

Figure: Representing the process of synthetic seed formation, germination and

acclimatization of in vitro plantlet in cactus.

a) and b) Encapsulation of callus and multiple shoots of the cactus. c) Germination of

artificial seed of multiple shoot (two shoots per explant). d) Germination of artificial seed

(one shoot per explant). e) In vitro plantlet from germinated artificial seed. f)

Acclimatization of in vitro plantlet. g- i) In vitro plantlets with various shapes of the cactus

(4, 5, 6 shoots per explant).

Source:http://umfacts.um.edu.my/gallery/index.php?menu=research_details&cid=119


Murashige (1977) was the first to define synthetic seed and accordingly only somatic

embryogenesis was possible. Later, technology was broadened to the encapsulation of

various in vitro derived propagules. Bapat and coworkers (1987) for the first time used

axillary buds of Morus indica encapsulation. Explants such as shoot tips, axillary buds and

somatic embryos are encapsulated in cryoprotectant material like hydrogel, alginate gel,

ethylene glycol, dimethylsulfoxide (DMSO) carragenan, guargum, nitrocellulose, sodium

pectate, etc. Redenbaugh and his coworkers (1998) for the first time proposed the use of

Na- alginate solution which becomes hardened in Ca- alginate gel in the presence of

Calcium chloride (CaCl2).

In vitro regeneration of haploids and triploids

Androgenesis and gynogenesis are the two major approaches employed for the production

of haploid plants in vitro. In 1922, Blackeslee and coworkers identified natural haploids in

Datura stramonium for the very first time. Thereafter, naturally occurring haploids have

been recognized in a number of species such as Antirrhinum, Crepis, Hordeum, Oenothera,

etc. but frequency of such incidences is very poor. For the experimental purposes, the

haploids were being produced artificially via distant hybridization, delayed pollination,

hormonal treatment, temperature shock treatment, etc. until 1964.

In 1964, a major breakthrough was established by two Indian botanists, Shipra Guha

Mukherjee and S. C. Maheshwari at Department of Botany, University of Delhi. They

succeed in developing pollen embryos from anther cultures of Datura innoxia and published

their research work in Nature. This discovery wrenched up lot of expectations among

scientists all over the world for the culture of excised floral organs like anther, pollen, ovary,

ovule, etc for raising haploid plants and subsequently for the obvious induction and

production of double haploids (DHs) or homozygous diploids by diploidization of the haploid

genome (duplication of haploid set of chromosomes).


Figure: S. Guha Mukherjee; She along with S. C. Maheshwari discovered the technique of

production of haploid plants via anther culture in Datura innoxia in 1964.

Source: www.ias.ac.in (cc)

Figure: S C Maheshwari; His group in 1964 made the landmark discovery of the anther

culture technique for the production of haploid plants, which leads to rapid production of

homozygous pure lines, a boon for agriculture. The technique has been adopted worldwide

for improvement of crop, horticultural and ornamental plants.

http://www.ias.ac.in/


Source: Authors

In 1967, Bourgin and Nitsch and later Nitsch and Norreel (1971) succeeded in the

development of complete haploid plants in Nicotiana tabaccum and N. sylvestris. Since then

in vitro androgenesis (production of androgenic haploid plants using anther or pollen

culture) has incessantly achieved in wide range of crop plants including both monocots

(barley, rice, wheat, etc.) and dicots (Atropa, Brassica, capsicum, Nicotiana, etc.).

Figure: androgenic cultures; In vitro production of androgenic haploid plants via

embryogenesis and callus formation.

Source: http://nptel.ac.in/courses/102103016/module1/lec9/images/d1.JPG (cc)

In vitro gynogenesis has also been confirmed in approximately 25- 30 plants, viz. Allium

cepa, Beta vulgaris, Brassica oleracea, Zea mays, etc. for the production of gynogenic

haploids, being valuable in various crop breeding programs. This technique was introduced

http://nptel.ac.in/courses/102103016/module1/lec9/images/d1.JPG


by San Noem in 1976 for raising the haploid plants in Hordeum vulgare. Excised unfertilized

ovules, ovaries and floral buds are the explants which are used for the production of

gynogenic haploids through direct embryogenesis or indirectly via callus phase. This

technique is of immense significance where androgenic haploid production is either not

applicable (dioecious crops and male sterile plants) or unsuccessful.

This in vitro technology of raising plants of altered ploidy (homozygous dihaploids) has

opened up facile routes for genetic and breeding studies and has proven beneficial

especially in case of trees, self incompatible and male sterile plants and greatly for

shortening the life cycle of the plants. Homozygous dihaploid plants (DHs) may be produced

in a single generation exploiting in vitro androgenesis or gynogenesis that are otherwise

produced via several back crosses (Fα) for haploid breeding schemes. This technology is

also useful for detecting the recessive mutants that otherwise do not express in

heterozygous diploid plants. In 1987, Neuhaus and coworkers and later Kuhlmann and

coworkers (1991) demonstrated the transformation of foreign DNA in pollen embryos and

microspore cultures of Brassica napus and Hordeum vulgare, respectively.

As far as triploids are concerned such plants are produced via endosperm culture. However,

triploids obtained via conventional approaches are already in the market especially where

the seedlessness is highly desirable in the economically important crops such as apple,

banana, mulberry, sugarbeat, watermelon, papaya, etc. Lampe and Mills (1933) attempted

the endosperm culture in maize for the first time to investigate the totipotent nature of the

triploid tissue endosperm but he could only obtain slight proliferation of cells. Later, La Rue

and his coworkers (1949) succeeded to grow and differentiate immature maize endosperm

in culture. Till 1960, several attempts were made employing immature and mature

endosperm culture in different crops such as Cucumis sativa, Hordeum vulgare, Zea mays,

etc. but the mature endosperm was not found amenable to induce divisions. Rangaswami

and Rao (1963) from India reported continuously growing callus cultures from mature

endosperm of Santalum album. At the same time in 1963 and 1965, another group of

Indian scientists, Mohan Ram and Satsangi demonstrated the division and proliferation of

mature endosperm cells in Ricinus communis.

Until 1965, none of the scientists could accomplish organogenic differentiation from

endosperm cultures. Finally, B. M. Johri and S. S. Bhojwani (1965) succeeded to obtain full

triploid shoots by culturing a mature endosperm of a root parasite, Exocarpus

cupressiformis. Since then, mature and immature endosperms have been cultured

successfully to regenerate shoots, embryo, plants, etc. in Jatropha panduraefolia, Putranjiva


roxburghii, Morus alba, Oryza sativa, Citrus gandis, C. sinensis, Leptomatria acida and

Carrica papaya, etc. However, this technology has not yet been exploited commercially.

In vitro pollination and fertilization

With the advent of ovule and ovary culture (Nitsch, 1951), botanists developed the

inquisitiveness for the formation of seeds in test tubes. This process of seed formation was

termed as in vitro pollination or test tube fertilization. In vitro pollination involves aseptic

pollination of cultured pistil or ovules to achieve pollen germination, pollen tube growth and

its entry into embryo sac, double fertilization and subsequent development of embryo,

endosperm and seed. However, if we go back to history, the in vitro pollination and

fertilization was initially carried out by Dahlgren (1926) who developed seeds in Codonopsis

ovata. Later, application of pollen grains to excised ovary (ovarian pollination) was

standardized at Department of Botany, University of Delhi, India in the members of

Papavaraceae and Brasicaceae by K. Kanta, P. Maheshwari and their co– workers in 1960s.

Figure: P. Maheshwari; He was one of the leading plant embryologists who successfully

established the technique of test-tube fertilization of angiosperms (1961).


Kanta in 1960, achieved first intra ovarian pollination in Papaver rhoeas and this research

was published in the journal Nature. Later, this group successfully obtained viable seeds in

Papaver somniferum, P. rhoeas, Argemone maxicana and A. ochroleuca. In this technique,

the pollen grains were directly injected into the ovary achieving pollen germination, entry of

pollen tubes into ovules and fertilization. Application of pollen grains to stigma, i.e. stigmatic

pollination was also successfully reported in 1965 by Rao, Shivanna and Usha in Nicotiana



rustica, Petunia violacea and Antirrhinum majus, respectively. In this technique, the whole

pistils along with short length of pedicel and calyx were excised, surface sterilized and

cultured on a regenerating medium. The compatible and aseptic pollen grains were

deposited on the stigma of the cultured pistils. Later, this technique was applied to two

tobacco species, i.e. N. tabacum and N. rustica by Duliieu (1966) and Rao and Rangaswami

(1972) to achieve in vitro pollination followed by fertilization, respectively. But this method

did not prove effective in overcoming sexual incompatibility because of the zone of

inhibition, i.e. stigma and style.

Application of pollen grains to ovules attached to the placenta is equally effective in self

pollination and cross pollination for inducing seeds. Rangaswami (1967) and Shivanna

(1971) devised placental pollination technique at Department of Botany, University of Delhi,

India. Thereafter, in vitro pollination followed by fertilization was also achieved in Dianthus

caryophyllus by Zenketeler in 1965 while working again at University of Delhi. Later on, this

technique was applied to raise the in vitro inter specific and inter generic hybrids by

different groups of scientists using 15 species as paternal parent and two species of

Melandrium, i.e. M. album and M. rubrum as maternal parent which were otherwise

unknown in nature. The 15 paternal parents were mainly the members of Caryophyllaceae,

viz. Cerastium arvense, Dianthus carthusianorum, D. serotinus, Lychnis coronaria,

Melandrium album, M. rubrum, Minuratialaricifolia, Silene alpine, S. freiwaldskyana, S.

schafta, S. tatrica, Vaccaria pyramidata and one each from Campanulaceae, Brasicaceae

and Solanaceae, i.e. Campanulapercisifolia, Hasparis matronalis and Datura stramonium,

respectively.

In vitro gamete fusions are performed without involving surrounding cells of mother tissue

or other cells of the embryo sac in the protoplast fusion manner. The sperm cells and egg

cells are isolated from the paternal and maternal tissues brought into contact with each

other under ideal conditions to ensure their fusion. Thus, starting with gamete fusion, the

development of a single zygote into a two celled and multicellular embryo and finally into a

higher plant can be traced in vitro. The key steps or micro- techniques involved in in vitro

fertilization are i) isolation of viable male gametes, ii) isolation of viable female gametes iii)

fusion of isolated male and female gametes and iv) single zygote culture. Isolation of viable,

high quality male and female gametes and maintenance of ultra- structural integrity of the

isolated gametes is an essential and sophisticated step for in vitro fertilization. Isolated

sperm cells should be rounded after isolation but should remain structurally intact, viable,

functional and metabolically active. The history of flowering plant‟s sperm cell isolation goes


back to 1920s, when Finn (1925) isolated sperm cells from Asclepias. It was Cass who

reported the first isolation of sperm cells in Barley (Hordeum vulgare) in 1973 by brushing

anthesis of pollens in 20% sucrose solution. He dealt with the cellular characteristics of male

gametes and also described that sperm cells became spherical in shape after its release

from the pollen grains. Since then, numerous attempts were made for isolation and

characterization of male gametophytic cells in various taxa, viz. Lolium, Triticum aestivum,

Zea mays, Brassica napus, Nicotiana tabacum, Plumbago zeylanica and Torenia fournieri,

etc. in different laboratories worldwide.

The female gametes are located in the embryo sac, deeply embedded in the ovule.

Therefore, for single cell manipulation and gametic fusion these cells have to be isolated.

Female gametes are isolated by mechanical means, i.e. micro dissection and squashing and

enzymatic maceration technique. Mechanical isolation is a simple and manual technique and

is also used in combination with enzymatic maceration technique. The enzymatic

degradation of cell walls is rapid and relatively easy but it may harm the isolating embryo

sac which is the negative aspect of this technique. When we go back to history, the first

isolation of embryo sac was done in the mid of 20th century by Bradley from Nicotiana and

Petunia using acid hydrolyzation followed by mechanical isolation from fixed plant material.

Forbes in 1960, isolated the female gametophyte from Paspalum using enzymatic

degradation of cell wall. Thereafter, Hu et al. (1985) and Huang and Russell (1989) reported

the isolation of living and viable embryo sac and central cells for the first time in Nicotiana

and Plumbago, respectively. Since then, female gametes have been isolated employing

squashing, dissection, enzymatic degradation and osmotic shock from maternal structures

of various plant taxa, viz. Antirrhinum majus, Arabidopsis thaliana,Brassica campastris,

Helianthus annuus, Hordeum vulgare, Lilium longiflorum, Nicotiana tabacum, Oenothera

odorata, Paspalum sp., Petunia hybrida, Plumbago zeylenicum, Poa sp., Rudheckia laciniata,

Santalum album, Sesamum indicum, Torenia fournieri, Triticum aestivum, Vicia faba and

Zea mays.

As far as fusion of isolated male and female gametes is concerned, Chasan in 1992 quoted

that “in vitro fertilization will give botanists the opportunity to observe gamete binding and

fusion in real time, possibly yielding new information about the dynamics of fertilization”.

Different microscopic techniques (electron microscopy, phase contrast) combined with serial

sectioning of electrofusion products along with molecular analysis of fertilization and embryo

development have given new insights to in vitro fertilization system. Because the gametes

are considered as true protoplast, lacking a cell wall, their fusion can be done


spontaneously, chemically or by electrofusion method available for the fusion of somatic

protoplast and hybridization.

Spontaneous or chemically induced fusion has been performed in various taxa by means of

calcium-mediated fusion or osmotic- shock mediated fusion, i.e. fusion of isolated male and

female gametes occurs in the presence of an osmoticum (mannitol, sorbitol, sucrose)

mediated by calcium at high concentration. It was Kranz and Lörz (1994) who fused the

male and female gametes in a microdroplet of a fusiogenic medium (pH- 11.0) containing

0.01- 0.05 M CaCl2. Electrofusions are performed under microscopic observation on a

coverslip in microdroplets consisting 2000 nl mannitol solution that are overlayered by

mineral oil (Kranz and his coworkers in 1991, Kranz and Lorz, 1993). The fusion is induced

by single or multiple negative direct current pulses after dielectrophoretic alignment on one

of the electrodes fixed to a support under the condenser of an inverted microscope. The

frequencies of electrofusion of egg and sperm protoplast were very high, even high fusion

frequencies were also observed on the combinations of different protoplasts, viz. sperm cell,

synergid, central cell and somatic cytoplast.

However, the electrically performed in vitro fertilization techniques are much more efficient

for obtaining artificial zygotes in comparison to the spontaneous fusion method. Electrically

induced fusions are forced, unspecific and complete in less than 1s, hence it is not

appropriate for investigating the initial events associated with membrane and cytoplasmic

fusion between eggs and sperms. Contrary to this, calcium-mediated fusions occur over a

few seconds in which the site and fusion time can be easily programmed by the

experimenter for each pair of gametes. This method proved better to study gamete

recognition and the molecular mechanism of adhesion and fusion and also for better

understanding of double fertilization.

In vitro pollination (IVP) and in vitro fertilization (IVF) provides very effective techniques in

fundamental and applied areas of fertilization and seed development. In vitro system with

its controlled conditions provides a much more convenient method for understanding many

fundamental aspects particularly of ovule physiology before, during and after fertilization

than in vivo system. It is proved as an effective tool for overcoming the self- incompatibility

in several species. It is also helpful in inducing the formation of haploid plants. The placental

pollination method is promising in studies of induced parthenogenesis and mutation

research. It also provides a better mean of treating only the female partner (ovules) with

any chemical or physical factor. Use of pollen tube cultures for in vitro pollination offers a

convenient method for treating only the male partner (gametes in pollen tubes) with


chemical and physical mutagens. In vitro fertilization may be used to overcome inter-

specific recognition barriers occurring during pollination. This is presumably a more

rewarding substitute to delicate operations such as style grafting and stump pollination

which are not always easy to accomplish nor always successful in field conditions. Cultured

in vitro zygotes overcome a number of the intrinsic limitations of somatic cell hybridization

and provide a number of advantages for wide hybridization. First unequal division of zygote

can be observed directly and two celled embryo can be used as materials for further

analysis of developmental fates. Advances in in vitro fertilization constitute a unique model

for studies of early stages of zygotic embryogenesis, endosperm development and has

opened a vista for mechanisms of recognition, adhesion and fusion of gametes,

parthenogenesis and Polyspermy. This IVP and IVF technology has also been proved

beneficial to study the cellular events which take place immediately after fertilization as well

as gene regulation and molecular analysis of embryogenesis. Overall, this led to molecular

understanding and bioengineering in combination of cell hybridization techniques with

transformation and regeneration has given an impetus to manipulate the living reproductive

cells to plant scientists.

In vitro production of somatic hybrids

Plant protoplast fusion technology comprises protoplast isolation from somatic cells and

culture of the protoplasts thereby facilitating to combine these naked cells with each other

from different cultivars, species or genera. Subsequently, the fusion products are

regenerated either via organogenesis or embryogenesis to raise new hybrids and cybrids.

Such novel hybrids are produced bypassing the sex or sexual crossings employed in

traditional breeding schemes targeting the genetic transformation using isolated protoplasts

from two desirable plants.

Edward C. Cocking (1960) was pioneer to this technology who isolated the viable protoplast

enzymatically and published his research in Nature. However, the protoplast isolation was

first attempted by Klercker in 1892 from higher plants by mechanical disruption of the cell

wall with the help of a fine knife while still incubating them in a plasmolysing solution but

the number of protopalsts isolated with this method was very low.

.


Figure: E.C. Cocking;Cocking was pioneer for the enzymatic isolation and culture of

protoplasts from living cells in 1960.


Cocking established the enzymatic isolation of protoplasts employing cellulase enzyme

obtained from a fungus, Myrothecium verucaria. The isolation of protoplast by enzymatic

digestion accelerated when several cell wall degrading enzymes beacame available

commercially by 1968. Cellulase Orozuka RS, Cellulase R-10, Macerozyme R-10, Pectolyase

Y- 23, Driselase are some of the effective cell wall digestive enzymes which are available

commercially to date. This enzymatic degradation of plant cells for the isolation of

protoplast is routinely employed widely from any kind of cells, i.e. from cotyledons,

hypocotyls, and leaves of seedling grown either in vivo or in vitro. But the leaf tissues

excised from in vitro grown shoots or seedlings are the preferred source of material for

protoplast isolation. The viable protoplast was also liberated from mesophyll cells of tobacco

for the first time using commercially available cell wall degrading enzymes of bacterial and

fungal origin by Takebe and coworkers in 1968. The tissue was macerated with macerozyme

to obtain single cells in the first step followed by cell wall digestion with cellulase in the next

step. At the same time in 1968, Power and Cocking reported single step protoplast isolation

protocol employing double digestion of the plant tissues; this double digestion method

suppressed the mechanical method of protoplast isolation considerably. Takebe and his co-

workers finally led to successful regeneration of tobacco leaf prtoplast into whole plants.



Any kind of isolated naked cell (protoplast) tend to fuse if brought together even for a very

short duration (few minutes). Carlson and coworkers (1972) put forwarded the application

of certain fusogens to the isolated somatic cells (protoplasts) which accelerate this fusion,

eventually combining such isolated cells and giving a way to somatic hybridization. They

produced first somatic hybids in tobacco (Nicotiana tabaccum X N. langsdorfii) employing

NaNO3 as fusogen. However, NaNO3 was already recognized as a fusogen by Power and

coworkers in 1970. Keller and Melcher (1973) further demonstrated that an alkaline solution

of CaCl2 (pH 10.5, 50 mM) induces the protoplast fusion at 37 oC for about 30 min. and

obtained somatic hybrids in tobacco. Later this group reported several intra- specific and

inter- specific somatic hybrids in tobacco. Thereafter, polyethylene glycol (PEG) was

reported to be a potent fusogen by Kao and Micheyluk (1974) to achieve high frequency

protoplast fusion. Dudits and coworkers (1976) proposed that PEG may induce inter-

generic or inter- kingdom somatic hybrids.

Since, the fusogens need to be removed before culture and sometimes chemical fusogens

may be toxic to the plants this fusogen induced somatic hybridization faced serious

competitive substitution with the advent of electrofusion. Electrofusion introduced by Sanda

and his co workers in 1979 is relatively fast, non toxic synchronous and more reproducible

with greater fusion frequency and applicable to diverse taxa. Zimmermann and Vienken

(1982) were also the pioneers to this electrofusion technology for inducing somatic hybrids

who developed first automatic electrofusion system (Zimmermann electrofusion system)

with GCA Corp., Precision Scientific Group, USA which was claimed to be 10,000 times more

efficient to any other kind of protoplast fusion technology. In 1983, Koop and his co workers

demonstrated approximately 50 one-to- one fusion of protoplasts by microdroplet method of

electrofusion. Since then, a large number of electrofusion apparatus have been fabricated

for induction of somatic hybrids in isolated protoplasts in versatile plant systems.

In 1995, Makankawkeyoon and his coworkers succeeded in developing a novel somatic

hybrid between Tobacco X Mouse where mouse immunoglobulin G (introduced animal trait)

was expressed in tobacco leaves.


Figure: Fusion of Protoplasts, Depicting the phenomenon of protoplast fusion carried out

among protoplasts of two distinct species of plants which are fused together to form a new

hybrid plant with the characteristics of both.

Source: http://en.wikipedia.org/wiki/Somatic_fusion#/media/File:Protoplast_fusion.jpg

Somaclonal Variations

Considerable variations are observed when explants excised from plants are cultivated in

vitro. Such variations exhibited in plant tissue culture may be temporary (certain

physiological changes) or permanent that may be inherited from one generation to next

generation (genetic) or may not be transmitted sexually (epigenetic). Thus the genetic

variations observed in callus cultures were named as calciclones by Skirvin (1978).

Likewise, protoclones were assigned to the genetic variants obtained from protoplast

cultures by Shepard and his co workers in 1980. Similarly, the genetic variants observed in

gametic cell cultures were named as gametoclones. In 1981, Larkin and Scowcroft assigned

a general term “somaclonal variation” for any induced genetic or epigenetic variation in

micopropagated plants from any somatic cells. However, it was Heinz and Mee (1971) who

probably reported first somaclonal variants in sugarcane. Since then, this phenomenon has

been proved to be of immense utility and several valuable somaclones of sugarcane, potato,

tomato, flax, banana, etc. have been released worldwide.

http://en.wikipedia.org/wiki/Somatic_fusion#/media/File:Protoplast_fusion.jpg


Figure: Somaclonal variations; Mechanism exhibiting the appearance of genetic variants

due to the regulation of various factors viz. plant growth regulators, light/ dark conditions,

temperature, nutrients, etc. during in vitro plant regeneration that involves

dedifferentiataion and differentiation either via organogenesis or somatic embryogenesis.

Source: http://jxb.oxfordjournals.org/content/early/2011/05/26/jxb.err155/F2.large.jpg

(cc)

In vitro production of secondary metabolites and

metabolic engineering

Plants produce a wide diversity of organic compounds that includes alkaloids, flavonoids,

terpenoids, phenylpropanoids, fatty acid and amino acid derivatives, etc as a consequence

of their biosynthetic pathways. Such versatile phytochemicals commonly known as

secondary metabolites are of immense utility and being used as dyes, insecticides,

antimicrobial agents and more significantly in pharmaceuticals, nutraceuticals and

cosmetics. The certain biotechnological approaches such as plant tissue culture and

metabolic engineering give a way for the optimized production of desired plant derived

compounds.

http://jxb.oxfordjournals.org/content/early/2011/05/26/jxb.err155/F2.large.jpg


Figure: Image showing a closed Bioreactor for scaling up of desired bioactive natural

compounds.

Source:http://upload.wikimedia.org/wikipedia/commons/thumb/c/c7/Pg166_bioreactor.jpg/

375px-Pg166_bioreactor.jpg (cc)

The metabolic engineering can be defined as directed modification of cellular metabolism

and properties through the introduction, deletion and/ or targeted genetic modification of

metabolic pathways by using recombinant DNA and other molecular biology tools and

techniques. The strategies for metabolic engineering for improved production of secondary

metabolite involves screening and selection, in vitro culture medium optimization and

manipulating its physical and chemical components, selection of high producing plant cell

lines, precursor feeding and biotransformation, elicitor and stress induced production,

Agrobacterium rhizogenes mediated transformation and further scale up of secondary

metabolites through bioreactors, conversion of an existing product into a new product and

decreasing the catabolism of the desired compound, enhancing expression or activity of all

genes involved in the biosynthetic pathway of the specific secondary metabolite. Thus,

metabolic engineering is an ideal strategy in bioprocess optimization by means of

integrating optimization of cellular metabolism and the use of molecular biology tools for the

specific manipulation of the metabolic pathways. Significant advances in technological

approaches to improve a wide range of desired plant traits have been observed till date. The

inter disciplinary efforts involving several disciplines including biochemistry, molecular

biology, cell physiology and biochemical engineering and bioinformatics are crucial in this


endeavor and will play a significant role for the future advanced utility of metabolic

engineering.

Plant cell and tissue culture being very basic but essential to metabolic engineering has

been successfully used in large number of species to enhance specific biomolecule

(metabolite) by manipulating the physical (pH, temperature, light intensity, incubation

period, etc.) and chemical components (mineral nutrients, hormones, etc.) of culture

medium or exposing the cultures to a variety of abiotic and biotic elicitors.

The production of polyhydroxyalkanoate via metabolic engineering in Arabidopsis thaliana

was reported by Poirier and coworkers in 1992. Paclitaxel, a potent anti-cancer compound

extracted from the bark of Taxus brevifolia by Jaziri and coworkers (1996) was another

significant contribution in this direction. A 10–20-fold increase in productivity of taxol

compared with the average Taxus cultures was reported. A significant increase in the

production of vinblastine and vincristine through gene over expression studies was reported

in Catharanthus roseus by Peebles and his group in 2011. Employing Agrobacterium

rhizogenes for hairy roots formation, Yun and coworkers (1992) reported the increased

production of scopolamine in hairy roots of Atropa belladonna nicotine in Nicotiana

sylvestris. Similarly, Hallard and coworkers 1997 reported the improved production of

quinoline alkaloids in Cinchona ledgeriana through hairy roots formation. Employing

elicitation techniques, Sato et al. (2001) reported the over production of berberine in

Coptisjaponica and sanguinarine in Eschscholtzia californica. The naphthoquinone pigment

shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) possessing

antibacterial, anti-inflammatory wound-healing acitivities was the first plant secondary

metabolite produced in vitro from plant cell cultures on industrial scale by Mitsui

Petrochemical Co., Japan in 1984.

The original concept of edible vaccines implied that transgenic fruit or vegetable expressing

an antigen from a virus or bacteria can be eaten raw without any previous processing and

act as a vaccine for launching sufficiently protective immune response against a particular

disease. Some vegetables are used as the receptor of plant-derived vaccines because many

of them are free of toxicant, full of nutrients and fresh edible. Potato (Solanum tuberosum)

against cholera by Arakawa and coworkers (1997), tomato (Lycopersicon esculentum)

against cholera and hepatitis by He and coworkers (2008), and carrot (Daucus carota)

against diarrhea (Rosales-Mendoza and coworkers (2008) have been reported to

successfully express vaccine candidates. Some other examples of possible edible vaccines

include maize against diarrhea by Chikwamba and coworkers (2002), rice against cholera


by Nochi and coworkers (2007); alfalfa (Medicago sativa) against viral gastroenteritis by

Dong and coworkers (2005), etc. Dow Agrosciences, USA received world‟s first regulatory

approval from USDA for a plant cell made vaccine.

Genetic Engineering: Production of transgenic crops

Genetic engineering is alternation of the genome of an organism by introducing one or a few

specific gene (s) isolated from any living being such as bacteria, fungi, animals, plants

viruses. Such genetically engineered organisms are known as genetically modified

organisms (GMOs) and the crops are termed as transgenic crops or genetically modified

(GM) crops. The gene of interest (GOI) if transferred to the desired crop is known as

transgene. Today, introduction of any foreign gene into the plant genome, especially of

commercially important crop plants is a well established process for improving the crop

performance for various agronomic traits. Genetic engineering thus supplements

conventional breeding for development of transgenic plants involving a number of

biochemical and molecular techniques along with cell/ tissue selection by in vitro plant

regeneration and acclimatization; thereby being referred as molecular breeding, precision

breeding, genetic transformation, etc. If the goal is to develop a commercial variety, the

transgenic plants and their progenies are subjected to a series of tests and molecular

analysis to check their genetic stability to assure their field performance. In addition, the

product quality and safety are checked in order to comply with both market demands and

the relevant regulatory process.

As far as insertion of gene of interest (GOI) is concerned, it is just beginning of the process

that is achieved employing one of the two approaches. First, being the most preferred

choice for plant biotechnologists is the vector mediated gene transfer to the host genome.

In this approach a gram negative soil bacterium, Agrobacterium tumefaciens is employed to

transfer the foreign gene to the host. Another alternate to transfer alien gene directly into

the plant genome is either via electroporation, imbibition, microinjection, particle

bombardment and ultrasound induced, PEG induced, laser mediated, silicon carbide fiber

mediated, pollen mediated DNA uptake, etc.

Over a century, it is known that the two strains of Agrobacterium, A. tumefaciens and A.

rhizogenes are pathogenic and cause crown gall and hairy root in the plants, respectively. In

1947, Brawn suggested that a tumor inducing principal (TiP) of A. tumefaciens may be

responsible for autonomous growth of crown gall. In 1950, he further reported that the

bacterium transport the Ti principal into the plant genome naturally. Later in 1974, Zaemin


and his co workers revealed that the bacterium Ti plasmids are responsible for crown gall

formation. In 1977, it was Chilton and his coworkers who demonstrated that a piece of DNA

(T- DNA) from the bacterium plasmid Ti (tumor inducing) is inserted into the plant naturally

and is ultimately responsible for crown gall formation in plants. Today, Agrobacterium is

considered as natural genetic engineer and is the only example of inter- kingdom gene

transfer that facilitates transfer of low number of copies of genes into the plant nucleus. In

1983, Hoekama and his coworkers created first disarmed vector of ~150 kb by removing a

wild type T- DNA or oncogenes for transformation of the plants. This led to the development

of the most important tool in gene transfer technique, the binary vector in mid 1990s. In

this progression, Horsch and his coworkers (1985) reported the co- cultivation or leaf disc

method of transformation in tobacco where the wounded cells at the cut ends of the explant

were infected with Agrobacterium which eventually developed shoots. In this approach

different seedling explants such as node, petiole, hypocotyle, cotyledons, etc are used as

target tissue that have facilitated transformation in versatile dicotyledonous crops. This

method has not proved much effective in case of monocotyledonous plants, especially

cereals. This happens due to the different biochemical responses of wound heal mechanism

in dicots and monocots. However, transformation in cereals and other recalcitrant crops has

now become possible by the addition of acetosyringone (causes over expression of vir

genes) to the co- cultivation medium. The first monocot plant to be transformed by

Agrobacterium mediated transformation method was rice and subsequently, this has now

been achieved in several monocot crops, viz. Cenchrus, maize, wheat, etc. Today,

transformation relies on the regeneration efficiency of the explants in the culture medium

then the regeneration capacity of the transformants.

Of the various direct gene transformation methods available, particle bombardment or

microprojectile bombardment or biolistic method has also been another one of the most

preferable choice for plant biotechnologists due to its high transformation efficiency,

versatility and ease of adaptability to diverse range of plant cells, tissues, organs such as

leaves, stems, embryos, inflorescence, microspore, meristem, callus, etc. Various

parameters such as size and density of microparticles, osmoticum treatment, time of

preculture of the target tissue and certain physical parameters, i.e. vaccum, pressure and

distance of target tissues from the macro carrier disc are the critical factors to achieve

maximum transformation efficiency by particle bombardment transfer method. The ability to

tune all these factors accordingly makes this direct approach much versatile. However, a

major drawback that persists with biolistic method is randomness of DNA integration and

high copy number of the introduced gene. Another limitation is that it requires expensive


equipment. Moreover, this method overcomes the host limitations of Agrobacterium

mediated transformation method and effectively avoids integration of prokaryotic vector

sequences into the targeted cell/ tissue genome. It also prevails over the technical difficulty

of protoplast mediated gene transfer. Besides, this is the only efficient system for gene

transfer to the chloroplast and mitochondria genome. If we look at its history, biolistic

method of gene transfer was first introduced by Sanford and his coworkers in 1987 to

deliver accelerated DNA coated tungsten particles directly into plant cell genome. They

employed the first particle delivery apparatus, Biolistic Device Model BPG marketed by

Biolistics Inc. In this device, macrocarrier was propelled by gun powder to bombard the

tungsten particles (DNA coated) through the openings in the stopping screen to penetrate

the plant tissue below the screen. Later, PDS- 1000 device was developed by DuPoint Inc.

and it was further advanced to PDS- 1000/ He apparatus by BioRad laboratories. PDS-

1000/ He apparatus utilizes helium (He) gas as the accelerating force and is the most

commonly used equipment for direct gene transfer in to plant genome thereby facilitating

the generation of several transgenic crops. BioRad (2002) also assembled a semi-portable

particle bombardment apparatus, helios gene gun for field or greenhouse application. This

device employ helium accelerated DNA coated gold particles but it did not prove much

effective for the production of transgenics. In 1992, particle inflow gun (PIG), a low

cost alternative apparatus to PDS- 1000/ He was introduced by Finer and his coworkers.

This instrument proved effective towards tissues that are very much susceptible to burst of

gas or acoustic shock. Sauter (1993) reported a microtargeting bombardment device for

transformation of the intact tissues. This apparatus applies pressurized nitrogen gas and

enables to target the actively dividing totipotent cells in the shoot meristem region as small

as 150 µm. But the transformation efficiency achieved by this device has been reported to

be very poor. In addition to this, an apparatus based on ACCEL technology with an added

advantage of controlling the penetration of target tissue has also been described by Christou

and McCabe in 1992 and 1993. The device employ high voltage electrical discharge for DNA

coated gold particles and has been used for transformation of several important crops,

accurately.

Today, genetic engineering that has proved a useful technique either to insert a novel trait

or manipulating a known biochemical pathway for the production of large number of

transgenic crops has been progressed to the stage of commercialization. The GM crops for

large scale cultivation were first introduced in the US in 1995-1996 and since then several

GM crops have been released for commercial cultivation. In 1994, a genetically modified

tomato, Flavr Savr became the first commercially grown genetically engineered food to be


granted a license for human consumption. However, it had to be withdrawn from the market

soon due to certain issues and controversy with it.

In this progression, „Roundup ready‟ soyabean and brassica were generated by inserting a

bacterial, CP4 EPSPS from Agrobaterium in 1987 and 1993, respectively by Hinchee and his

teammates. Fitch et al., 1992 developed the virus resistant papaya plants which includes a

gene that made the papaya plants resistant to the ringspot virus. The Rainbow papaya is an

F-1 hybrid variety of papaya produced by crossing Hawaii‟s standard yellow-flesh export

variety, Kapoho Solo with the red-flesh SunUp, the first genetically engineered papaya with

resistance to papaya ringspot virus. The genetically altered papaya were generated and

brought to market (including 'SunUp' and 'Rainbow') that have some PRV DNA incorporated

into the DNA of the plant. Commercialized in 1998, the Rainbow papaya produced

immediate results. Within four years, the genetic improvement had not only stopped the

rapid decline of the Hawaii papaya industry, but production had actually returned to levels

near where they were before the papaya ringspot virus invasion.

In 1997, Ronald inserted the R gene Xa21 resistant to bacterial blight into domesticated rice

from wild type of rice (Oryza lagistamminata). Since then Xa21 has been introduced into

several important rice varieties but none of them appeared under large scale cultivation.

Rice is the major staple food worldwide but it is very poor in protein and vitamins.

Therefore, Ingo Potrycus and Peter Bayer in 1999 successfully engineered a rice variety,

japonica for the tissue specific expression of β- carotene (a precursor of vitamin A) in rice

endosperms and named it golden rice. The Golden rice was created by transforming rice

with two beta-carotene biosynthesis genes: psy (phytoene synthase) from daffodil

(Narcissus pseudonarcissus) and crtI from the soil bacterium, Erwinia uredovora. The total

carotenoids content was approximately 1.6 µg/gram of dry weight of grain. Subsequently,

the Golden rice2 was created by Jacqueline A. Paine & his team (Syngenta:

Biotechnology Company) in 2005. They combined the phytoene synthase gene

from maize (Zea mays) with crt1 from the soil bacterium, Erwinia uredovora. The total

carotenoids content was approximately 37 µg/gram of dry weight of grain and the beta-

carotene content was up to 31 µg/g of the 37 µg/g of carotenoids.

The Roundup Ready alfalfa (Medicago sativa), a genetically modified variety was released by

Forage Genetics International, USA in 2005. This was developed through the insertion of a

gene owned by Monsanto Company, USA that confers resistance to glyphosate, a broad-

spectrum herbicide. Although most grassy and broad leaf plants, including ordinary alfalfa,


are killed by Roundup, growers can spray fields of Roundup Ready alfalfa with the

glyphosate herbicide and kill the weeds without harming the alfalfa crop.

Different strains of gram positive bacterium, Bacillus thuringiensis (Bt) comprises over 240

insecticidal cry proteins. These cry proteins are potent bioinsecticides but are non- toxic to

human consumption or other animals. Therefore, the sprays based on such protoxins have

been employed successfully over 50 years. Apart from this approximately 10 cry genes

encoding different protoxin have been transformed into 26 plant species including maize,

cotton, soyabean, alfalfa, brinjal, broccoli, cabbage, tobacco, etc. Today, Bt- cotton, Bt-

maize, Bt- soyabean are under large scale cultivation worldwide. Nonetheless, Bt- cotton is

the only GM crop so far which has been approved by Government of India for commercial

cultivation in 2000. The Bt brinjal was created by inserting a crystal protein gene (Cry1Ac)

from the soil bacterium Bacillus thuringiensis into the genome of various brinjal cultivars in

the year 2000. The insertion of the gene, along with other genetic elements such as

promoters, terminators and an antibiotic resistance marker gene into the brinjal plant was

accomplished using Agrobacterium-mediated genetic transformation. The Bt brinjal has

been developed to give resistance against lepidopteron insects, in particular the brinjal fruit

and shoot borer (Leucinodes orbonalis). In India Mahyco in Jalna, Maharashtra has

developed the Bt brinjal but it could not through with the controversies with it for its

commercialization.

The Bt toxin gene were first engineered in 1987 in tobacco by different groups (Adang and

co workers, Benton and coworkers, Vavch and co workers) and tomato (Fischoff and his

coworkers). Bt- cotton with cry1Ac gene was the first commercially cultivated transgenic

crop in 1996.

A major setback in the commercialization of GM crops has been the apprehension of public.

Critics have objected to use of genetic engineering due to ethical, ecological and economic

concerns raised by the fact that GM techniques and GM organisms are subject to intellectual

property law. GMOs also are involved in controversies over GM food with respect to whether

food produced from GM crops is safe, whether it should be labeled and whether GM crops

are needed to address the world's food needs. Although, the USA and many developing

countries have accepted GM crops for large scale cultivation, most of the European countries

including the UK have not accepted the introduction of GM crops or their products. These

controversies have led to litigation, international trade disputes and protests and to

restrictive regulation of commercial products in most countries.


Exercise

Fill in the blanks

1. First Gynogenic haploids were raised in Hordeum vulgare.

2. Protoplasts are the cells devoid of cell wall.

3. Protoplast fusion breeding method uses a chemical to strip the cell wall of plant cells of

two sexually incompatible species.

4. Enzymatic isolation and culture of protoplasts was achieved by Edward C. Cocking.

5. Cellular totipotency is the property of plants.

6. Pioneer to Plant tissue culture technique in India wasPanchanan Maheshwari.

7. The pioneers of in vitro haploid culture were Guha and Maheshwari (1964).

8. F.C. Steward and J. Reinert (1958) were the pioneers in developing somatic embryos

from the somatic cells of carrot (somatic embryogenesis).

9. T. Murashige and F. Skoog (1962) formulated the most widely used plant tissue culture,

MS medium.

10. The first transgenic crop was tobacco.

11. First automatic electrofusion system was invented by Zimmerman and Vienken.

12. White and Nobecourt reported first callus culture in tobacco.

13. Stimulatory effect of coconut milk was first demonstrated by Van Overbeek (1941).

14. Woody Plant Medium(WPM) was formulated by Lloyd and McCown.

15. Agrobacterium is a gram negative soil bacterium.

16. Bt toxins are produced by a gram positive soil bacterium Bacillus thuringiensis.

17. ACCEL technology was first described by Christou and McCabe.

18. First ovarion pollination was standardized at University of Delhi, Delhi.

19. Kogl and his co workers discovered indole-3-acetic acid (IAA).

20. Genetic variants observed in gametic cells are referred as gametoclones.

21. Cocking isolated protoplast using an enzyme extracted from afugus.

22. Pollen embryos from anther cultures ofDatura innoxiawas the first major breakthrough in

androgenesis.

23. Agrobacterium tumefaciens causes crown gall disease and Agrobacterium rhizogenes

causes hairy roots in plants.

24. Self incompatibility can be overcome by in vitro pollination and fertilization.

25. First intra-ovarian pollination was seen in Papaver rhoeas.

26. Guha and Maheshwari (1964) are pioneers for anther culture.


27. Agrobacterium mediated transformation method in monocots was first demonstrated in

rice plants.

28. Skoog and Miller (1995) isolated first cytokinin which plays an important role in plant

tissue culture.

29. First successful plant regeneration from protoplasts was seen in tobacco.

30. Liabach achieved first embryo culture in Linum.

Multiple Choice Questions

1. The most common solidifying agent used in micropropagation is

a) Agar

b) Dextran

c) Mannan

d) All of these

2. Which of the following is best suited method for production of virus free plants

a) embryo culture

b) meristem culture

c) ovule culture

d) anther culture

3. The culturing of cells in liquid agitated medium is called

a) liquid culture

b) micropropagation

c) Agar culture

d) suspension culture

4. Artificial seeds are

a) seeds produced in laboratory condition

b) seeds encapsulated in a gel

c) somatic embryos encapsulated in a gel

d) zygotic embryos encapsulated in a gel

5. Hairy root cultures for secondary metabolite production are induced by transforming

plant cells with

a) virus

b) Agrobacterium tumefaciens

c) Bacillus thuringiensis

d) Agrobacterium rhizogenes

6. The variation in in vitro culture is called as

a) in vitro variation

b) mutation

c) somaclonal variation

d) all of these


7.Haploid plants are produced in large numbers by

a) anther culture

b) Ovary culture

c) both a and b

d) embryo culture

8. Who is the father of tissue culture

a) Bonner

b) Haberlandt

c) Laibach

d) Gautheret

9. The most widely used chemical for protoplast fusogens is

a) Manitol

b) Sorbitol

c) Mannol

d) Poly ethylene glycol (PEG)

10. Cybrids are produced by

a) Fusion of two different nuclei from two different species

b) Fusion of two same nuclei from same species

c) Nucleus of one species but cytoplasm from both the parent species

d) None of the above

11. Hormone pair required for a callus to differentiate are

a) auxin and cytokinin

b) auxin and ethylene

c) auxin and absiccic acid

d) cytokinins and gibberllin

12. Syntheticseedisproducedbyencapsulatingsomatic embryo with

a) sodium chloride

b) sodium alginate

c) sodium acetate

d) sodium nitrate

13. Callus is

a) Tissue that forms embryo

b) An insoluble carbohydrate

c) Tissue that grows to form embryoid

d) Unorganised actively dividing mass of cells maintained under in vitro conditions

14. The most important feature of the transgenic crop „Golden rice‟ is

a) insect resistence

b) disease resistence


c) high protein content

d) high vitamin A content

15. Haploids are important in crop improvement programme because

a) they can grow better in adverse conditions

b) are useful in the study of meiosis

c) require less of fertilizers as compared to diploids

d) give homozygous lines on diploidisation

16. First organ cutur ewas demonstrated by

a) White (1993)

b) Robbins and Kotte (1922)

c) Laibach (1929)

d) Gautheret and Nobecourt

17. Father of micropropagation is

a) E. Ball

b) G. Morel

c) G. Haberlandt

d) R. J. Gautheret

18. First embryo culture was achieved by

a) Libach (1925)

b) Hanning (1904)

c) Steward (1948)

d) Morel (1950)

19. Totipotency of endosperm cells was first demonstrated by

a) Kanta and Maheshwari (1960)

b) Johri and Bhojwani (1965)

c) Fujita and Tabata (1987)

d) Gautheret and Nobecourt (1939)

20. Chemical control of in vitro morphogenesis was postulated by

a) Skoog and Miller(1957)

b) Murashige and Skoog (1962)

c) Morel and Martin (1952)


21. First anther culture was achieved by

a) Guha and Maheshwari (1964)

b) Blackeslee and coworkers (1922)

c) Kasha and Kao (1970)

d) Kao and Micheyluk (1974)

22. In Golden rice 1 the psy (phytoene synthase) gene was obtained from


a) Erwinia uredovora

b) Narcissus pseudonarcisus

c) Zea maize

d) None

23. First GM crop which was granted license for human consumption

a) bt- brinjal

b) Roundup Soyabean

c) Flavr Savr Tomato

d) Bt- corn

24. Golden rice was created by

a) Peter Bayer

b) Ingo Potrycus

c) both a and b


25. Crystal protein gene (cry) was isolated from

a) Agrobacterium rhizogenesis

b) Agrobacterium tumefaciens

c) Leucinodes orbonalis

d) Bacillus thuringiensis

26. First disarmed vector was generated by

a) Hoekama and coworkers

b) Horsch and coworkers

c) Ingo Potrycus


27. Co- cultivation method of transformation is

a) Leaf disc method

b) Floral dip method

c) both a and b


28. Somaclonal variations are

a) Genetic changes

b) epigenetic changes

c) physiological changes and biochemical changes

d) both a and b

29. Name the first particle biolistic delivery apparatus

a) PDS- 1000/ He

b) Biolistic Device Model BPG

c) Illumina



30. Totipotency means

a) The ability of a cell to form an organ

b) The ability of a cell to form a new cell

c) Theability of a cell to form complete plant

d) The ability of a cell to undergo rapid cell division

31. Somatic embryogenesis is

a) A process in which zygotic embryos resemble somatic cells

b) A process in which somatic cells resemble zygotic embryos

c) A process in which somatic cells undergoes division like zygotic embryos and

forms new plants

d) A process in which zygotic embryos undergoes division to from only somatic

cells

32. Triploid culture means

a) Culture of three cells together

b) Culture of three nuclei

c) Culture of endosperm

d) Culture of megasporangium

33. The concept of artificial seeds was first defined by

a) Murashige and Skoog

b) Murashige

c) Murashige, Skoog and Miller

d) Bapat and Co- workers

References

Bhojwani S. S. and Dantu P. K. 2013. Plant Tissue Culture: An Introductory Text.

Springer India.

Bhojwani S. S. and Razdan M. K. 2005. Plant Tissue Culture: Theory and Practice.

Elsevier, New Delhi, India.

George E. F., Hall M. A. and De Klerk G. J. 2008. Plant Propagation by Tissue Culture

3rd Edition Vol. 1. The Background. Springer Netherlands.

Trigiano R. N. and Gray D. J. (eds). 1996. Plant Tissue Culture Concepts and

Laboratory Exercises. CRC Press, USA.


Street H. E. (ed.) 1977. Plant Tissue and Cell Culture. Blackswell Scientific Publications

Ltd. Australia.

Evans D. A., Sharp W. R., Ammirato P. V. and Yamada Y. 2009. Handbook of Plant Cell

Culture, Vol. 1, Techniques for Propagation and Breeding. Panima Publishing Corp.

New Delhi, India.

Web links

http://agriinfo.in/default.aspx?page=topic&superid=3&topicid=1881

http://www.slideshare.net/shivam_hayabusa/1plant-tissue-culture-history

http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&ved=0CEQ

QFjAF&url=http%3A%2F%2Fagridr.in%2FtnauEAgri%2Feagri50%2FGPBR311%2Flec

02.pdf&ei=lzJDVYyTJaHDmAWcu4H4Aw&usg=AFQjCNHmmeIwcSbkuKcUddTmB5plYU

yi2w&sig2=LVbMQzMxmcTn4EdQPKr7bQ&bvm=bv.92189499,d.dGY

http://agriinfo.in/default.aspx?page=topic&superid=3&topicid=1881

http://www.slideshare.net/shivam_hayabusa/1plant-tissue-culture-history

http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&ved=0CEQQFjAF&url=http%3A%2F%2Fagridr.in%2FtnauEAgri%2Feagri50%2FGPBR311%2Flec02.pdf&ei=lzJDVYyTJaHDmAWcu4H4Aw&usg=AFQjCNHmmeIwcSbkuKcUddTmB5plYUyi2w&sig2=LVbMQzMxmcTn4EdQPKr7bQ&bvm=bv.92189499,d.dGY




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